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Zhou SD, Zhou Q, Cui YD, Zhong X, Chen X, Lin XR, Yang ZN, Zhu J. Identification of Nuclear Localization Sequence (NLS) Sites in R2R3-MYB Transcription Factor Involved in Anther Development. Cells 2025; 14:470. [PMID: 40214424 PMCID: PMC11987959 DOI: 10.3390/cells14070470] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2025] [Revised: 03/01/2025] [Accepted: 03/19/2025] [Indexed: 04/14/2025] Open
Abstract
The R2R3-MYB family of transcription factors (TFs) plays a crucial role in cell specification and secondary metabolism regulation during plant development. In Arabidopsis, MS188, a typical R2R3-MYB protein, is essential for tapetal development and pollen wall formation. However, the nuclear localization sequence (NLS) responsible for directing MS188 into the nucleus has not been fully elucidated. In this study, the subcellular localization of the NLS-containing proteins was determined by GFP tagging in tobacco leaves, and three NLS regions within MS188 were identified: two located at the N-terminus of R2-MYB and one at the C-terminus of R3-MYB. We further narrowed the NLSs located at amino acids (AAs) 12-15, 18-22, and 96-107 via point mutation analysis. Combined with the cytoplasmic protein FBA6, these NLSs fusion proteins could localize in the nucleus. Importantly, the proteins with mutations in AAs 18-22 exhibited completely cytoplasmic signals, whereas other mutated sites partially abolished the nuclear signals. These findings suggest that the NLS at AAs 18-22 is sufficient for nuclear localization. To confirm the NLS functions in vivo, we constructed the vectors including the MS188 gene without the NLS sites, which failed to complement the male sterile phenotype of ms188. We also searched the highly conserved NLSs in other R2R3-MYB TFs and showed they are required for nuclear localization. Collectively, these findings revealed the specific NLS regions within R2R3-MYB transcription factors and highlighted their critical role for subcellular localization in plant developmental regulation.
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Affiliation(s)
- Si-Da Zhou
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
- Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany
| | - Que Zhou
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
| | - Yan-Dan Cui
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
| | - Xiang Zhong
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
| | - Xing Chen
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
| | - Xue-Rong Lin
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
| | - Zhong-Nan Yang
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
| | - Jun Zhu
- Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China; (S.-D.Z.); (Q.Z.); (Y.-D.C.); (X.Z.)
- Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
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Panagiotopoulos AA, Kalyvianaki K, Angelidaki A, Dellis D, Panagiotidis CA, Kampa M, Castanas E. The Sequence [RRKLPVGRS] Is a Nuclear Localization Signal for Importin 8 Binding (NLS8): A Chemical Biology and Bioinformatics Study. Int J Mol Sci 2025; 26:2814. [PMID: 40141456 PMCID: PMC11942892 DOI: 10.3390/ijms26062814] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2024] [Revised: 03/17/2025] [Accepted: 03/18/2025] [Indexed: 03/28/2025] Open
Abstract
Karyopherins, carrier proteins that recognize particular cargo protein patterns known as nuclear localization signals (NLSs), mediate the nuclear translocation of big proteins. In order to better understand the process of nuclear transport of proteins and create the groundwork for the development of innovative treatments that specifically target importins, it is imperative to clarify the intricate interactions between nuclear transporters and their cargo proteins. Until recently, very few NLSs have been documented. In the current work, an in silico method was used to identify NLSs for importin 8. It was determined that the sequence RRKLPVGRS serves as a recognition motif for importin 8 binding a karyopherin that is involved in the nuclear transportation of several important proteins like AGOs, SMADs, RPL23A, and TFE3. The sequence was validated in vitro in the breast cancer cell line T47D. This work subscribes to the effort to clarify the intricate relationships between nuclear transporters and their cargo proteins, in order to better understand the mechanism of nuclear transport of proteins and lay the groundwork for the development of novel therapeutics that target particular importins and have an immediate translational impact.
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Affiliation(s)
- Athanasios A. Panagiotopoulos
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, Voutes Campus, 71013 Heraklion, Greece; (K.K.); (A.A.); (M.K.); (E.C.)
| | - Konstantina Kalyvianaki
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, Voutes Campus, 71013 Heraklion, Greece; (K.K.); (A.A.); (M.K.); (E.C.)
| | - Aikaterini Angelidaki
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, Voutes Campus, 71013 Heraklion, Greece; (K.K.); (A.A.); (M.K.); (E.C.)
| | - Dimitris Dellis
- National Infrastructures for Research and Technology, 11523 Athens, Greece;
| | - Christos A. Panagiotidis
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Marilena Kampa
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, Voutes Campus, 71013 Heraklion, Greece; (K.K.); (A.A.); (M.K.); (E.C.)
| | - Elias Castanas
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, Voutes Campus, 71013 Heraklion, Greece; (K.K.); (A.A.); (M.K.); (E.C.)
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3
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Yi X, Hussain I, Zhang P, Xiao C. Nuclear-Targeting Peptides for Cancer Therapy. Chembiochem 2024; 25:e202400596. [PMID: 39215136 DOI: 10.1002/cbic.202400596] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 08/19/2024] [Accepted: 08/30/2024] [Indexed: 09/04/2024]
Abstract
Nucleus is the central regulator of cells that controls cell proliferation, metabolism, and cell cycle, and is considered the most important organelle in cells. The precision medicine that can achieve nuclear targeting has achieved good therapeutic effects in anti-tumor therapy. However, the presence of biological barriers such as cell membranes and nuclear membranes in cells limit the delivery of therapeutic agents to the nucleus. Therefore, developing effective nuclear-targeting drug delivery strategies is particularly important. Nuclear-targeting peptides are a class of functional peptides that can penetrate cell membranes and target the nucleus. They mainly recognize and bind to the nuclear transport molecules (such as Importin-α/β) and transport the therapeutic agents to the nucleus through nuclear pore complexes (NPC). This review summarizes the most recent developments of strategies for anti-tumor therapy utilizing nuclear-targeting peptides, which will ultimately contribute to the development of more effective nuclear-targeting strategies to achieve better anti-tumor outcomes.
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Affiliation(s)
- Xuan Yi
- Key Laboratory of Polymer Ecomaterials, Jilin Biomedical Polymers Engineering Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P.R. China
| | - Irshad Hussain
- Department of Chemistry and Chemical Engineering, SBA School of Science & Engineering, Lahore University of Management Sciences (LUMS). DHA, Lahore, 54792, Pakistan
| | - Peng Zhang
- Key Laboratory of Polymer Ecomaterials, Jilin Biomedical Polymers Engineering Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P.R. China
| | - Chunsheng Xiao
- Key Laboratory of Polymer Ecomaterials, Jilin Biomedical Polymers Engineering Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P.R. China
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Hua Y, Zhang J, Yang MY, Ren JY, Suo F, Liang L, Dong MQ, Ye K, Du LL. Structural duality enables a single protein to act as a toxin-antidote pair for meiotic drive. Proc Natl Acad Sci U S A 2024; 121:e2408618121. [PMID: 39485800 PMCID: PMC11551426 DOI: 10.1073/pnas.2408618121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Accepted: 09/05/2024] [Indexed: 11/03/2024] Open
Abstract
In sexual reproduction, selfish genetic elements known as killer meiotic drivers (KMDs) bias inheritance by eliminating gametes that do not carry them. The selective killing behavior of most KMDs can be explained by a toxin-antidote model, where a toxin harms all gametes while an antidote provides resistance to the toxin in carriers. This study investigates whether and how the KMD element tdk1 in the fission yeast Schizosaccharomyces pombe deploys this strategy. Intriguingly, tdk1 relies on a single protein product, Tdk1, for both killing and resistance. We show that Tdk1 exists in a nontoxic tetrameric form during vegetative growth and meiosis but transforms into a distinct toxic form in spores. This toxic form acquires the ability to interact with the histone reader Bdf1 and assembles into supramolecular foci that disrupt mitosis in noncarriers after spore germination. In contrast, Tdk1 synthesized during germination of carrier spores is nontoxic and acts as an antidote, dismantling the preformed toxic Tdk1 assemblies. Replacement of the N-terminal region of Tdk1 with a tetramer-forming peptide reveals its dual roles in imposing an autoinhibited tetrameric conformation and facilitating the assembly of supramolecular foci when autoinhibition is released. Moreover, we successfully reconstituted a functional KMD element by combining a construct that exclusively expresses Tdk1 during meiosis ("toxin-only") with another construct that expresses Tdk1 specifically during germination ("antidote-only"). This work uncovers a remarkable example of a single protein employing structural duality to form a toxin-antidote pair, expanding our understanding of the mechanisms underlying toxin-antidote systems.
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Affiliation(s)
- Yu Hua
- National Institute of Biological Sciences, Beijing102206, China
| | - Jianxiu Zhang
- Key Laboratory of RNA Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing100101, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing100049, China
| | - Man-Yun Yang
- National Institute of Biological Sciences, Beijing102206, China
| | - Jing-Yi Ren
- National Institute of Biological Sciences, Beijing102206, China
| | - Fang Suo
- National Institute of Biological Sciences, Beijing102206, China
| | - Lingfei Liang
- Key Laboratory of RNA Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing100101, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing100049, China
| | - Meng-Qiu Dong
- National Institute of Biological Sciences, Beijing102206, China
- Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing102206, China
| | - Keqiong Ye
- Key Laboratory of RNA Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing100101, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing100049, China
| | - Li-Lin Du
- National Institute of Biological Sciences, Beijing102206, China
- Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing102206, China
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Ahmad B, Lerma-Reyes R, Mukherjee T, Nguyen HV, Weber AL, Cummings EE, Schulze WX, Comer JR, Schrick K. Nuclear localization of Arabidopsis HD-Zip IV transcription factor GLABRA2 is driven by importin α. JOURNAL OF EXPERIMENTAL BOTANY 2024; 75:6441-6461. [PMID: 39058342 DOI: 10.1093/jxb/erae326] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/06/2024] [Accepted: 07/24/2024] [Indexed: 07/28/2024]
Abstract
GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to enhanced yellow fluorescent protein (EYFP) show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent with tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell type differentiation of the epidermis.
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Affiliation(s)
- Bilal Ahmad
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - Ruben Lerma-Reyes
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - Thiya Mukherjee
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
- Donald Danforth Plant Science Center, Olivette, MO 63132, USA
| | - Hieu V Nguyen
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - Audra L Weber
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - Emily E Cummings
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - Waltraud X Schulze
- Department of Plant Systems Biology, University of Hohenheim, Stuttgart, Germany
| | - Jeffrey R Comer
- Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA
| | - Kathrin Schrick
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
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6
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Liu H, Lu C, Liu XQ, Zhuo CJ, Luo RJ, Huang QT, Tang Z, Zhao CQ, Guerinot ML, Salt DE, Zhao FJ, Huang XY. A chloroplast localized heavy metal-associated domain containing protein regulates grain calcium accumulation in rice. Nat Commun 2024; 15:9265. [PMID: 39462135 PMCID: PMC11513116 DOI: 10.1038/s41467-024-53648-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Accepted: 10/16/2024] [Indexed: 10/28/2024] Open
Abstract
Calcium (Ca) is an essential mineral nutrient and plays a crucial signaling role in all living organisms. Increasing Ca content in staple foods such as rice is vital for improving Ca nutrition of humans. Here we map a quantitative trait locus that controls Ca concentration in rice grains and identify the causal gene as GCSC1 (Grain Ca and Sr Concentrations 1), which encodes a chloroplast vesicle localized homo-oligomeric protein. GCSC1 exhibits Ca2+ transport activity in heterologous assays in yeast and Xenopus laevis oocytes and is involved in the efflux of Ca2+ from the chloroplast to the cytosol. Knockout of GCSC1 results in increased chloroplast Ca concentration, lower stomatal conductance in leaves and enhanced Ca allocation to grains. Natural variation in grain Ca concentration is attributed to the variable expression of GCSC1 resulting from its promoter sequence variation. Our study identifies a chloroplast localized heavy metal-associated domain containing protein that regulates chloroplast Ca2+ efflux and provides a way to biofortify Ca in rice to benefit human nutrition.
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Affiliation(s)
- Huan Liu
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
| | - Cun Lu
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
| | - Xiang-Qian Liu
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
| | - Chen-Jin Zhuo
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
- Sanya Institute of Nanjing Agricultural University, Sanya, China
| | - Rong-Jian Luo
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
| | - Qiu-Tang Huang
- Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
| | - Zhong Tang
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
| | - Chun-Qing Zhao
- Key Laboratory of Integrated Pest Management on Crops in East China, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
| | - Mary Lou Guerinot
- Department of Biological Sciences, Dartmouth College, Hanover, NH, USA
| | - David E Salt
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, UK
| | - Fang-Jie Zhao
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China
| | - Xin-Yuan Huang
- State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China.
- Sanya Institute of Nanjing Agricultural University, Sanya, China.
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Ewerling A, May-Simera HL. Evolutionary trajectory for nuclear functions of ciliary transport complex proteins. Microbiol Mol Biol Rev 2024; 88:e0000624. [PMID: 38995044 PMCID: PMC11426024 DOI: 10.1128/mmbr.00006-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/13/2024] Open
Abstract
SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.
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Affiliation(s)
- Alexander Ewerling
- Faculty of Biology, Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany
| | - Helen Louise May-Simera
- Faculty of Biology, Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany
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8
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Nematollahzadeh S, Athukorala A, Donnelly CM, Pavan S, Atelie-Djossou V, Di Iorio E, Nath B, Helbig KJ, McSharry BP, Forwood JK, Sarker S, Alvisi G. Mechanistic Insights Into an Ancient Adenovirus Precursor Protein VII Show Multiple Nuclear Import Receptor Pathways. Traffic 2024; 25:e12953. [PMID: 39301720 DOI: 10.1111/tra.12953] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Revised: 07/10/2024] [Accepted: 07/30/2024] [Indexed: 09/22/2024]
Abstract
Adenoviral pVII proteins are multifunctional, highly basic, histone-like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus (HAdV)2, the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre-) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1), using a combination of structural, functional, and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)β1 and IMPα, respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting and intranuclear binding. Expression of wild-type and NLS defective derivatives Pre-pVII in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre-pVII nuclear import is dependent on IMPα/β1 and IMPβ1, but not on transportin-1 (IMPβ2). Clearly, AdVs evolved to maximize the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery.
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Affiliation(s)
| | - Ajani Athukorala
- Department of Microbiology, Anatomy, Physiology, and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
| | - Camilla M Donnelly
- School of Dentistry and Medical Sciences Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia
- Gulbali Institute, Charles Sturt University, Wagga Wagga, New South Wales, Australia
| | - Silvia Pavan
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | | | - Enzo Di Iorio
- Department of Molecular Medicine, University of Padua, Padua, Italy
| | - Babu Nath
- School of Dentistry and Medical Sciences Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia
| | - Karla J Helbig
- Department of Microbiology, Anatomy, Physiology, and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
| | - Brian P McSharry
- School of Dentistry and Medical Sciences Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia
- Gulbali Institute, Charles Sturt University, Wagga Wagga, New South Wales, Australia
| | - Jade K Forwood
- School of Dentistry and Medical Sciences Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia
- Gulbali Institute, Charles Sturt University, Wagga Wagga, New South Wales, Australia
| | - Subir Sarker
- Department of Microbiology, Anatomy, Physiology, and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
- Biomedical Sciences and Molecular Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Queensland, Australia
- Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, Queensland, Australia
| | - Gualtiero Alvisi
- Department of Molecular Medicine, University of Padua, Padua, Italy
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9
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Racca JD, Chen YS, Brabender AR, Battistin U, Weiss MA, Georgiadis MM. Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY. J Biol Chem 2024; 300:107683. [PMID: 39168182 PMCID: PMC11458547 DOI: 10.1016/j.jbc.2024.107683] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2024] [Revised: 08/04/2024] [Accepted: 08/06/2024] [Indexed: 08/23/2024] Open
Abstract
Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high-mobility group box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY high-mobility group domain bound to a DNA site containing consensus element 5'-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5'-CATTGT/ACAATG-3', shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever) and Arg20, Asn32, and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles (69-84°) of the distinct box-DNA complexes are similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36, or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10, or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective Ala substitutions exhibited significantly decreased transcriptional activation of SRY's principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.
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Affiliation(s)
- Joseph D Racca
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Yen-Shan Chen
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Adam R Brabender
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Umberto Battistin
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Michael A Weiss
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
| | - Millie M Georgiadis
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
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10
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Thevenin KR, Tieche IS, Di Benedetto CE, Schrager M, Dye KN. The small tumor antigen of Merkel cell polyomavirus accomplishes cellular transformation by uniquely localizing to the nucleus despite the absence of a known nuclear localization signal. Virol J 2024; 21:125. [PMID: 38831469 PMCID: PMC11149282 DOI: 10.1186/s12985-024-02395-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Accepted: 05/22/2024] [Indexed: 06/05/2024] Open
Abstract
BACKGROUND Merkel Cell Carcinoma (MCC) is an aggressive skin cancer that is three times deadlier than melanoma. In 2008, it was found that 80% of MCC cases are caused by the genomic integration of a novel polyomavirus, Merkel Cell Polyomavirus (MCPyV), and the expression of its small and truncated large tumor antigens (ST and LT-t, respectively). MCPyV belongs to a family of human polyomaviruses; however, it is the only one with a clear association to cancer. METHODS To investigate the role and mechanisms of various polyomavirus tumor antigens in cellular transformation, Rat-2 and 293A cells were transduced with pLENTI MCPyV LT-t, MCPyV ST, TSPyV ST, HPyV7 ST, or empty pLENTI and assessed through multiple transformation assays, and subcellular fractionations. One-way ANOVA tests were used to assess statistical significance. RESULTS Soft agar, proliferation, doubling time, glucose uptake, and serum dependence assays confirmed ST to be the dominant transforming protein of MCPyV. Furthermore, it was found that MCPyV ST is uniquely transforming, as the ST antigens of other non-oncogenic human polyomaviruses such as Trichodysplasia Spinulosa-Associated Polyomavirus (TSPyV) and Human Polyomavirus 7 (HPyV7) were not transforming when similarly assessed. Identification of structural dissimilarities between transforming and non-transforming tumor antigens revealed that the uniquely transforming domain(s) of MCPyV ST are likely located within the structurally dissimilar loops of the MCPyV ST unique region. Of all known MCPyV ST cellular interactors, 62% are exclusively or transiently nuclear, suggesting that MCPyV ST localizes to the nucleus despite the absence of a canonical nuclear localization signal. Indeed, subcellular fractionations confirmed that MCPyV ST could achieve nuclear localization through a currently unknown, regulated mechanism independent of its small size, as HPyV7 and TSPyV ST proteins were incapable of nuclear translocation. Although nuclear localization was found to be important for several transforming properties of MCPyV ST, some properties were also performed by a cytoplasmic sequestered MCPyV ST, suggesting that MCPyV ST may perform different transforming functions in individual subcellular compartments. CONCLUSIONS Together, these data further elucidate the unique differences between MCPyV ST and other polyomavirus ST proteins necessary to understand MCPyV as the only known human oncogenic polyomavirus.
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Affiliation(s)
- Kaira R Thevenin
- Department of Health Sciences, Stetson University, 421 N Woodland Blvd, DeLand, FL, 32723, USA
| | - Isabella S Tieche
- Department of Health Sciences, Stetson University, 421 N Woodland Blvd, DeLand, FL, 32723, USA
| | - Cody E Di Benedetto
- Department of Health Sciences, Stetson University, 421 N Woodland Blvd, DeLand, FL, 32723, USA
| | - Matt Schrager
- Department of Health Sciences, Stetson University, 421 N Woodland Blvd, DeLand, FL, 32723, USA
| | - Kristine N Dye
- Department of Health Sciences, Stetson University, 421 N Woodland Blvd, DeLand, FL, 32723, USA.
- Department of Biology, Stetson University, DeLand, FL, 32723, USA.
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11
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Yoshinari A, Isoda R, Yagi N, Sato Y, Lindeboom JJ, Ehrhardt DW, Frommer WB, Nakamura M. Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2024; 118:1699-1712. [PMID: 38509728 DOI: 10.1111/tpj.16699] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Revised: 01/06/2024] [Accepted: 02/14/2024] [Indexed: 03/22/2024]
Abstract
Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.
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Affiliation(s)
- Akira Yoshinari
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8601, Japan
- Institute of Advanced Research, Nagoya University, Nagoya, 464-0814, Japan
| | - Reika Isoda
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8601, Japan
| | - Noriyoshi Yagi
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8601, Japan
| | - Yoshikatsu Sato
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8601, Japan
| | - Jelmer J Lindeboom
- Department of Plant Biology, Carnegie Institution for Science, Stanford, California, 94305, USA
| | - David W Ehrhardt
- Department of Plant Biology, Carnegie Institution for Science, Stanford, California, 94305, USA
- Department of Biology, Stanford University, Stanford, California, 94305, USA
| | - Wolf B Frommer
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8601, Japan
- Institute for Molecular Physiology, Düsseldorf, 40225, Germany
| | - Masayoshi Nakamura
- Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8601, Japan
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12
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Schiefer S, Hale BG. Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2. Nat Commun 2024; 15:4484. [PMID: 38802340 PMCID: PMC11130243 DOI: 10.1038/s41467-024-48800-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Accepted: 05/15/2024] [Indexed: 05/29/2024] Open
Abstract
Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leverage TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map the proximal human proteomes of all seven canonical type I IFN signaling cascade members under basal and IFN-stimulated conditions. This uncovers a network of 103 high-confidence proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validates several known constitutive protein assemblies, while also revealing novel stimulus-dependent and -independent associations between key signaling molecules. Functional screening further identifies PJA2 as a negative regulator of IFN signaling via its E3 ubiquitin ligase activity. Mechanistically, PJA2 interacts with TYK2 and JAK1, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2 thereby restraining downstream STAT signaling. Our high-resolution proximal protein landscapes provide global insights into the type I IFN signaling network, and serve as a valuable resource for future exploration of its functional complexities.
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Affiliation(s)
- Samira Schiefer
- Institute of Medical Virology, University of Zurich, 8057, Zurich, Switzerland
- Life Science Zurich Graduate School, ETH and University of Zurich, 8057, Zurich, Switzerland
| | - Benjamin G Hale
- Institute of Medical Virology, University of Zurich, 8057, Zurich, Switzerland.
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13
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Yu S, Pan H, Yang H, Zhuang H, Yang H, Yu X, Zhang S, Fang M, Li T, Ge S, Xia N. A non-viral DNA delivery system consisting of multifunctional chimeric peptide fused with zinc-finger protein. iScience 2024; 27:109464. [PMID: 38558940 PMCID: PMC10981093 DOI: 10.1016/j.isci.2024.109464] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2023] [Revised: 02/06/2024] [Accepted: 03/07/2024] [Indexed: 04/04/2024] Open
Abstract
Non-viral gene delivery systems have received sustained attention as a promising alternative to viral vectors for disease treatment and prevention in recent years. Numerous methods have been developed to enhance gene uptake and delivery in the cytoplasm; however, due to technical difficulties and delivery efficiency, these systems still face challenges in a range of biological applications, especially in vivo. To alleviate this challenge, we devised a novel system for gene delivery based on a recombinant protein eTAT-ZF9-NLS, which consisted of a multifunctional chimeric peptide and a zinc-finger protein with sequence-specific DNA-binding activity. High transfection efficiency was observed in several mammalian cells after intracellular delivery of plasmid containing ZF9-binding sites mediated by eTAT-ZF9-NLS. Our new approach provides a novel transfection strategy and the transfection efficiency was confirmed both in vitro and in vivo, making it a preferential transfection reagent for possible gene therapy.
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Affiliation(s)
- Siyuan Yu
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Haifeng Pan
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Han Yang
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Haoyun Zhuang
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Haihui Yang
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Xuan Yu
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Shiyin Zhang
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Mujin Fang
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Tingdong Li
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Shengxiang Ge
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
| | - Ningshao Xia
- State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, NMPA Key Laboratory for Research and Evaluation of Infectious Disease Diagnostic Technology, the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University, Xiamen 361102, China
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14
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Giltrap A, Yuan Y, Davis BG. Late-Stage Functionalization of Living Organisms: Rethinking Selectivity in Biology. Chem Rev 2024; 124:889-928. [PMID: 38231473 PMCID: PMC10870719 DOI: 10.1021/acs.chemrev.3c00579] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2023] [Revised: 11/14/2023] [Accepted: 11/15/2023] [Indexed: 01/18/2024]
Abstract
With unlimited selectivity, full post-translational chemical control of biology would circumvent the dogma of genetic control. The resulting direct manipulation of organisms would enable atomic-level precision in "editing" of function. We argue that a key aspect that is still missing in our ability to do this (at least with a high degree of control) is the selectivity of a given chemical reaction in a living organism. In this Review, we systematize existing illustrative examples of chemical selectivity, as well as identify needed chemical selectivities set in a hierarchy of anatomical complexity: organismo- (selectivity for a given organism over another), tissuo- (selectivity for a given tissue type in a living organism), cellulo- (selectivity for a given cell type in an organism or tissue), and organelloselectivity (selectivity for a given organelle or discrete body within a cell). Finally, we analyze more traditional concepts such as regio-, chemo-, and stereoselective reactions where additionally appropriate. This survey of late-stage biomolecule methods emphasizes, where possible, functional consequences (i.e., biological function). In this way, we explore a concept of late-stage functionalization of living organisms (where "late" is taken to mean at a given state of an organism in time) in which programmed and selective chemical reactions take place in life. By building on precisely analyzed notions (e.g., mechanism and selectivity) we believe that the logic of chemical methodology might ultimately be applied to increasingly complex molecular constructs in biology. This could allow principles developed at the simple, small-molecule level to progress hierarchically even to manipulation of physiology.
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Affiliation(s)
- Andrew
M. Giltrap
- The
Rosalind Franklin Institute, Oxfordshire OX11 0FA, U.K.
- Department
of Pharmacology, University of Oxford, Oxford OX1 3QT, U.K.
| | - Yizhi Yuan
- The
Rosalind Franklin Institute, Oxfordshire OX11 0FA, U.K.
- Department
of Pharmacology, University of Oxford, Oxford OX1 3QT, U.K.
| | - Benjamin G. Davis
- The
Rosalind Franklin Institute, Oxfordshire OX11 0FA, U.K.
- Department
of Pharmacology, University of Oxford, Oxford OX1 3QT, U.K.
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15
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Cross EM, Akbari N, Ghassabian H, Hoad M, Pavan S, Ariawan D, Donnelly CM, Lavezzo E, Petersen GF, Forwood JK, Alvisi G. A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α-dependent nuclear localization signals. Protein Sci 2024; 33:e4876. [PMID: 38108201 PMCID: PMC10807245 DOI: 10.1002/pro.4876] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 12/12/2023] [Accepted: 12/13/2023] [Indexed: 12/19/2023]
Abstract
Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPβ1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, while bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) large tumor antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, merkel cell PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site.
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Affiliation(s)
- Emily M. Cross
- School of Dentistry and Medical SciencesCharles Sturt UniversityWagga WaggaAustralia
- Diamond Light SourceHarwell Science and Innovation CampusDidcotUnited Kingdom
| | - Nasim Akbari
- Department of Molecular MedicineUniversity of PadovaPadovaItaly
| | | | - Mikayla Hoad
- School of Dentistry and Medical SciencesCharles Sturt UniversityWagga WaggaAustralia
| | - Silvia Pavan
- Department of Molecular MedicineUniversity of PadovaPadovaItaly
| | - Daryl Ariawan
- Dementia Research CentreMacquarie UniversitySydneyAustralia
| | - Camilla M. Donnelly
- School of Dentistry and Medical SciencesCharles Sturt UniversityWagga WaggaAustralia
| | - Enrico Lavezzo
- Department of Molecular MedicineUniversity of PadovaPadovaItaly
| | | | - Jade K. Forwood
- School of Dentistry and Medical SciencesCharles Sturt UniversityWagga WaggaAustralia
- Gulbali InstituteCharles Sturt UniversityWagga WaggaAustralia
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16
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Yamada A, Wake K, Imaoka S, Motoyoshi M, Yamamoto T, Asano M. Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells. Sci Rep 2024; 14:1322. [PMID: 38225348 PMCID: PMC10789739 DOI: 10.1038/s41598-024-51521-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2023] [Accepted: 01/06/2024] [Indexed: 01/17/2024] Open
Abstract
Interleukin-1α (IL-1α), a cytokine released by necrotic cells, causes sterile inflammation. On the other hand, IL-1α is present in the nucleus and also regulates the expression of many proteins. A protein substrate containing a classical nuclear localization signal (cNLS) typically forms a substrate/importin α/β complex, which is subsequently transported to the nucleus. To the best of our knowledge, no study has directly investigated whether IL-1α-which includes cNLS-is imported into the nucleus in an importin α/β-dependent manner. In this study, we noted that all detected importin α subtypes interacted with IL-1α. In HeLa cells, importin α1-mediated nuclear translocation of IL-1α occurred at steady state and was independent of importin β1. Importin α1 not only was engaged in IL-1α nuclear transport but also concurrently functioned as a molecule that regulated IL-1α protein level in the cell. Furthermore, we discussed the underlying mechanism of IL-1α nuclear translocation by importin α1 based on our findings.
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Affiliation(s)
- Akiko Yamada
- Department of Pathology, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan.
- Division of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan.
| | - Kiyotaka Wake
- Department of Orthodontics, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
- Division of Oral Structural and Functional Biology, Nihon University Graduate School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
| | - Saya Imaoka
- Department of Pathology, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
- Division of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
| | - Mitsuru Motoyoshi
- Department of Orthodontics, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
- Division of Clinical Research, Dental Research Center, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
| | - Takenori Yamamoto
- Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-9501, Japan
- Institute for Genome Research, Tokushima University, Kuramotocho-3, Tokushima, 770-8503, Japan
| | - Masatake Asano
- Department of Pathology, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
- Division of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan
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17
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Goswami R, Gupta A, Bednova O, Coulombe G, Patel D, Rotello VM, Leyton JV. Nuclear localization signal-tagged systems: relevant nuclear import principles in the context of current therapeutic design. Chem Soc Rev 2024; 53:204-226. [PMID: 38031452 PMCID: PMC10798298 DOI: 10.1039/d1cs00269d] [Citation(s) in RCA: 10] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2023]
Abstract
Nuclear targeting of therapeutics provides a strategy for enhancing efficacy of molecules active in the nucleus and minimizing off-target effects. 'Active' nuclear-directed transport and efficient translocations across nuclear pore complexes provide the most effective means of maximizing nuclear localization. Nuclear-targeting systems based on nuclear localization signal (NLS) motifs have progressed significantly since the beginning of the current millennium. Here, we offer a roadmap for understanding the basic mechanisms of nuclear import in the context of actionable therapeutic design for developing NLS-therapeutics with improved treatment efficacy.
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Affiliation(s)
- Ritabrita Goswami
- Department of Chemistry, University of Massachusetts, Massachusetts, USA.
| | - Aarohi Gupta
- Department of Chemistry, University of Massachusetts, Massachusetts, USA.
| | - Olga Bednova
- Département de médecine nucléaire et radiobiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Québec, Canada
| | - Gaël Coulombe
- Service des stages et du développement professionnel, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Dipika Patel
- Service des stages et du développement professionnel, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Vincent M Rotello
- Department of Chemistry, University of Massachusetts, Massachusetts, USA.
| | - Jeffrey V Leyton
- École des sciences pharmaceutiques, Université d'Ottawa, Ottawa, Ontario, Canada.
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
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18
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Cai W, Feng Q, Wang L, Su S, Hou Z, Liu D, Kang X, Xu J, Pan Z, Tao J. Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix. Front Cell Infect Microbiol 2023; 13:1305727. [PMID: 38116134 PMCID: PMC10728482 DOI: 10.3389/fcimb.2023.1305727] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2023] [Accepted: 11/20/2023] [Indexed: 12/21/2023] Open
Abstract
Introduction Apicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2). Methods The cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein. Results The EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel β-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (P<0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of E. necatrix. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein. Discussion EnApiAP2 protein encoded by ENH_00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study.
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Affiliation(s)
- Weimin Cai
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Qianqian Feng
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Liyue Wang
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Shijie Su
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Zhaofeng Hou
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Dandan Liu
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Xilong Kang
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
| | - Jinjun Xu
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
| | - Zhiming Pan
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Principal's Office, Suqian University, Suqian, China
| | - Jianping Tao
- College of Veterinary Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China
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Moens U, Passerini S, Falquet M, Sveinbjørnsson B, Pietropaolo V. Phosphorylation of Human Polyomavirus Large and Small T Antigens: An Ignored Research Field. Viruses 2023; 15:2235. [PMID: 38005912 PMCID: PMC10674619 DOI: 10.3390/v15112235] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2023] [Revised: 11/02/2023] [Accepted: 11/07/2023] [Indexed: 11/26/2023] Open
Abstract
Protein phosphorylation and dephosphorylation are the most common post-translational modifications mediated by protein kinases and protein phosphatases, respectively. These reversible processes can modulate the function of the target protein, such as its activity, subcellular localization, stability, and interaction with other proteins. Phosphorylation of viral proteins plays an important role in the life cycle of a virus. In this review, we highlight biological implications of the phosphorylation of the monkey polyomavirus SV40 large T and small t antigens, summarize our current knowledge of the phosphorylation of these proteins of human polyomaviruses, and conclude with gaps in the knowledge and a proposal for future research directions.
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Affiliation(s)
- Ugo Moens
- Department of Medical Biology, Faculty of Health Sciences, University of Tromsø—The Arctic University of Norway, 9037 Tromsø, Norway; (M.F.); (B.S.)
| | - Sara Passerini
- Department of Public Health and Infectious Diseases, “Sapienza” University of Rome, 00185 Rome, Italy;
| | - Mar Falquet
- Department of Medical Biology, Faculty of Health Sciences, University of Tromsø—The Arctic University of Norway, 9037 Tromsø, Norway; (M.F.); (B.S.)
| | - Baldur Sveinbjørnsson
- Department of Medical Biology, Faculty of Health Sciences, University of Tromsø—The Arctic University of Norway, 9037 Tromsø, Norway; (M.F.); (B.S.)
| | - Valeria Pietropaolo
- Department of Public Health and Infectious Diseases, “Sapienza” University of Rome, 00185 Rome, Italy;
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20
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Ahmad B, Lerma-Reyes R, Mukherjee T, Nguyen HV, Weber AL, Schulze WX, Comer JR, Schrick K. Nuclear localization of HD-Zip IV transcription factor GLABRA2 is driven by Importin α. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.03.565550. [PMID: 37961624 PMCID: PMC10635128 DOI: 10.1101/2023.11.03.565550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2023]
Abstract
GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis , is a developmental regulator of specialized cell types in the epidermis. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain (HD). We demonstrate that NLS deletion or alanine substitution of its basic residues (KRKRKK) affects nuclear localization and results in a loss-of-function phenotype. Fusion of the predicted NLS (GTNKRKRKKYHRH) to the fluorescent protein EYFP is sufficient for its nuclear localization in roots and trichomes. The functional NLS is evolutionarily conserved in a distinct subset of HD-Zip IV members including PROTODERMAL FACTOR2 (PDF2). Despite partial overlap of the NLS with the HD, genetic dissection of the NLS from PDF2 indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plant tissues followed by mass spectrometry-based proteomics identified Importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Split-ubiquitin cytosolic yeast two-hybrid assays suggest interaction between GL2 and four IMPα isoforms from Arabidopsis. Direct interactions were verified in vitro by co-immunoprecipitation with recombinant proteins. IMPα triple mutants ( impα- 1,2,3 ) exhibit defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific and redundant functions of IMPα isoforms in Arabidopsis . Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 and other HD-Zip IV TFs in plants. One sentence summary GLABRA2, a representative HD-Zip IV transcription factor from Arabidopsis , contains an evolutionarily conserved monopartite nuclear localization sequence that is recognized by Importin α for translocation to the nucleus, a process that is necessary for cell-type differentiation of the epidermis.
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21
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Hill AC, Becker JP, Slominski D, Halloy F, Søndergaard C, Ravn J, Hall J. Peptide Conjugates of a 2'- O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes. ACS OMEGA 2023; 8:40463-40481. [PMID: 37929104 PMCID: PMC10620785 DOI: 10.1021/acsomega.3c05144] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Accepted: 09/26/2023] [Indexed: 11/07/2023]
Abstract
Antisense oligonucleotides (ASOs) are short, single-stranded nucleic acid molecules that alter gene expression. However, their transport into appropriate cellular compartments is a limiting factor in their potency. Here, we synthesized splice-switching oligonucleotides (SSOs) previously developed to treat the rare disease erythropoietic protoporphyria. Using chemical ligation-quantitative polymerase chain reaction (CL-qPCR), we quantified the SSOs in cells and subcellular compartments following free uptake. To drive nuclear localization, we covalently conjugated nuclear localization signal (NLS) peptides to a lead 2'-O-methoxyethyl phosphorothioate SSO using thiol-maleimide chemistry. The conjugates and parent SSO displayed similar RNA target-binding affinities. CL-qPCR quantification of the conjugates in cells and subcellular compartments following free uptake revealed one conjugate with better nuclear accumulation relative to the parent SSO. However, compared to the parent SSO, which altered the splicing of the target pre-mRNA, the conjugates were inactive at splice correction under free uptake conditions in vitro. Splice-switching activity could be conferred on the conjugates by delivering them into cells via cationic lipid-mediated transfection or by treating the cells into which the conjugates had been freely taken up with chloroquine, an endosome-disrupting agent. Our results identify the major barrier to the activity of the peptide-oligonucleotide conjugates as endosomal entrapment.
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Affiliation(s)
- Alyssa C. Hill
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule Zürich
(ETH Zürich), Zürich 8093, Switzerland
| | - J. Philipp Becker
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule Zürich
(ETH Zürich), Zürich 8093, Switzerland
| | - Daria Slominski
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule Zürich
(ETH Zürich), Zürich 8093, Switzerland
| | - François Halloy
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule Zürich
(ETH Zürich), Zürich 8093, Switzerland
| | | | - Jacob Ravn
- Roche
Innovation Center Copenhagen (RICC), Hørsholm 2970, Denmark
| | - Jonathan Hall
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule Zürich
(ETH Zürich), Zürich 8093, Switzerland
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22
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Spriggs CC. Viruses unveil the mechanisms of nuclear entry. Nat Rev Microbiol 2023; 21:632. [PMID: 37700053 DOI: 10.1038/s41579-023-00940-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/14/2023]
Affiliation(s)
- Chelsey C Spriggs
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA.
- Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
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23
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Lee RS, Geronimo CL, Liu L, Twarowski JM, Malkova A, Zakian VA. Identification of the nuclear localization signal in the Saccharomyces cerevisiae Pif1 DNA helicase. PLoS Genet 2023; 19:e1010853. [PMID: 37486934 PMCID: PMC10399864 DOI: 10.1371/journal.pgen.1010853] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2023] [Revised: 08/03/2023] [Accepted: 07/02/2023] [Indexed: 07/26/2023] Open
Abstract
Saccharomyces cerevisiae Pif1 is a multi-functional DNA helicase that plays diverse roles in the maintenance of the nuclear and mitochondrial genomes. Two isoforms of Pif1 are generated from a single open reading frame by the use of alternative translational start sites. The Mitochondrial Targeting Signal (MTS) of Pif1 is located between the two start sites, but a Nuclear Localization Signal (NLS) has not been identified. Here we used sequence and functional analysis to identify an NLS element. A mutant allele of PIF1 (pif1-NLSΔ) that lacks four basic amino acids (781KKRK784) in the carboxyl-terminal domain of the 859 amino acid Pif1 was expressed at wild type levels and retained wild type mitochondrial function. However, pif1-NLSΔ cells were defective in four tests for nuclear function: telomere length maintenance, Okazaki fragment processing, break-induced replication (BIR), and binding to nuclear target sites. Fusing the NLS from the simian virus 40 (SV40) T-antigen to the Pif1-NLSΔ protein reduced the nuclear defects of pif1-NLSΔ cells. Thus, four basic amino acids near the carboxyl end of Pif1 are required for the vast majority of nuclear Pif1 function. Our study also reveals phenotypic differences between the previously described loss of function pif1-m2 allele and three other pif1 mutant alleles generated in this work, which will be useful to study nuclear Pif1 functions.
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Affiliation(s)
- Rosemary S. Lee
- Department of Biology, University of Iowa, Iowa City, Iowa, United States of America
| | - Carly L. Geronimo
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | - Liping Liu
- Department of Biology, University of Iowa, Iowa City, Iowa, United States of America
| | - Jerzy M. Twarowski
- Department of Biology, University of Iowa, Iowa City, Iowa, United States of America
| | - Anna Malkova
- Department of Biology, University of Iowa, Iowa City, Iowa, United States of America
| | - Virginia A. Zakian
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
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24
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Gu J, Ye Y, Zheng ZW, Luo W, Gong YJ, Feng QL, Li S, Huang LH. Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2023; 157:103964. [PMID: 37230333 DOI: 10.1016/j.ibmb.2023.103964] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/25/2022] [Revised: 05/21/2023] [Accepted: 05/22/2023] [Indexed: 05/27/2023]
Abstract
Metamorphosis is one of the most important physiological processes in insects, which is coordinated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Ecdysone receptor (EcR) is a steroid receptor (SR), which usually presents in cytoplasm and transfers into nucleus after binding to 20E. Heat shock proteins (Hsps) are suggested to be important members of the SR complex. However, their role in nucleocytoplasmic shuttle of the EcR remains unclear. In the present study, we found that apoptozole (Hsp70 inhibitor) suppressed the larval molting by decreasing the expression of ecdysone signaling genes. Two cytoplasmic (Cy) Hsp70s (Hsp72 and Hsp73) interacted with both EcR and ultraspiracle (USP, the heterodimer partner of EcR). By immunohistochemistry experiments, we revealed that CyHsp70 co-localized with EcR in the cytoplasm, and that both apoptozole and interfering of CyHsp70 significantly inhibited the process of EcR entering the nucleus under 20E induction, while reducing the expression of ecdysone signaling genes. Interestingly, the nuclear localization of EcR was also promoted by two other stimuli, including JH and heat stress, and this promotion was inhibited by apoptozole. This implies that various stimuli can induce EcR entry into the nucleus, and that this process is mediated by CyHsp70. Curiously, neither JH nor heat stress activated the ecdysone signaling genes; instead, they have a significant inhibitory effect on them. Taken together, it seems that Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli, and that the biological effects of various stimuli passing through the EcR are different. Thus, our data provide a new viewpoint to understand the mechanism of nucleocytoplasmic shuttle of EcR.
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Affiliation(s)
- Jun Gu
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Yan Ye
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Zi-Wen Zheng
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Wei Luo
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Yan-Jun Gong
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Qi-Li Feng
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China
| | - Sheng Li
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China
| | - Li-Hua Huang
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China.
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25
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Jibiki K, Kodama TS, Yasuhara N. Importin alpha family NAAT/IBB domain: Functions of a pleiotropic long chameleon sequence. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2023; 134:175-209. [PMID: 36858734 DOI: 10.1016/bs.apcsb.2022.11.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Nuclear transport is essential for eukaryotic cell survival and regulates the movement of functional molecules in and out of the nucleus via the nuclear pore. Transport is facilitated by protein-protein interactions between cargo and transport receptors, which contribute to the expression and regulation of downstream genetic information. This chapter focuses on the molecular basis of the multifunctional nature of the importin α family, the representative transport receptors that bring proteins into the nucleus. Importin α performs multiple functions during the nuclear transport cycle through interactions with multiple molecules by a single domain called the IBB domain. This domain is a long chameleon sequence, which can change its conformation and binding mode depending on the interaction partners. By considering the evolutionarily conserved biochemical/physicochemical propensities of the amino acids constituting the functional complex interfaces, together with their structural properties, the mechanisms of switching between multiple complexes formed via IBB and the regulation of downstream functions are examined in detail. The mechanism of regulation by IBB indicates that the time has come for a paradigm shift in the way we view the molecular mechanisms by which proteins regulate downstream functions through their interactions with other molecules.
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Affiliation(s)
- Kazuya Jibiki
- Graduate School of Integrated Basic Sciences, Nihon University, Setagaya-ku, Tokyo, Japan
| | - Takashi S Kodama
- Laboratory of Molecular Biophysics, Institute for Protein Research, Osaka University, Osaka, Japan.
| | - Noriko Yasuhara
- Graduate School of Integrated Basic Sciences, Nihon University, Setagaya-ku, Tokyo, Japan.
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26
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Yuan Z, Geng Y, Dai Y, Li J, Lv M, Liao Q, Xie L, Zhang H. A fijiviral nonstructural protein triggers cell death in plant and bacterial cells via its transmembrane domain. MOLECULAR PLANT PATHOLOGY 2023; 24:59-70. [PMID: 36305370 PMCID: PMC9742498 DOI: 10.1111/mpp.13277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 10/06/2022] [Accepted: 10/07/2022] [Indexed: 05/10/2023]
Abstract
Southern rice black-streaked dwarf virus (SRBSDV; Fijivirus, Reoviridae) has become a threat to cereal production in East Asia in recent years. Our previous cytopathologic studies have suggested that SRBSDV induces a process resembling programmed cell death in infected tissues that results in distinctive growth abnormalities. The viral product responsible for the cell death, however, remains unknown. Here P9-2 protein, but not its RNA, was shown to induce cell death in Escherichia coli and plant cells when expressed either locally with a transient expression vector or systemically using a heterologous virus. Both computer prediction and fluorescent assays indicated that the viral nonstructural protein was targeted to the plasma membrane (PM) and further modification of its subcellular localization abolished its ability to induce cell death, indicating that its PM localization was required for the cell death induction. P9-2 was predicted to harbour two transmembrane helices within its central hydrophobic domain. A series of mutation assays further showed that its central transmembrane hydrophobic domain was crucial for cell death induction and that its conserved F90, Y101, and L103 amino acid residues could play synergistic roles in maintaining its ability to induce cell death. Its homologues in other fijiviruses also induced cell death in plant and bacterial cells, implying that the fijiviral nonstructural protein may trigger cell death by targeting conserved cellular factors or via a highly conserved mechanism.
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Affiliation(s)
- Zhengjie Yuan
- Laboratory of Virology, Institute of Virology and BiotechnologyZhejiang Academy of Agricultural SciencesHangzhouChina
| | - Yanfei Geng
- Laboratory of Virology, Institute of Virology and BiotechnologyZhejiang Academy of Agricultural SciencesHangzhouChina
| | - Yuanxing Dai
- Laboratory of Virology, Institute of Virology and BiotechnologyZhejiang Academy of Agricultural SciencesHangzhouChina
- College of Chemistry and Life ScienceZhejiang Normal UniversityJinhuaChina
| | - Jing Li
- Laboratory of Virology, Institute of Virology and BiotechnologyZhejiang Academy of Agricultural SciencesHangzhouChina
| | - Mingfang Lv
- Laboratory of Virology, Institute of Virology and BiotechnologyZhejiang Academy of Agricultural SciencesHangzhouChina
| | - Qiansheng Liao
- College of Life ScienceZhejiang Sci‐Tech UniversityHangzhouChina
| | - Li Xie
- Analysis Center of Agrobiology and Environmental SciencesZhejiang UniversityHangzhouChina
| | - Heng‐Mu Zhang
- Laboratory of Virology, Institute of Virology and BiotechnologyZhejiang Academy of Agricultural SciencesHangzhouChina
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27
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Yang J, Griffin A, Qiang Z, Ren J. Organelle-targeted therapies: a comprehensive review on system design for enabling precision oncology. Signal Transduct Target Ther 2022; 7:379. [PMID: 36402753 PMCID: PMC9675787 DOI: 10.1038/s41392-022-01243-0] [Citation(s) in RCA: 67] [Impact Index Per Article: 22.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 10/19/2022] [Accepted: 10/25/2022] [Indexed: 11/21/2022] Open
Abstract
Cancer is a major threat to human health. Among various treatment methods, precision therapy has received significant attention since the inception, due to its ability to efficiently inhibit tumor growth, while curtailing common shortcomings from conventional cancer treatment, leading towards enhanced survival rates. Particularly, organelle-targeted strategies enable precise accumulation of therapeutic agents in organelles, locally triggering organelle-mediated cell death signals which can greatly reduce the therapeutic threshold dosage and minimize side-effects. In this review, we comprehensively discuss history and recent advances in targeted therapies on organelles, specifically including nucleus, mitochondria, lysosomes and endoplasmic reticulum, while focusing on organelle structures, organelle-mediated cell death signal pathways, and design guidelines of organelle-targeted nanomedicines based on intervention mechanisms. Furthermore, a perspective on future research and clinical opportunities and potential challenges in precision oncology is presented. Through demonstrating recent developments in organelle-targeted therapies, we believe this article can further stimulate broader interests in multidisciplinary research and technology development for enabling advanced organelle-targeted nanomedicines and their corresponding clinic translations.
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Affiliation(s)
- Jingjing Yang
- grid.24516.340000000123704535Institute of Nano and Biopolymeric Materials, School of Materials Science and Engineering, Tongji University, 201804 Shanghai, China
| | - Anthony Griffin
- grid.267193.80000 0001 2295 628XSchool of Polymer Science and Engineering, University of Southern Mississippi, Hattiesburg, MS 39406 USA
| | - Zhe Qiang
- grid.267193.80000 0001 2295 628XSchool of Polymer Science and Engineering, University of Southern Mississippi, Hattiesburg, MS 39406 USA
| | - Jie Ren
- grid.24516.340000000123704535Institute of Nano and Biopolymeric Materials, School of Materials Science and Engineering, Tongji University, 201804 Shanghai, China
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Gao C, Lu S, Zhou R, Ding J, Fan J, Han B, Chen M, Wang B, Cao Y. Phylogenetic analysis and stress response of the plant U2 small nuclear ribonucleoprotein B″ gene family. BMC Genomics 2022; 23:744. [DOI: 10.1186/s12864-022-08956-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Accepted: 10/19/2022] [Indexed: 11/10/2022] Open
Abstract
Abstract
Background
Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear.
Result
Phylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein–protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins.
Conclusion
U2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.
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Varzandeh M, Labbaf S, Varshosaz J, Laurent S. An overview of the intracellular localization of high-Z nanoradiosensitizers. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2022; 175:14-30. [PMID: 36029849 DOI: 10.1016/j.pbiomolbio.2022.08.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/27/2021] [Revised: 07/17/2022] [Accepted: 08/19/2022] [Indexed: 06/15/2023]
Abstract
Radiation therapy (RT) is a method commonly used for cancer treatment worldwide. Commonly, RT utilizes two routes for combating cancers: 1) high-energy radiation to generate toxic reactive oxygen species (ROS) (through the dissociation of water molecules) for damaging the deoxyribonucleic acid (DNA) inside the nucleus 2) direct degradation of the DNA. However, cancer cells have mechanisms to survive under intense RT, which can considerably decrease its therapeutic efficacy. Excessive radiation energy damages healthy tissues, and hence, low doses are applied for cancer treatment. Additionally, different radiosensitizers were used to sensitize cancer cells towards RT through individual mechanisms. Following this route, nanoparticle-based radiosensitizers (herein called nanoradiosensitizers) have recently gained attention owing to their ability to produce massive electrons which leads to the production of a huge amount of ROS. The success of the nanoradiosensitizer effect is closely correlated to its interaction with cells and its localization within the cells. In other words, tumor treatment is affected from the chain of events which is started from cell-nanoparticle interaction followed by the nanoparticles direction and homing inside the cell. Therefore, passive or active targeting of the nanoradiosensitizers in the subcellular level and the cell-nano interaction would determine the efficacy of the radiation therapy. The importance of the nanoradiosensitizer's targeting is increased while the organelles beyond nucleus are recently recognized as the mediators of the cancer cell death or resistance under RT. In this review, the principals of cell-nanomaterial interactions and which dominate nanoradiosensitizer efficiency in cancer therapy, are thoroughly discussed.
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Affiliation(s)
- Mohammad Varzandeh
- Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran.
| | - Sheyda Labbaf
- Department of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran.
| | - Jaleh Varshosaz
- Novel Drug Delivery Systems Research Center and Department of Pharmaceutics, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
| | - Sophie Laurent
- Laboratory of NMR and Molecular Imaging, Department of General, Organic Chemistry and Biomedical, University of Mons, Mons, Belgium.
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30
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Panagiotopoulos AA, Kalyvianaki K, Tsodoulou PK, Darivianaki MN, Dellis D, Notas G, Daskalakis V, Theodoropoulos PA, Panagiotidis CΑ, Castanas E, Kampa M. Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation. Comput Struct Biotechnol J 2022; 20:5952-5961. [PMID: 36382187 PMCID: PMC9646746 DOI: 10.1016/j.csbj.2022.10.015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Revised: 10/10/2022] [Accepted: 10/11/2022] [Indexed: 01/21/2023] Open
Abstract
Nuclear translocation of large proteins is mediated through karyopherins, carrier proteins recognizing specific motifs of cargo proteins, known as nuclear localization signals (NLS). However, only few NLS signals have been reported until now. In the present work, NLS signals for Importins 4 and 5 were identified through an unsupervised in silico approach, followed by experimental in vitro validation. The sequences LPPRS(G/P)P and KP(K/Y)LV were identified and are proposed as recognition motifs for Importins 4 and 5 binding, respectively. They are involved in the trafficking of important proteins into the nucleus. These sequences were validated in the breast cancer cell line T47D, which expresses both Importins 4 and 5. Elucidating the complex relationships of the nuclear transporters and their cargo proteins is very important in better understanding the mechanism of nuclear transport of proteins and laying the foundation for the development of novel therapeutics, targeting specific importins.
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Affiliation(s)
| | - Konstantina Kalyvianaki
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece
| | - Paraskevi K. Tsodoulou
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
| | - Maria N. Darivianaki
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
| | - Dimitris Dellis
- National Infrastructures for Research and Technology, Athens 11523, Greece
| | - George Notas
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece
| | - Vangelis Daskalakis
- Department of Chemical Engineering, Cyprus University of Technology, Limassol, Cyprus
| | | | - Christos Α. Panagiotidis
- Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
| | - Elias Castanas
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece,Corresponding authors.
| | - Marilena Kampa
- Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece,Corresponding authors.
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31
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Bioinformatics and Functional Analysis of a New Nuclear Localization Sequence of the Influenza A Virus Nucleoprotein. Cells 2022; 11:cells11192957. [PMID: 36230922 PMCID: PMC9563117 DOI: 10.3390/cells11192957] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2022] [Revised: 09/16/2022] [Accepted: 09/19/2022] [Indexed: 11/30/2022] Open
Abstract
Influenza viruses deliver their genome into the nucleus of infected cells for replication. This process is mediated by the viral nucleoprotein (NP), which contains two nuclear localization sequences (NLSs): NLS1 at the N-terminus and a recently identified NLS2 (212GRKTR216). Through mutagenesis and functional studies, we demonstrated that NP must have both NLSs for an efficient nuclear import. As with other NLSs, there may be variations in the basic residues of NLS2 in different strains of the virus, which may affect the nuclear import of the viral genome. Although all NLS2 variants fused to the GFP mediated nuclear import of GFP, bioinformatics showed that 98.8% of reported NP sequences contained either the wild-type sequence 212GRKTR216 or 212GRRTR216. Bioinformatics analyses used to study the presence of NLS2 variants in other viral and nuclear proteins resulted in very low hits, with only 0.4% of human nuclear proteins containing putative NLS2. From these, we studied the nucleolar protein 14 (NOP14) and found that NLS2 does not play a role in the nuclear import of this protein but in its nucleolar localization. We also discovered a functional NLS at the C-terminus of NOP14. Our findings indicate that NLS2 is a highly conserved influenza A NP sequence.
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Wang N, Tang C, Fan X, He M, Gan P, Zhang S, Hu Z, Wang X, Yan T, Shu W, Yu L, Zhao J, He J, Li L, Wang J, Huang X, Huang L, Zhou JM, Kang Z, Wang X. Inactivation of a wheat protein kinase gene confers broad-spectrum resistance to rust fungi. Cell 2022; 185:2961-2974.e19. [PMID: 35839760 DOI: 10.1016/j.cell.2022.06.027] [Citation(s) in RCA: 102] [Impact Index Per Article: 34.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 05/10/2022] [Accepted: 06/14/2022] [Indexed: 01/21/2023]
Abstract
Wheat crops are frequently devastated by pandemic stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). Here, we identify and characterize a wheat receptor-like cytoplasmic kinase gene, TaPsIPK1, that confers susceptibility to this pathogen. PsSpg1, a secreted fungal effector vital for Pst virulence, can bind TaPsIPK1, enhance its kinase activity, and promote its nuclear localization, where it phosphorylates the transcription factor TaCBF1d for gene regulation. The phosphorylation of TaCBF1d switches its transcriptional activity on the downstream genes. CRISPR-Cas9 inactivation of TaPsIPK1 in wheat confers broad-spectrum resistance against Pst without impacting important agronomic traits in two years of field tests. The disruption of TaPsIPK1 leads to immune priming without constitutive activation of defense responses. Taken together, TaPsIPK1 is a susceptibility gene known to be targeted by rust effectors, and it has great potential for developing durable resistance against rust by genetic modifications.
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Affiliation(s)
- Ning Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China; State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Science, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Chunlei Tang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Xin Fan
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Mengying He
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Pengfei Gan
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Shan Zhang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Zeyu Hu
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Xiaodong Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Tong Yan
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Weixue Shu
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Ligang Yu
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Jinren Zhao
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Jiani He
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Lili Li
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Jianfeng Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Xueling Huang
- State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, China
| | - Lili Huang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Jian-Min Zhou
- State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology and the Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing 100101, China
| | - Zhensheng Kang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China.
| | - Xiaojie Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Pioneering Innovation Center for Wheat Stress Tolerance Improvement, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi 712100, China.
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Li H, Yao Y, Chen Y, Zhang S, Deng Z, Qiao W, Tan J. TRAF3IP3 Is Cleaved by EV71 3C Protease and Exhibits Antiviral Activity. Front Microbiol 2022; 13:914971. [PMID: 35814660 PMCID: PMC9260427 DOI: 10.3389/fmicb.2022.914971] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 05/17/2022] [Indexed: 11/27/2022] Open
Abstract
Enterovirus 71 (EV71) is one of the major pathogens of hand, foot, and mouth disease, which poses a major risk to public health and infant safety. 3C protease (3Cpro), a non-structural protein of EV71, promotes viral protein maturation by cleaving polyprotein precursors and facilitates viral immune escape by cleaving host proteins. In this study, we screened for human proteins that could interact with EV71 3Cpro using a yeast two-hybrid assay. Immune-associated protein TRAF3 Interacting Protein 3 (TRAF3IP3) was selected for further study. The results of co-immunoprecipitation and immunofluorescence demonstrated the interaction between TRAF3IP3 and EV71 3Cpro. A cleavage band was detected, indicating that both transfected 3Cpro and EV71 infection could cleave TRAF3IP3. 87Q-88G was identified as the only 3Cpro cleavage site in TRAF3IP3. In Jurkat and rhabdomyosarcoma (RD) cells, TRAF3IP3 inhibited EV71 replication, and 3Cpro cleavage partially resisted TRAF3IP3-induced inhibition. Additionally, the nuclear localization signal (NLS) and nuclear export signal (NES) of TRAF3IP3 were identified. The NES contributed to TRAF3IP3 alteration of 3Cpro localization and inhibition of EV71 replication. Together, these results indicate that TRAF3IP3 inhibits EV71 replication and 3Cpro resists such inhibition via proteolytic cleavage, providing a new example of virus-host interaction.
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34
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Delvaux NA, Mathew B, Rice KG. Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size. Anal Biochem 2022; 644:113895. [PMID: 32783899 PMCID: PMC7870724 DOI: 10.1016/j.ab.2020.113895] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Accepted: 07/27/2020] [Indexed: 11/16/2022]
Abstract
Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.
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Affiliation(s)
- Nathan A Delvaux
- Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, 52242, Iowa City, IA, USA
| | - Basil Mathew
- Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, 52242, Iowa City, IA, USA
| | - Kevin G Rice
- Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, 52242, Iowa City, IA, USA.
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35
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Zhang L, Sun K, Li Y, Ma T, Zhang Y, Yin Y, Zhang S, Shao W. The Importin FgPse1 Is Required for Vegetative Development, Virulence, and Deoxynivalenol Production by Interacting with the Nuclear Polyadenylated RNA-Binding Protein FgNab2 in Fusarium graminearum. PHYTOPATHOLOGY 2022; 112:1072-1080. [PMID: 34784736 DOI: 10.1094/phyto-08-21-0357-r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Karyopherins are involved in transport through nuclear pore complexes. Karyopherins are necessary for nuclear import and export pathways and bind to their cargos. Polyadenylation of messenger RNA (mRNA) is necessary for various biological processes, regulating gene expression in eukaryotes. Until now, the association of karyopherin with mRNA polyadenylation has been less understood in plant pathogenic fungi. In our study, we focused on the biological functions of the karyopherin FgPse1 in Fusarium graminearum. The results showed that FgPse1 is involved in mycelial growth, asexual reproduction, virulence, and deoxynivalenol (DON) production. Co-immunoprecipitation and bimolecular fluorescence complementation showed that FgPse1 interacts with the nuclear polyadenylated RNA-binding protein FgNab2. Moreover, a fluorescence localization assay indicated that FgPse1 is necessary for the nuclear import of FgNab2. The nuclear import of FgNab2 regulates the expression of FgTri4, FgTri5, and FgTri6, which are essential for DON production. Thus, ΔFgPse1 and ΔFgNab2 showed consistent defects in DON production. In summary, our data indicated that FgPse1 is necessary for mycelial growth, virulence, and DON production, interacting with FgNab2 in F. graminearum. These results contribute to our understanding of the functions of importins in phytopathogenic fungi.
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Affiliation(s)
- Lixin Zhang
- State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
| | - Kewei Sun
- State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
| | - Yiqing Li
- State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
| | - Tianling Ma
- State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
| | - Yu Zhang
- Department of Crop Protection, Zhejiang Agriculture and Forest University, Hangzhou 311300, China
| | - Yanni Yin
- State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
| | - Shuai Zhang
- National Agro-technology Extension and Service Center, Beijing 100125, China
| | - Wenyong Shao
- State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
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36
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Kim HJ, Mohassel P, Donkervoort S, Guo L, O'Donovan K, Coughlin M, Lornage X, Foulds N, Hammans SR, Foley AR, Fare CM, Ford AF, Ogasawara M, Sato A, Iida A, Munot P, Ambegaonkar G, Phadke R, O'Donovan DG, Buchert R, Grimmel M, Töpf A, Zaharieva IT, Brady L, Hu Y, Lloyd TE, Klein A, Steinlin M, Kuster A, Mercier S, Marcorelles P, Péréon Y, Fleurence E, Manzur A, Ennis S, Upstill-Goddard R, Bello L, Bertolin C, Pegoraro E, Salviati L, French CE, Shatillo A, Raymond FL, Haack TB, Quijano-Roy S, Böhm J, Nelson I, Stojkovic T, Evangelista T, Straub V, Romero NB, Laporte J, Muntoni F, Nishino I, Tarnopolsky MA, Shorter J, Bönnemann CG, Taylor JP. Heterozygous frameshift variants in HNRNPA2B1 cause early-onset oculopharyngeal muscular dystrophy. Nat Commun 2022; 13:2306. [PMID: 35484142 PMCID: PMC9050844 DOI: 10.1038/s41467-022-30015-1] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Accepted: 03/25/2022] [Indexed: 01/05/2023] Open
Abstract
Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics.
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Affiliation(s)
- Hong Joo Kim
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, United States
| | - Payam Mohassel
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States
| | - Sandra Donkervoort
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States
| | - Lin Guo
- Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, United States
| | - Kevin O'Donovan
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, United States
| | - Maura Coughlin
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, United States
| | - Xaviere Lornage
- Département Médecine Translationnelle et Neurogénétique, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U1258, Centre National de la Recherche Scientifique UMR7104, Université de Strasbourg, Illkirch, France
| | - Nicola Foulds
- Wessex Clinical Genetics Services, Princess Anne Hospital, Academic Unit of Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, England
| | - Simon R Hammans
- Wessex Neurological Centre, University Hospital Southampton, Southampton, UK
| | - A Reghan Foley
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States
| | - Charlotte M Fare
- Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States
| | - Alice F Ford
- Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States
| | - Masashi Ogasawara
- Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi, Kodaira, Tokyo, 187-8502, Japan
- Medical Genome Center, NCNP, Kodaira, Tokyo, Japan
| | - Aki Sato
- Department of Neurology, Niigata City General Hospital, Niigata, Japan
| | | | - Pinki Munot
- The Dubowitz Neuromuscular Centre, NIHR Great Ormond Street Hospital Biomedical Research Centre, Great Ormond Street Institute of Child Health, University College London, & Great Ormond Street Hospital Trust, London, UK
| | - Gautam Ambegaonkar
- Department of Paediatric Neurology, Cambridge University Hospital NHS Trust, Addenbrookes Hospital, Cambridge, CB2 0QQ, UK
| | - Rahul Phadke
- Division of Neuropathology, University College London Hospitals NHS Foundation Trust National Hospital for Neurology and Neurosurgery London, UK and Division of Neuropathology, UCL Institute of Neurology, Dubowitz Neuromuscular Centre, London, UK
| | - Dominic G O'Donovan
- Department of Histopathology Box 235, Level 5 John Bonnett Clinical Laboratories Addenbrooke's Hospital, Cambridge, UK
| | - Rebecca Buchert
- Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany
| | - Mona Grimmel
- Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany
| | - Ana Töpf
- John Walton Muscular Dystrophy Research Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK
| | - Irina T Zaharieva
- The Dubowitz Neuromuscular Centre, NIHR Great Ormond Street Hospital Biomedical Research Centre, Great Ormond Street Institute of Child Health, University College London, & Great Ormond Street Hospital Trust, London, UK
| | - Lauren Brady
- Division of Neuromuscular & Neurometabolic Disorders, Department of Pediatrics, McMaster University, Hamilton Health Sciences Centre, Hamilton, ON, Canada
| | - Ying Hu
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States
| | - Thomas E Lloyd
- Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Andrea Klein
- Division of Neuropaediatrics, Development and Rehabilitation, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
- Pediatric Neurology, University Children's Hospital Basel, University of Basel, Basel, Switzerland
| | - Maja Steinlin
- Division of Neuropaediatrics, Development and Rehabilitation, Department of Pediatrics, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
| | - Alice Kuster
- Department of Neurometabolism, University Hospital of Nantes, Nantes, France
| | - Sandra Mercier
- CHU Nantes, Service de génétique médicale, Centre de Référence des Maladies Neuromusculaires AOC, 44000, Nantes, France
- Université de Nantes, CNRS, INSERM, l'institut du thorax, 44000, Nantes, France
| | - Pascale Marcorelles
- Service d'anatomopathologie, CHU Brest and EA 4685 LIEN, Université de Bretagne Occidentale, Brest, France
| | - Yann Péréon
- CHU de Nantes, Centre de Référence des Maladies Neuromusculaires, Filnemus, Euro-NMD, Hôtel-Dieu, Nantes, France
| | - Emmanuelle Fleurence
- Etablissement de Santé pour Enfants et Adolescents de la région Nantaise, Nantes, France
| | - Adnan Manzur
- The Dubowitz Neuromuscular Centre, NIHR Great Ormond Street Hospital Biomedical Research Centre, Great Ormond Street Institute of Child Health, University College London, & Great Ormond Street Hospital Trust, London, UK
| | - Sarah Ennis
- Human Genetics and Genomic Medicine, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Rosanna Upstill-Goddard
- Human Genetics and Genomic Medicine, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Luca Bello
- Department of Neurosciences, DNS, University of Padova, Padova, Italy
| | - Cinzia Bertolin
- Clinical Genetics Unit, Department of Women and Children's Health, University of Padova, IRP Città della Speranza, Padova, Italy
| | - Elena Pegoraro
- Department of Neurosciences, DNS, University of Padova, Padova, Italy
| | - Leonardo Salviati
- Clinical Genetics Unit, Department of Women and Children's Health, CIR-Myo Myology Center, University of Padova, IRP Città della Speranza, Padova, Italy
| | | | - Andriy Shatillo
- Institute of Neurology, Psychiatry and Narcology of NAMS of Ukraine, Kharkiv, Ukraine
| | - F Lucy Raymond
- Cambridge Institute of Medical Research, University of Cambridge, Cambridge, UK
| | - Tobias B Haack
- Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany
| | - Susana Quijano-Roy
- Neuromuscular Unit, Pediatric Neurology and ICU Department, Raymond Poincaré Hospital (UVSQ), AP-HP Université Paris-Saclay, Garches, France
| | - Johann Böhm
- Département Médecine Translationnelle et Neurogénétique, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U1258, Centre National de la Recherche Scientifique UMR7104, Université de Strasbourg, Illkirch, France
| | - Isabelle Nelson
- Sorbonne Université, INSERM, Centre of Research in Myology, UMRS974, Paris, France
| | - Tanya Stojkovic
- APHP, Centre de Référence des Maladies Neuromusculaires Nord/Est/Ile de France, Institut de Myologie, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France
| | - Teresinha Evangelista
- Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France
| | - Volker Straub
- John Walton Muscular Dystrophy Research Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK
| | - Norma B Romero
- APHP, Centre de Référence des Maladies Neuromusculaires Nord/Est/Ile de France, Institut de Myologie, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France
- Unité de Morphologie Neuromusculaire, Institut de Myologie, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France
| | - Jocelyn Laporte
- Département Médecine Translationnelle et Neurogénétique, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U1258, Centre National de la Recherche Scientifique UMR7104, Université de Strasbourg, Illkirch, France
| | - Francesco Muntoni
- The Dubowitz Neuromuscular Centre, NIHR Great Ormond Street Hospital Biomedical Research Centre, Great Ormond Street Institute of Child Health, University College London, & Great Ormond Street Hospital Trust, London, UK
| | - Ichizo Nishino
- Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi, Kodaira, Tokyo, 187-8502, Japan
- Medical Genome Center, NCNP, Kodaira, Tokyo, Japan
| | - Mark A Tarnopolsky
- Division of Neuromuscular & Neurometabolic Disorders, Department of Pediatrics, McMaster University, Hamilton Health Sciences Centre, Hamilton, ON, Canada
| | - James Shorter
- Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States
| | - Carsten G Bönnemann
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States.
| | - J Paul Taylor
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, United States.
- Howard Hughes Medical Institute, Chevy Chase, MD, United States.
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Mondal B, Mahadik NS, Banerjee R, Sen Gupta S. Design and Synthesis of Shikimoylated-Polypeptides for Nuclear Specific Internalization. ACS Macro Lett 2022; 11:289-295. [PMID: 35575367 DOI: 10.1021/acsmacrolett.1c00740] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Targeted delivery of therapeutics such as small molecule drugs or nucleic acids exclusively to the nucleus of diseased mammalian cells poses a significant challenge. The development of targeting ligands that can specifically enter certain cancer cells via a specific receptor-mediated endocytosis and then traffic exclusively to the nucleus to deliver the cargo inside it can achieve this goal. We have developed an end-functionalized shikimoylated-polypeptide with pendant shikimoyl moieties that can enter mammalian cells via the mannose receptors and are then exclusively trafficked into the nucleus. The presence of the shikimoyl group in the polypeptide, which traffics it exclusively to the nucleus, contrasts with the mannosylated or galactosylated glycopolypeptides that are distributed all over the cytoplasm or the mannose-6-phosphate containing polypeptide that is exclusively trafficked to the lysosome. Using challenge experiments, we demonstrate that these polypeptides can enter both dendritic and cancer cells through mannose-receptors and subsequently enter the cell nucleus via the interaction with a nuclear pore complex (NPC) protein importin-α/β1. To the best of our knowledge, this represents the first example of a synthetic polyvalent glycopolypeptide mimic that performs the dual function of entering mammalian cells through specific receptors and subsequently traffics into the nucleus. The conjugation of these end-functionalized shikimoylated-polypeptides to other biological entities, such as recombinant anticancer drugs, DNA, RNA, and CRISPR-Cas9, may be a suitable alternative for delivery of these biological entities into cells affected by cancer and other genetic diseases.
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Affiliation(s)
- Basudeb Mondal
- Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, Mohanpur Campus, Nadia, West Bengal-741246, India
| | - Namita S. Mahadik
- Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India
- Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Ghaziabad-201002, Uttar Pradesh, India
| | - Rajkumar Banerjee
- Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana-500007, India
- Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Ghaziabad-201002, Uttar Pradesh, India
| | - Sayam Sen Gupta
- Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, Mohanpur Campus, Nadia, West Bengal-741246, India
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38
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Abstract
This review discusses peptide epitopes used as antigens in the development of vaccines in clinical trials as well as future vaccine candidates. It covers peptides used in potential immunotherapies for infectious diseases including SARS-CoV-2, influenza, hepatitis B and C, HIV, malaria, and others. In addition, peptides for cancer vaccines that target examples of overexpressed proteins are summarized, including human epidermal growth factor receptor 2 (HER-2), mucin 1 (MUC1), folate receptor, and others. The uses of peptides to target cancers caused by infective agents, for example, cervical cancer caused by human papilloma virus (HPV), are also discussed. This review also provides an overview of model peptide epitopes used to stimulate non-specific immune responses, and of self-adjuvanting peptides, as well as the influence of other adjuvants on peptide formulations. As highlighted in this review, several peptide immunotherapies are in advanced clinical trials as vaccines, and there is great potential for future therapies due the specificity of the response that can be achieved using peptide epitopes.
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Affiliation(s)
- Ian W Hamley
- Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, U.K
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39
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Wing CE, Fung HYJ, Chook YM. Karyopherin-mediated nucleocytoplasmic transport. Nat Rev Mol Cell Biol 2022; 23:307-328. [PMID: 35058649 PMCID: PMC10101760 DOI: 10.1038/s41580-021-00446-7] [Citation(s) in RCA: 157] [Impact Index Per Article: 52.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/09/2021] [Indexed: 12/25/2022]
Abstract
Efficient and regulated nucleocytoplasmic trafficking of macromolecules to the correct subcellular compartment is critical for proper functions of the eukaryotic cell. The majority of the macromolecular traffic across the nuclear pores is mediated by the Karyopherin-β (or Kap) family of nuclear transport receptors. Work over more than two decades has shed considerable light on how the different Kap family members bring their respective cargoes into the nucleus or the cytoplasm in efficient and highly regulated manners. In this Review, we overview the main features and established functions of Kap family members, describe how Kaps recognize their cargoes and discuss the different ways in which these Kap-cargo interactions can be regulated, highlighting new findings and open questions. We also describe current knowledge of the import and export of the components of three large gene expression machines - the core replisome, RNA polymerase II and the ribosome - pointing out the questions that persist about how such large macromolecular complexes are trafficked to serve their function in a designated subcellular location.
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40
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AlDaif BA, Mercer AA, Fleming SB. The parapoxvirus Orf virus ORF116 gene encodes an antagonist of the interferon response. J Gen Virol 2021; 102. [PMID: 34890310 DOI: 10.1099/jgv.0.001695] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Orf virus (ORFV) is the type species of the Parapoxvirus genus of the Poxviridae family. Genetic and functional studies have revealed ORFV has multiple immunomodulatory genes that manipulate innate immune responses, during the early stage of infection. ORF116 is a novel gene of ORFV with hitherto unknown function. Characterization of an ORF116 deletion mutant showed that it replicated in primary lamb testis cells with reduced levels compared to the wild-type and produced a smaller plaque phenotype. ORF116 was shown to be expressed prior to DNA replication. The potential function of ORF116 was investigated by gene-expression microarray analysis in HeLa cells infected with wild-type ORFV or the ORF116 deletion mutant. The analysis of differential cellular gene expression revealed a number of interferon-stimulated genes (ISGs) differentially expressed at either 4 or 6 h post infection. IFI44 showed the greatest differential expression (4.17-fold) between wild-type and knockout virus. Other ISGs that were upregulated in the knockout included RIG-I, IFIT2, MDA5, OAS1, OASL, DDX60, ISG20 and IFIT1 and in addition the inflammatory cytokine IL-8. These findings were validated by infecting HeLa cells with an ORF116 revertant recombinant virus and analysis of transcript expression by quantitative real time-PCR (qRT-PCR). These observations suggested a role for the ORFV gene ORF116 in modulating the IFN response and inflammatory cytokines. This study represents the first functional analysis of ORF116.
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Affiliation(s)
- Basheer A AlDaif
- Virus Research Unit, Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
| | - Andrew A Mercer
- Virus Research Unit, Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
| | - Stephen B Fleming
- Virus Research Unit, Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
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41
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Shelton SN, Smith SE, Unruh JR, Jaspersen SL. A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes. G3 (BETHESDA, MD.) 2021; 11:6400631. [PMID: 34849801 PMCID: PMC8664494 DOI: 10.1093/g3journal/jkab345] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Accepted: 09/10/2021] [Indexed: 01/24/2023]
Abstract
The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport; however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. We conducted a screen to determine how the INM proteome differs between mitotic cells and gametes. In addition, we used a strategy that allowed us to determine if spores synthesize their INM proteins de novo, rather than inheriting their INM proteins from the parental cell. This screen used a split-GFP complementation system, where we were able to compare the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation, and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis. Taken together, our data provide evidence for INM changes during gametogenesis and shed light on mechanisms used to shape the INM proteome of spores.
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Affiliation(s)
- Shary N Shelton
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Sarah E Smith
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Jay R Unruh
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Sue L Jaspersen
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA.,Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA
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42
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Ingram Z, Fischer DK, Ambrose Z. Disassembling the Nature of Capsid: Biochemical, Genetic, and Imaging Approaches to Assess HIV-1 Capsid Functions. Viruses 2021; 13:v13112237. [PMID: 34835043 PMCID: PMC8618418 DOI: 10.3390/v13112237] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Revised: 10/31/2021] [Accepted: 11/01/2021] [Indexed: 12/20/2022] Open
Abstract
The human immunodeficiency virus type 1 (HIV-1) capsid and its disassembly, or capsid uncoating, has remained an active area of study over the past several decades. Our understanding of the HIV-1 capsid as solely a protective shell has since shifted with discoveries linking it to other complex replication events. The interplay of the HIV-1 capsid with reverse transcription, nuclear import, and integration has led to an expansion of knowledge of capsid functionality. Coincident with advances in microscopy, cell, and biochemistry assays, several models of capsid disassembly have been proposed, in which it occurs in either the cytoplasmic, nuclear envelope, or nuclear regions of the cell. Here, we discuss how the understanding of the HIV-1 capsid has evolved and the key methods that made these discoveries possible.
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Affiliation(s)
- Zachary Ingram
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; (Z.I.); (D.K.F.)
- Pittsburgh Center for HIV Protein Interactions, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Douglas K. Fischer
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; (Z.I.); (D.K.F.)
- Pittsburgh Center for HIV Protein Interactions, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Zandrea Ambrose
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; (Z.I.); (D.K.F.)
- Pittsburgh Center for HIV Protein Interactions, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260, USA
- Correspondence:
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43
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Jibiki K, Kodama TS, Suenaga A, Kawase Y, Shibazaki N, Nomoto S, Nagasawa S, Nagashima M, Shimodan S, Kikuchi R, Okayasu M, Takashita R, Mehmood R, Saitoh N, Yoneda Y, Akagi KI, Yasuhara N. Importin α2 association with chromatin: Direct DNA binding via a novel DNA-binding domain. Genes Cells 2021; 26:945-966. [PMID: 34519142 DOI: 10.1111/gtc.12896] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 09/10/2021] [Accepted: 09/11/2021] [Indexed: 12/18/2022]
Abstract
The nuclear transport of proteins is important for facilitating appropriate nuclear functions. The importin α family proteins play key roles in nuclear transport as transport receptors for copious nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation. However, these nontransport functions of importin α are not yet fully understood, especially their molecular-level mechanisms and consequences for functioning with chromatin. Here, we report the novel molecular characteristics of importin α binding to diverse DNA sequences in chromatin. We newly identified and characterized a DNA-binding domain-the Nucleic Acid Associating Trolley pole domain (NAAT domain)-in the N-terminal region of importin α within the conventional importin β binding (IBB) domain that is necessary for nuclear transport of cargo proteins. Furthermore, we found that the DNA binding of importin α synergistically coupled the recruitment of its cargo protein to DNA. This is the first study to delineate the interaction between importin α and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin α N-terminal region for nuclear transport and chromatin association.
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Affiliation(s)
- Kazuya Jibiki
- Graduate School of Integrated Basic Sciences, Nihon University, Tokyo, Japan
| | - Takashi S Kodama
- National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan
| | - Atsushi Suenaga
- Graduate School of Integrated Basic Sciences, Nihon University, Tokyo, Japan.,Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Yota Kawase
- Graduate School of Integrated Basic Sciences, Nihon University, Tokyo, Japan
| | - Noriko Shibazaki
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Shin Nomoto
- Graduate School of Integrated Basic Sciences, Nihon University, Tokyo, Japan
| | - Seiya Nagasawa
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Misaki Nagashima
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Shieri Shimodan
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Renan Kikuchi
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Mina Okayasu
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Ruka Takashita
- Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
| | - Rashid Mehmood
- Department of Life Sciences, College of Science and General Studies, Alfaisal University, Riyadh, Saudi Arabia
| | - Noriko Saitoh
- Division of Cancer Biology, The Cancer Institute of JFCR, Tokyo, Japan
| | - Yoshihiro Yoneda
- National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan
| | - Ken-Ichi Akagi
- National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan.,Environmental Metabolic Analysis Research Team, RIKEN Center for Sustainable Resource Science, Yokohama, Japan
| | - Noriko Yasuhara
- Graduate School of Integrated Basic Sciences, Nihon University, Tokyo, Japan.,Department of Biosciences, College of Humanities and Sciences, Nihon University, Tokyo, Japan
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44
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Miyake T, McDermott JC. Nucleolar localization of c-Jun. FEBS J 2021; 289:748-765. [PMID: 34499807 DOI: 10.1111/febs.16187] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Revised: 08/09/2021] [Accepted: 09/06/2021] [Indexed: 01/20/2023]
Abstract
Nucleoli are well defined for their function in ribosome biogenesis, but only a small fraction of the nucleolar proteome has been characterized. Here, we report that the proto-oncogene, c-Jun, is targeted to the nucleolus. Using live cell imaging in myogenic cells, we document that the c-Jun basic domain contains a unique, evolutionarily conserved motif that determines nucleolar targeting. Fos family Jun dimer partners, such as Fra2, while nuclear, do not co-localize with c-Jun in the nucleolus. A point mutation in c-Jun that mimics Fra2 (M260E) in its Nucleolar Localization sequence (NoLS) results in loss of c-Jun nucleolar targeting while still preserving nuclear localization. Fra2 can sequester c-Jun in the nucleoplasm, indicating that the stoichiometric ratio of heterodimeric partners regulates c-Jun nucleolar targeting. Finally, nucleolar localization of c-Jun modulates nucleolar architecture and ribosomal RNA accumulation. These studies highlight a novel role for Jun family proteins in the nucleolus, having potential implications for a diverse array of AP-1-regulated cellular processes.
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Affiliation(s)
- Tetsuaki Miyake
- Department of Biology, York University, Toronto, ON, Canada.,Muscle Health Research Centre (MHRC), York University, Toronto, ON, Canada.,Centre for Research in Biomolecular Interactions (CRBI), York University, Toronto, ON, Canada
| | - John C McDermott
- Department of Biology, York University, Toronto, ON, Canada.,Muscle Health Research Centre (MHRC), York University, Toronto, ON, Canada.,Centre for Research in Biomolecular Interactions (CRBI), York University, Toronto, ON, Canada.,Centre for Research in Mass Spectrometry (CRMS), York University, Toronto, ON, Canada
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45
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Yang X, Dong W, Ren W, Zhao Q, Wu F, He Y. Cytoplasmic HYL1 modulates miRNA-mediated translational repression. THE PLANT CELL 2021; 33:1980-1996. [PMID: 33764452 PMCID: PMC8290291 DOI: 10.1093/plcell/koab090] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Accepted: 03/19/2021] [Indexed: 05/05/2023]
Abstract
MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation.
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Affiliation(s)
- Xi Yang
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
- University of the Chinese Academy of Sciences, Beijing 100049, China
| | - Weiguo Dong
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
- University of the Chinese Academy of Sciences, Beijing 100049, China
- School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
| | - Wenqing Ren
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
- University of the Chinese Academy of Sciences, Beijing 100049, China
| | - Qiuxia Zhao
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
| | - Feijie Wu
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
| | - Yuke He
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
- Author for correspondence:
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46
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Structural and calorimetric studies reveal specific determinants for the binding of a high-affinity NLS to mammalian importin-alpha. Biochem J 2021; 478:2715-2732. [PMID: 34195786 DOI: 10.1042/bcj20210401] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 06/21/2021] [Accepted: 06/25/2021] [Indexed: 11/17/2022]
Abstract
The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its nuclear localization sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high-affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3, the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.
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47
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Shin S, Kim SH, Lee JS, Lee GM. Streamlined Human Cell-Based Recombinase-Mediated Cassette Exchange Platform Enables Multigene Expression for the Production of Therapeutic Proteins. ACS Synth Biol 2021; 10:1715-1727. [PMID: 34133132 DOI: 10.1021/acssynbio.1c00113] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
A platform, based on targeted integration of transgenes using recombinase-mediated cassette exchange (RMCE) coupled with CRISPR/Cas9, is increasingly being used for the development of mammalian cell lines that produce therapeutic proteins, because of reduced clonal variation and predictable transgene expression. However, low efficiency of the RMCE process has hampered its application in multicopy or multisite integration of transgenes. To improve RMCE efficiency, nuclear transport of RMCE components such as site-specific recombinase and donor plasmid was accelerated by incorporation of nuclear localization signal and DNA nuclear-targeting sequence, respectively. Consequently, the efficiency of RMCE in dual-landing pad human embryonic kidney 293 (HEK293) cell lines harboring identical or orthogonal pairs of recombination sites at two well-known human safe harbors (AAVS1 and ROSA26 loci), increased 6.7- and 8.1-fold, respectively. This platform with enhanced RMCE efficiency enabled simultaneous integration of transgenes at the two sites using a single transfection without performing selection and enrichment processes. The use of a homotypic dual-landing pad HEK293 cell line capable of incorporating the same transgenes at two sites resulted in a 2-fold increase in the transgene expression level compared to a single-landing pad HEK293 cell line. In addition, the use of a heterotypic dual-landing pad HEK293 cell line, which can incorporate transgenes for a recombinant protein at one site and an effector transgene for cell engineering at another site, increased recombinant protein production. Overall, a streamlined RMCE platform can be a versatile tool for mammalian cell line development by facilitating multigene expression at genomic safe harbors.
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Affiliation(s)
- Seunghyeon Shin
- Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea
| | - Su Hyun Kim
- Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea
| | - Jae Seong Lee
- Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea
| | - Gyun Min Lee
- Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea
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Sajidah ES, Lim K, Wong RW. How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System. Cells 2021; 10:1424. [PMID: 34200500 PMCID: PMC8230057 DOI: 10.3390/cells10061424] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2021] [Revised: 05/31/2021] [Accepted: 06/03/2021] [Indexed: 12/14/2022] Open
Abstract
The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.
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Affiliation(s)
- Elma Sakinatus Sajidah
- Division of Nano Life Science in the Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
| | - Keesiang Lim
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan
| | - Richard W. Wong
- Division of Nano Life Science in the Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
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Sharma S, Jan R, Riar CS, Bansal V. Analyzing the effect of germination on the pasting, rheological, morphological and in- vitro antioxidant characteristics of kodo millet flour and extracts. Food Chem 2021; 361:130073. [PMID: 34029901 DOI: 10.1016/j.foodchem.2021.130073] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 04/05/2021] [Accepted: 05/08/2021] [Indexed: 11/17/2022]
Abstract
The present investigationwas carried out to determine the effect of germination on pasting, rheological, morphological properties of Kodo millet flour and in-vitroantioxidant characteristics of its phenolic and γ-amino butyric acid extracts. Rheological analysis depicted complex flour viscosity decreased with an improvement in shear intensity, symbolizing the shear-thinning action of flour upon germination. The frequency and temperature sweep demonstrated a decrease in visco-elasticity as a result of germination wherein, SEM revealed destruction in the continuous composite structure of starch which got entangled in dense protein matrix following germination. The in-vitroantioxidant activities such as total antioxidant capacity, DPPH*, FRAP, metal chelating ability and hydrogen peroxide scavenging activities of both the extracts increased significantly. There was a decrease in pasting properties and gelatinization behaviour whereas, visco-elastic solid behaviour changed to visco-elastic fluid. This research explores the potential of germinated Kodo millet flour for valuable bioactive compounds extraction and its utilization in product development.
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50
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Kalita J, Kapinos LE, Lim RYH. On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions. J Cell Sci 2021; 134:239102. [PMID: 33912945 DOI: 10.1242/jcs.240382] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the β-karyopherin (Kapβ) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapβs, cargoes and Ran to establish the asymmetry of NCT.
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Affiliation(s)
- Joanna Kalita
- Biozentrum and the Swiss Nanoscience Institute, University of Basel, Basel CH4056, Switzerland
| | - Larisa E Kapinos
- Biozentrum and the Swiss Nanoscience Institute, University of Basel, Basel CH4056, Switzerland
| | - Roderick Y H Lim
- Biozentrum and the Swiss Nanoscience Institute, University of Basel, Basel CH4056, Switzerland
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