|
1
|
Enenkel C, Ernst OP. Proteasome dynamics in response to metabolic changes. Front Cell Dev Biol 2025; 13:1523382. [PMID: 40099196 PMCID: PMC11911490 DOI: 10.3389/fcell.2025.1523382] [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: 11/05/2024] [Accepted: 02/03/2025] [Indexed: 03/19/2025] Open
Abstract
Proteasomes, essential protease complexes in protein homeostasis, adapt to metabolic changes through intracellular movements. As the executive arm of the ubiquitin-proteasome system, they selectively degrade poly-ubiquitinated proteins in an ATP-dependent process. The primary proteasome configuration involved in this degradation is the 26S proteasome, which is composed of a proteolytically active core particle flanked by two regulatory particles. In metabolically active cells, such as proliferating yeast and mammalian cancer cells, 26S proteasomes are predominantly nuclear and actively engaged in protein degradation. However, during nutrient deprivation or stress-induced quiescence, proteasome localization changes. In quiescent yeast, proteasomes initially accumulate at the nuclear envelope. During prolonged quiescence with decreased ATP levels, proteasomes exit the nucleus and are sequestered into cytoplasmic membraneless organelles, so-called proteasome storage granules (PSGs). In mammalian cells, starvation and stress trigger formation of membraneless organelles containing proteasomes and poly-ubiquitinated substrates. The proteasome condensates are motile, reversible, and contribute to stress resistance and improved fitness during aging. Proteasome condensation may involve liquid-liquid phase separation, a mechanism underlying the assembly of membraneless organelles.
Collapse
Affiliation(s)
- Cordula Enenkel
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Oliver P. Ernst
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| |
Collapse
|
|
2
|
Liu S, Atkinson E, Paulucci-Holthauzen A, Wang B. A CK2 and SUMO-dependent, PML NB-involved regulatory mechanism controlling BLM ubiquitination and G-quadruplex resolution. Nat Commun 2023; 14:6111. [PMID: 37777511 PMCID: PMC10542384 DOI: 10.1038/s41467-023-41705-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Accepted: 09/14/2023] [Indexed: 10/02/2023] Open
Abstract
The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex.
Collapse
Affiliation(s)
- Shichang Liu
- Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA
| | - Erin Atkinson
- Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA
- Genetics and Epigenetics Program, The MD Anderson Cancer Center and UT Health Graduate School of Biomedical Sciences, Houston, TX, 77030, USA
| | | | - Bin Wang
- Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA.
- Genetics and Epigenetics Program, The MD Anderson Cancer Center and UT Health Graduate School of Biomedical Sciences, Houston, TX, 77030, USA.
| |
Collapse
|
|
3
|
Iriki T, Iio H, Yasuda S, Masuta S, Kato M, Kosako H, Hirayama S, Endo A, Ohtake F, Kamiya M, Urano Y, Saeki Y, Hamazaki J, Murata S. Senescent cells form nuclear foci that contain the 26S proteasome. Cell Rep 2023; 42:112880. [PMID: 37541257 DOI: 10.1016/j.celrep.2023.112880] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Revised: 05/26/2023] [Accepted: 07/13/2023] [Indexed: 08/06/2023] Open
Abstract
The proteasome plays a central role in intracellular protein degradation. Age-dependent decline in proteasome activity is associated with cellular senescence and organismal aging; however, the mechanism by which the proteasome plays a role in senescent cells remains elusive. Here, we show that nuclear foci that contain the proteasome and exhibit liquid-like properties are formed in senescent cells. The formation of senescence-associated nuclear proteasome foci (SANPs) is dependent on ubiquitination and RAD23B, similar to previously known nuclear proteasome foci, but also requires proteasome activity. RAD23B knockdown suppresses SANP formation and increases mitochondrial activity, leading to reactive oxygen species production without affecting other senescence traits such as cell-cycle arrest and cell morphology. These findings suggest that SANPs are an important feature of senescent cells and uncover a mechanism by which the proteasome plays a role in senescent cells.
Collapse
Affiliation(s)
- Tomohiro Iriki
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Hiroaki Iio
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Shu Yasuda
- Department of Hygienic Chemistry and Medical Research Laboratories, School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 1088641, Japan
| | - Shun Masuta
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Masakazu Kato
- Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo 1648530, Japan
| | - Hidetaka Kosako
- Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Kuramoto-cho, Tokushima 7708503, Japan
| | - Shoshiro Hirayama
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Akinori Endo
- Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 1568506, Japan
| | - Fumiaki Ohtake
- Institute for Advanced Life Sciences, Hoshi University, Shinagawa-ku, Tokyo 1428501, Japan
| | - Mako Kamiya
- Department of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 2268501, Japan
| | - Yasuteru Urano
- Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan; Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Yasushi Saeki
- Division of Protein Metabolism, the Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 1088639, Japan
| | - Jun Hamazaki
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
| | - Shigeo Murata
- Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan.
| |
Collapse
|
|
4
|
Oxidative-Stress-Associated Proteostasis Disturbances and Increased DNA Damage in the Hippocampal Granule Cells of the Ts65Dn Model of Down Syndrome. Antioxidants (Basel) 2022; 11:antiox11122438. [PMID: 36552646 PMCID: PMC9774833 DOI: 10.3390/antiox11122438] [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: 08/23/2022] [Revised: 11/30/2022] [Accepted: 12/02/2022] [Indexed: 12/14/2022] Open
Abstract
Oxidative stress (OS) is one of the neuropathological mechanisms responsible for the deficits in cognition and neuronal function in Down syndrome (DS). The Ts65Dn (TS) mouse replicates multiple DS phenotypes including hippocampal-dependent learning and memory deficits and similar brain oxidative status. To better understand the hippocampal oxidative profile in the adult TS mouse, we analyzed cellular OS-associated alterations in hippocampal granule cells (GCs), a neuronal population that plays an important role in memory formation and that is particularly affected in DS. For this purpose, we used biochemical, molecular, immunohistochemical, and electron microscopy techniques. Our results indicate that TS GCs show important OS-associated alterations in the systems essential for neuronal homeostasis: DNA damage response and proteostasis, particularly of the proteasome and lysosomal system. Specifically, TS GCs showed: (i) increased DNA damage, (ii) reorganization of nuclear proteolytic factories accompanied by a decline in proteasome activity and cytoplasmic aggregation of ubiquitinated proteins, (iii) formation of lysosomal-related structures containing lipid droplets of cytotoxic peroxidation products, and (iv) mitochondrial ultrastructural defects. These alterations could be implicated in enhanced cellular senescence, accelerated aging and neurodegeneration, and the early development of Alzheimer's disease neuropathology present in TS mice and the DS population.
Collapse
|
|
5
|
Mandic R, Marquardt A, Terhorst P, Ali U, Nowak-Rossmann A, Cai C, Rodepeter FR, Stiewe T, Wezorke B, Wanzel M, Neff A, Stuck BA, Bette M. The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC. BMC Cancer 2022; 22:785. [PMID: 35850701 PMCID: PMC9290296 DOI: 10.1186/s12885-022-09854-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Accepted: 06/29/2022] [Indexed: 11/10/2022] Open
Abstract
Background More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. Methods We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. Results RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. Conclusions Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09854-0.
Collapse
Affiliation(s)
- Robert Mandic
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany.
| | - André Marquardt
- Comprehensive Cancer Center Mainfranken, University Hospital Würzburg, Würzburg, Germany.,Institute of Pathology, University of Würzburg, Würzburg, Germany.,Bavarian Cancer Research Center (BZKF), Würzburg, Germany
| | - Philip Terhorst
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany
| | - Uzma Ali
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany.,Institute for Pharmaceutical Technology & Biopharmacy, Philipps-Universität Marburg, Marburg, Germany
| | - Annette Nowak-Rossmann
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany.,Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps-Universität Marburg, Marburg, Germany
| | - Chengzhong Cai
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany
| | - Fiona R Rodepeter
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany.,Institute of Pathology, University Hospital Giessen and Marburg, Campus Marburg, Marburg, Germany
| | - Thorsten Stiewe
- Institute of Molecular Oncology, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Philipps-Universität Marburg, Marburg, Germany
| | - Bernadette Wezorke
- Institute of Molecular Oncology, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Philipps-Universität Marburg, Marburg, Germany
| | - Michael Wanzel
- Institute of Molecular Oncology, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Philipps-Universität Marburg, Marburg, Germany
| | - Andreas Neff
- Department of Oro- and Maxillofacial Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, Marburg, Germany
| | - Boris A Stuck
- Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Philipps-Universität Marburg, 3. BA, +3/08070, Marburg, Germany
| | - Michael Bette
- Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps-Universität Marburg, Marburg, Germany
| |
Collapse
|
|
6
|
Mee Hayes E, Sirvio L, Ye Y. A Potential Mechanism for Targeting Aggregates With Proteasomes and Disaggregases in Liquid Droplets. Front Aging Neurosci 2022; 14:854380. [PMID: 35517053 PMCID: PMC9062979 DOI: 10.3389/fnagi.2022.854380] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Accepted: 02/18/2022] [Indexed: 01/26/2023] Open
Abstract
Insoluble protein deposits are hallmarks of neurodegenerative disorders and common forms of dementia. The aberrant aggregation of misfolded proteins involves a complex cascade of events that occur over time, from the cellular to the clinical phase of neurodegeneration. Declining neuronal health through increased cell stress and loss of protein homeostasis (proteostasis) functions correlate with the accumulation of aggregates. On the cellular level, increasing evidence supports that misfolded proteins may undergo liquid-liquid phase separation (LLPS), which is emerging as an important process to drive protein aggregation. Studying, the reverse process of aggregate disassembly and degradation has only recently gained momentum, following reports of enzymes with distinct aggregate-disassembly activities. In this review, we will discuss how the ubiquitin-proteasome system and disaggregation machineries such as VCP/p97 and HSP70 system may disassemble and/or degrade protein aggregates. In addition to their canonically associated functions, these enzymes appear to share a common feature: reversibly assembling into liquid droplets in an LLPS-driven manner. We review the role of LLPS in enhancing the disassembly of aggregates through locally increasing the concentration of these enzymes and their co-proteins together within droplet structures. We propose that such activity may be achieved through the concerted actions of disaggregase machineries, the ubiquitin-proteasome system and their co-proteins, all of which are condensed within transient aggregate-associated droplets (TAADs), ultimately resulting in aggregate clearance. We further speculate that sustained engagement of these enzymatic activities within TAADs will be detrimental to normal cellular functions, where these activities are required. The possibility of facilitating endogenous disaggregation and degradation activities within TAADs potentially represents a novel target for therapeutic intervention to restore protein homeostasis at the early stages of neurodegeneration.
Collapse
Affiliation(s)
- Emma Mee Hayes
- Division of Neuroscience, Department of Brain Sciences, Imperial College London, London, United Kingdom
- UK Dementia Research Institute at Imperial College London, London, United Kingdom
| | - Liina Sirvio
- Division of Neuroscience, Department of Brain Sciences, Imperial College London, London, United Kingdom
- UK Dementia Research Institute at Imperial College London, London, United Kingdom
| | - Yu Ye
- Division of Neuroscience, Department of Brain Sciences, Imperial College London, London, United Kingdom
- UK Dementia Research Institute at Imperial College London, London, United Kingdom
- *Correspondence: Yu Ye,
| |
Collapse
|
|
7
|
Xu Y, Qiao H. A Hypothesis: Linking Phase Separation to Meiotic Sex Chromosome Inactivation and Sex-Body Formation. Front Cell Dev Biol 2021; 9:674203. [PMID: 34485277 PMCID: PMC8415632 DOI: 10.3389/fcell.2021.674203] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Accepted: 07/22/2021] [Indexed: 01/12/2023] Open
Abstract
During meiotic prophase I, X and Y chromosomes in mammalian spermatocytes only stably pair at a small homologous region called the pseudoautosomal region (PAR). However, the rest of the sex chromosomes remain largely unsynapsed. The extensive asynapsis triggers transcriptional silencing - meiotic sex chromosome inactivation (MSCI). Along with MSCI, a special nuclear territory, sex body or XY body, forms. In the early steps of MSCI, DNA damage response (DDR) factors, such as BRCA1, ATR, and γH2AX, function as sensors and effectors of the silencing signals. Downstream canonical repressive histone modifications, including methylation, acetylation, ubiquitylation, and SUMOylation, are responsible for the transcriptional repression of the sex chromosomes. Nevertheless, mechanisms of the sex-body formation remain unclear. Liquid-liquid phase separation (LLPS) may drive the formation of several chromatin subcompartments, such as pericentric heterochromatin, nucleoli, inactive X chromosomes. Although several proteins involved in phase separation are found in the sex bodies, when and whether these proteins exert functions in the sex-body formation and MSCI is still unknown. Here, we reviewed recent publications on the mechanisms of MSCI and LLPS, pointed out the potential link between LLPS and the formation of sex bodies, and discussed its implications for future research.
Collapse
Affiliation(s)
| | - Huanyu Qiao
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States
| |
Collapse
|
|
8
|
Kemp JP, Yang XC, Dominski Z, Marzluff WF, Duronio RJ. Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes. Mol Biol Cell 2021; 32:942-955. [PMID: 33788585 PMCID: PMC8108526 DOI: 10.1091/mbc.e20-10-0645] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a “core–shell” organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core–shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core–shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.
Collapse
Affiliation(s)
- James P Kemp
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Xiao-Cui Yang
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Zbigniew Dominski
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - William F Marzluff
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Robert J Duronio
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| |
Collapse
|
|
9
|
Pham K, Masoudi N, Leyva-Díaz E, Hobert O. A nervous system-specific subnuclear organelle in Caenorhabditis elegans. Genetics 2021; 217:1-17. [PMID: 33683371 PMCID: PMC8045701 DOI: 10.1093/genetics/iyaa016] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Accepted: 11/12/2020] [Indexed: 12/26/2022] Open
Abstract
We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4-10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.
Collapse
Affiliation(s)
- Kenneth Pham
- Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, NY 10027, USA
| | - Neda Masoudi
- Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, NY 10027, USA
| | - Eduardo Leyva-Díaz
- Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, NY 10027, USA
| | - Oliver Hobert
- Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, NY 10027, USA
| |
Collapse
|
|
10
|
Hsp40 proteins phase separate to chaperone the assembly and maintenance of membraneless organelles. Proc Natl Acad Sci U S A 2020; 117:31123-31133. [PMID: 33229560 DOI: 10.1073/pnas.2002437117] [Citation(s) in RCA: 50] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Membraneless organelles contain a wide spectrum of molecular chaperones, indicating their important roles in modulating the metastable conformation and biological function of membraneless organelles. Here we report that class I and II Hsp40 (DNAJ) proteins possess a high ability of phase separation rendered by the flexible G/F-rich region. Different Hsp40 proteins localize in different membraneless organelles. Specifically, human Hdj1 (DNAJB1), a class II Hsp40 protein, condenses in ubiquitin (Ub)-rich nuclear bodies, while Hdj2 (DNAJA1), a class I Hsp40 protein, condenses in nucleoli. Upon stress, both Hsp40 proteins incorporate into stress granules (SGs). Mutations of the G/F-rich region not only markedly impaired Hdj1 phase separation and SG involvement and disrupted the synergistic phase separation and colocalization of Hdj1 and fused in sarcoma (FUS) in cells. Being cophase separated with FUS, Hdj1 stabilized the liquid phase of FUS against proceeding into amyloid aggregation in vitro and alleviated abnormal FUS aggregation in cells. Moreover, Hdj1 uses different domains to chaperone FUS phase separation and amyloid aggregation. This paper suggests that phase separation is an intrinsic property of Hsp40 proteins, which enables efficient incorporation and function of Hsp40 in membraneless organelles and may further mediate the buildup of chaperone network in membraneless organelles.
Collapse
|
|
11
|
Shim MS, Nettesheim A, Hirt J, Liton PB. The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress. Autophagy 2020; 16:1248-1261. [PMID: 31476975 PMCID: PMC7469449 DOI: 10.1080/15548627.2019.1662584] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Revised: 08/18/2019] [Accepted: 08/28/2019] [Indexed: 12/19/2022] Open
Abstract
The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. ABBREVIATION AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.
Collapse
Affiliation(s)
- Myoung Sup Shim
- Department of Ophthalmology, Duke Eye Center, Duke University, Durham, NC, USA
| | - April Nettesheim
- Department of Ophthalmology, Duke Eye Center, Duke University, Durham, NC, USA
| | - Joshua Hirt
- Department of Ophthalmology, Duke Eye Center, Duke University, Durham, NC, USA
| | - Paloma B. Liton
- Department of Ophthalmology, Duke Eye Center, Duke University, Durham, NC, USA
| |
Collapse
|
|
12
|
Boulpicante M, Darrigrand R, Pierson A, Salgues V, Rouillon M, Gaudineau B, Khaled M, Cattaneo A, Bachi A, Cascio P, Apcher S. Tumors escape immunosurveillance by overexpressing the proteasome activator PSME3. Oncoimmunology 2020; 9:1761205. [PMID: 32923122 PMCID: PMC7458623 DOI: 10.1080/2162402x.2020.1761205] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Accepted: 04/03/2020] [Indexed: 11/09/2022] Open
Abstract
The success of CD8+ T cell-based cancer immunotherapy emphasizes the importance of understanding the mechanisms of generation of MHC-I peptide ligands and the possible pathways of tumor cell escape from immunosurveillance. Recently, we showed that peptides generated in the nucleus during a pioneer round of mRNA translation (pioneer translation products, or PTPs) are an important source of tumor specific peptides which correlates with the aberrant splicing and transcription events associated with oncogenesis. Here we show that up-regulation of PSME3 proteasome activator in cancer cells results in increased destruction of PTP-derived peptides in the nucleus thus enabling cancer cell to subvert immunosurveillance. These findings unveil a previously unexpected role for PSME3 in antigen processing and identify PSME3 as a druggable target to improve the efficacy of cancer immunotherapy.
Collapse
Affiliation(s)
- Mathilde Boulpicante
- Immunologie des Tumeurs et Immunothérapie, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Romain Darrigrand
- Immunologie des Tumeurs et Immunothérapie, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Alison Pierson
- Immunologie des Tumeurs et Immunothérapie, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Valérie Salgues
- Immunologie des Tumeurs et Immunothérapie, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Marine Rouillon
- Immunologie des Tumeurs et Immunothérapie, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Benoit Gaudineau
- Dynamique des Cellules Tumorales, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Mehdi Khaled
- Dynamique des Cellules Tumorales, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| | - Angela Cattaneo
- IFOM, The FIRC Institute of Molecular Oncology, Milano, Italy
| | - Angela Bachi
- IFOM, The FIRC Institute of Molecular Oncology, Milano, Italy
| | - Paolo Cascio
- Department of Veterinary Sciences, University of Turin, 10095, Grugliasco, Turin, Italy
| | - Sébastien Apcher
- Immunologie des Tumeurs et Immunothérapie, Université Paris-Saclay, Institut Gustave Roussy, Inserm, Villejuif, France
| |
Collapse
|
|
13
|
Marinello M, Werner A, Giannone M, Tahiri K, Alves S, Tesson C, den Dunnen W, Seeler JS, Brice A, Sittler A. SUMOylation by SUMO2 is implicated in the degradation of misfolded ataxin-7 via RNF4 in SCA7 models. Dis Model Mech 2019; 12:dmm.036145. [PMID: 30559154 PMCID: PMC6361149 DOI: 10.1242/dmm.036145] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2018] [Accepted: 12/04/2018] [Indexed: 01/10/2023] Open
Abstract
Perturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.
Collapse
Affiliation(s)
- Martina Marinello
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France.,Ecole Pratique des Hautes Etudes (EPHE), Paris Sciences et Lettres (PSL) Research University, Neurogenetics Group, 75013 Paris, France
| | - Andreas Werner
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany
| | - Mariagiovanna Giannone
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France.,Ecole Pratique des Hautes Etudes (EPHE), Paris Sciences et Lettres (PSL) Research University, Neurogenetics Group, 75013 Paris, France
| | - Khadija Tahiri
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France
| | - Sandro Alves
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France
| | - Christelle Tesson
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France.,Ecole Pratique des Hautes Etudes (EPHE), Paris Sciences et Lettres (PSL) Research University, Neurogenetics Group, 75013 Paris, France
| | - Wilfred den Dunnen
- Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands
| | - Jacob-S Seeler
- Nuclear Organization and Oncogenesis Unit, INSERM U.993, Department of Cell Biology and Infection, Institut Pasteur, F-75015 Paris, France
| | - Alexis Brice
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France.,AP-HP, Genetic Department, Pitié-Salpêtrière University Hospital, F-75013 Paris, France
| | - Annie Sittler
- Sorbonne Universités, UPMC, Univ Paris 06 UMRS 1127, INSERM U 1127, CNRS UMR 7225, ICM (Brain and Spine Institute) Pitié-Salpêtrière Hospital, 75013 Paris, France
| |
Collapse
|
|
14
|
Follo MY, Ratti S, Manzoli L, Ramazzotti G, Faenza I, Fiume R, Mongiorgi S, Suh PG, McCubrey JA, Cocco L. Inositide-Dependent Nuclear Signalling in Health and Disease. Handb Exp Pharmacol 2019; 259:291-308. [PMID: 31889219 DOI: 10.1007/164_2019_321] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Nuclear inositides have a specific subcellular distribution that is linked to specific functions; thus their regulation is fundamental both in health and disease. Emerging evidence shows that alterations in multiple inositide signalling pathways are involved in pathophysiology, not only in cancer but also in other diseases. Here, we give an overview of the main features of inositides in the cell, and we discuss their potential as new molecular therapeutic targets.
Collapse
Affiliation(s)
- Matilde Y Follo
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Stefano Ratti
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Lucia Manzoli
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Giulia Ramazzotti
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Irene Faenza
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Roberta Fiume
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Sara Mongiorgi
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Pann Ghill Suh
- Korea Brain Research Institute, Daegu, Republic of Korea.,School of Life Sciences, UNIST, Ulsan, Republic of Korea
| | - James A McCubrey
- Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA
| | - Lucio Cocco
- Cellular Signalling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy.
| |
Collapse
|
|
15
|
Narcís JO, Tapia O, Tarabal O, Piedrafita L, Calderó J, Berciano MT, Lafarga M. Accumulation of poly(A) RNA in nuclear granules enriched in Sam68 in motor neurons from the SMNΔ7 mouse model of SMA. Sci Rep 2018; 8:9646. [PMID: 29941967 PMCID: PMC6018117 DOI: 10.1038/s41598-018-27821-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Accepted: 06/11/2018] [Indexed: 01/07/2023] Open
Abstract
Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene, which results in reduced levels of the SMN protein and the selective degeneration of lower MNs. The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the pre-mRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN∆7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal nuclear accumulation in euchromatin domains of poly(A) RNA granules (PARGs) enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were accompanied by changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. PARG-containing MNs were observed at presymptomatic SMA stage, increasing their number during the symptomatic stage. Moreover, the massive accumulations of poly(A) RNA granules in MNs was accompanied by the cytoplasmic depletion of polyadenylated mRNAs for their translation. We suggest that the SMN-dependent abnormal accumulation of polyadenylated mRNAs and Sam68 in PARGs reflects a severe dysfunction of both mRNA processing and translation, which could contribute to SMA pathogenesis.
Collapse
Affiliation(s)
- J Oriol Narcís
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Santander, Spain
| | - Olga Tapia
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Santander, Spain
| | - Olga Tarabal
- Department of Experimental Medicine, School of Medicine, University of Lleida and "Institut de Recerca Biomèdica de Lleida" (IRBLLEIDA), Lleida, Spain
| | - Lídia Piedrafita
- Department of Experimental Medicine, School of Medicine, University of Lleida and "Institut de Recerca Biomèdica de Lleida" (IRBLLEIDA), Lleida, Spain
| | - Jordi Calderó
- Department of Experimental Medicine, School of Medicine, University of Lleida and "Institut de Recerca Biomèdica de Lleida" (IRBLLEIDA), Lleida, Spain
| | - Maria T Berciano
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Santander, Spain.,Department of Molecular Biology and CIBERNED, University of Cantabria-IDIVAL, Santander, Spain
| | - Miguel Lafarga
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Santander, Spain.
| |
Collapse
|
|
16
|
Korzhevskii DE, Gusel’nikova VV, Kirik OV, Sukhorukova EG, Grigorev IP. The Spatial Organization of the Intranuclear Structures of Human Brain Dopaminergic Neurons. Acta Naturae 2017; 9:81-88. [PMID: 29104779 PMCID: PMC5662277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2017] [Indexed: 10/24/2022] Open
Abstract
We studied the intranuclear localization of protein nucleophosmin (B23) and ubiquitin in the dopaminergic neurons of human substantia nigra (n = 6, age of 25-87 years) using immunohistochemistry and confocal laser microscopy. Intranuclear ubiquitin-immunopositive bodies that morphologically correspond to Marinesco bodies were found to be present in substantia nigra dopaminergic (tyrosine hydroxylase-immunopositive) neurons but absent in non-dopaminergic neurons. The number of bodies varied from 0 to 6 per cell nucleus. Nucleophosmin (B23) was found in the neuronal nucleolus, with the nucleolus size being constant in the nigral neurons of each individual brain. All the observed neurons had only one large nucleolus with intense nucleophosmin immunoreactivity and a lightly stained region (1-2 μm in diameter) that apparently represents the giant fibrillar center (GFC). An intensely immunostained nucleophosmin-containing granule was often observed at the GFC periphery. Double labeling demonstrated that nucleophosmin-immunoreactive nucleolus and ubiquitin-immunoreactive Marinesco bodies can occur both closely to and remotely from each other. Three-dimensional reconstruction indicates that rounded Marinesco bodies are polymorphic and often have a complex shape, with some flattening and concavities, which may be associated with contact not only with the nucleolus, but also, presumably, with other intranuclear structures free of ubiquitin or nucleophosmin. Ubiquitin-immunoreactive structures with a relatively small size (up to 1 μm in length) and various clastosome-like shapes (Lafarga et al., 2002) often occur near Marinesco bodies. There were no cases of detection of ubiquitin in the nucleoli of dopaminergic neurons and nucleophosmin/B23 in typical Marinesco bodies. The obtained information may be helpful in unraveling the molecular mechanisms of the selective vulnerability of substantia nigra dopaminergic neurons to damaging factors.
Collapse
Affiliation(s)
- D. E. Korzhevskii
- Federal State Budgetary Research Institution «Institute of Experimental Medicine», akad. Pavlov Str. 12, St. Petersburg, 197376, Russia
| | - V. V. Gusel’nikova
- Federal State Budgetary Research Institution «Institute of Experimental Medicine», akad. Pavlov Str. 12, St. Petersburg, 197376, Russia
| | - O. V. Kirik
- Federal State Budgetary Research Institution «Institute of Experimental Medicine», akad. Pavlov Str. 12, St. Petersburg, 197376, Russia
| | - E. G. Sukhorukova
- Federal State Budgetary Research Institution «Institute of Experimental Medicine», akad. Pavlov Str. 12, St. Petersburg, 197376, Russia
| | - I. P. Grigorev
- Federal State Budgetary Research Institution «Institute of Experimental Medicine», akad. Pavlov Str. 12, St. Petersburg, 197376, Russia
| |
Collapse
|
|
17
|
Sampuda KM, Riley M, Boyd L. Stress induced nuclear granules form in response to accumulation of misfolded proteins in Caenorhabditis elegans. BMC Cell Biol 2017; 18:18. [PMID: 28424053 PMCID: PMC5395811 DOI: 10.1186/s12860-017-0136-x] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Accepted: 04/07/2017] [Indexed: 01/30/2023] Open
Abstract
Background Environmental stress can affect the viability or fecundity of an organism. Environmental stressors may affect the genome or the proteome and can cause cellular distress by contributing to protein damage or misfolding. This study examines the cellular response to environmental stress in the germline of the nematode, C. elegans. Results Salt stress, oxidative stress, and starvation, but not heat shock, induce the relocalization of ubiquitin, proteasome, and the TIAR-2 protein into distinct subnuclear regions referred to as stress induced nuclear granules (SINGs). The SINGs form within 1 h of stress initiation and do not require intertissue signaling. K48-linked polyubiquitin chains but not K63 chains are enriched in SINGs. Worms with a mutation in the conjugating enzyme, ubc-18, do not form SINGs. Additionally, knockdown of ubc-20 and ubc-22 reduces the level of SING formation as does knockdown of the ubiquitin ligase chn-1, a CHIP homolog. The nuclear import machinery is required for SING formation. Stressed embryos containing SINGs fail to hatch and cell division in these embryos is halted. The formation of SINGs can be prevented by pre-exposure to a brief period of heat shock before stress exposure. Heat shock inhibition of SINGs is dependent upon the HSF-1 transcription factor. Conclusions The heat shock results suggest that chaperone expression can prevent SING formation and that the accumulation of damaged or misfolded proteins is a necessary precursor to SING formation. Thus, SINGs may be part of a novel protein quality control system. The data suggest an interesting model where SINGs represent sites of localized protein degradation for nuclear or cytosolic proteins. Thus, the physiological impacts of environmental stress may begin at the cellular level with the formation of stress induced nuclear granules. Electronic supplementary material The online version of this article (doi:10.1186/s12860-017-0136-x) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Katherine M Sampuda
- Department of Biology, Middle Tennessee State University, 1301 E. Main Street, Murfreesboro, TN, 37132, USA
| | - Mason Riley
- Department of Biology, Middle Tennessee State University, 1301 E. Main Street, Murfreesboro, TN, 37132, USA
| | - Lynn Boyd
- Department of Biology, Middle Tennessee State University, 1301 E. Main Street, Murfreesboro, TN, 37132, USA.
| |
Collapse
|
|
18
|
Casafont I, Palanca A, Lafarga V, Mata-Garrido J, Berciano MT, Lafarga M. Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation. Mol Neurobiol 2015; 53:6799-6808. [PMID: 26660115 DOI: 10.1007/s12035-015-9565-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Accepted: 11/29/2015] [Indexed: 12/20/2022]
Abstract
Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4 Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30 min post-IR; their number progressively reduced at 3 h, 1 day, and 3 days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.
Collapse
Affiliation(s)
- Iñigo Casafont
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Cardenal Herrera Oria s/N, Santander, 39011, Spain
| | - Ana Palanca
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Cardenal Herrera Oria s/N, Santander, 39011, Spain
| | - Vanesa Lafarga
- Laboratorio de Inestabilidad Genómica, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
| | - Jorge Mata-Garrido
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Cardenal Herrera Oria s/N, Santander, 39011, Spain
| | - Maria T Berciano
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Cardenal Herrera Oria s/N, Santander, 39011, Spain
| | - Miguel Lafarga
- Department of Anatomy and Cell Biology and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", University of Cantabria-IDIVAL, Cardenal Herrera Oria s/N, Santander, 39011, Spain.
| |
Collapse
|
|
19
|
Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing. Acta Neuropathol 2015; 130:537-55. [PMID: 26085200 PMCID: PMC4575390 DOI: 10.1007/s00401-015-1450-z] [Citation(s) in RCA: 153] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Revised: 05/25/2015] [Accepted: 06/03/2015] [Indexed: 12/12/2022]
Abstract
A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly-PR inclusions were rare throughout the brain but significantly more abundant in the CA3/4 region of FTLD cases than in MND cases. Thus, although DPR distribution is not correlated with neurodegeneration spatially, it correlates with neuropathological subtypes.
Collapse
|
|
20
|
Hall MH, Magalska A, Malinowska M, Ruszczycki B, Czaban I, Patel S, Ambrożek-Latecka M, Zołocińska E, Broszkiewicz H, Parobczak K, Nair RR, Rylski M, Pawlak R, Bramham CR, Wilczyński GM. Localization and regulation of PML bodies in the adult mouse brain. Brain Struct Funct 2015; 221:2511-25. [PMID: 25956166 DOI: 10.1007/s00429-015-1053-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2014] [Accepted: 04/28/2015] [Indexed: 01/19/2023]
Abstract
PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.
Collapse
Affiliation(s)
- Małgorzata H Hall
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Adriana Magalska
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Monika Malinowska
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Błażej Ruszczycki
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Iwona Czaban
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Satyam Patel
- Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 7RH, UK
| | - Magdalena Ambrożek-Latecka
- Department of Clinical Cytology, Center of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland
| | - Ewa Zołocińska
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Hanna Broszkiewicz
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Kamil Parobczak
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland
| | - Rajeevkumar R Nair
- Neuroscience Research Group, Department of Biomedicine and KG Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway
| | - Marcin Rylski
- Department of Clinical Cytology, Center of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland
| | - Robert Pawlak
- Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 7RH, UK.,Hatherley Laboratories, University of Exeter Medical School, Prince of Wales Road, Exeter, EX4 4PS, UK
| | - Clive R Bramham
- Neuroscience Research Group, Department of Biomedicine and KG Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway
| | - Grzegorz M Wilczyński
- Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland.
| |
Collapse
|
|
21
|
Miki Y, Tanji K, Mori F, Wakabayashi K. Sigma-1 receptor is involved in degradation of intranuclear inclusions in a cellular model of Huntington's disease. Neurobiol Dis 2015; 74:25-31. [PMID: 25449906 DOI: 10.1016/j.nbd.2014.11.005] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2014] [Accepted: 11/04/2014] [Indexed: 02/07/2023] Open
Abstract
The sigma-1 receptor (SIGMAR1) is one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins via the ER-related degradation machinery linked to the ubiquitin-proteasome pathway. ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. Recently, we demonstrated that accumulation of SIGMAR1 was common to neuronal nuclear inclusions in polyglutamine diseases including Huntington's disease. Our study also indicated that SIGMAR1 might shuttle between the cytoplasm and the nucleus. In the present study, we investigated the role of SIGMAR1 in nuclear inclusion (NI) formation, using HeLa cells transfected with N-terminal mutant huntingtin. Cell harboring the mutant huntingtin produced SIGMAR1-positive NIs. SIGMAR1 siRNA and a specific inhibitor of the proteasome (epoxomicin) caused significant accumulation of aggregates in the cytoplasm and nucleus. A specific inhibitor of exportin 1 (leptomycin B) also caused NIs. Huntingtin became insolubilized in Western blot analysis after treatments with SIGMAR1 siRNA and epoxomicin. Furthermore, proteasome activity increased chronologically along with the accumulation of mutant huntingtin, but was significantly reduced in cells transfected with SIGMAR1 siRNA. By contrast, overexpression of SIGMAR1 reduced the accumulation of NIs containing mutant huntingtin. Although the LC3-I level was decreased in cells treated with both SIGMAR1 siRNA and control siRNA, the levels of LC3-II and p62 were unchanged. SIGMAR1 agonist and antagonist had no effect on cellular viability and proteasome activity. These findings suggest that the ubiquitin-proteasome pathway is implicated in NI formation, and that SIGMAR1 degrades aberrant proteins in the nucleus via the ER-related degradation machinery. SIGMAR1 might be a promising candidate for therapy of Huntington's disease.
Collapse
Affiliation(s)
- Yasuo Miki
- Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Japan.
| | - Kunikazu Tanji
- Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Japan
| | - Fumiaki Mori
- Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Japan
| | - Koichi Wakabayashi
- Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Japan
| |
Collapse
|
|
22
|
Souquere S, Weil D, Pierron G. Comparative ultrastructure of CRM1-Nucleolar bodies (CNoBs), Intranucleolar bodies (INBs) and hybrid PML/p62 bodies uncovers new facets of nuclear body dynamic and diversity. Nucleus 2015; 6:326-38. [PMID: 26275159 PMCID: PMC4615761 DOI: 10.1080/19491034.2015.1082695] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2015] [Revised: 08/07/2015] [Accepted: 08/10/2015] [Indexed: 12/24/2022] Open
Abstract
In order to gain insights on the nuclear organization in mammalian cells, we characterized ultrastructurally nuclear bodies (NBs) previously described as fluorescent foci. Using high resolution immunoelectron microscopy (I-EM), we provide evidence that CNoBs (CRM1-Nucleolar bodies) and INBs (Intranucleolar bodies) are distinct genuine nucleolar structures in untreated HeLa cells. INBs are fibrillar and concentrate the post-translational modifiers SUMO1 and SUMO-2/3 as strongly as PML bodies. In contrast, the smallest CRM1-labeled CNoBs are vitreous, preferentially located at the periphery of the nucleolus and, intricately linked to the chromatin network. Upon blockage of the CRM1-dependent nuclear export by leptomycin B (LMB), CNoBs disappear while p62/SQSTM1-containing fibrillar nuclear bodies are induced. These p62 bodies are enriched in ubiquitinated proteins. They progressively associate with PML bodies to form hybrid bodies of which PML decorates the periphery while p62/SQSTM1 is centrally-located. Our study is expanding the repertoire of nuclear bodies; revealing a previously unrecognized composite nucleolar landscape and a new mode of interactions between ubiquitous (PML) and stress-induced (p62) nuclear bodies, resulting in the formation of hybrid bodies.
Collapse
Affiliation(s)
- Sylvie Souquere
- Functional Organization of the Cell; CNRS UMR-9196; Institut Gustave Roussy; Villejuif, France
| | - Dominique Weil
- UPMC Univ Paris 06; Institut de Biologie Paris-Seine (IBPS); CNRS UMR-7622; Paris, France
| | - Gérard Pierron
- Functional Organization of the Cell; CNRS UMR-9196; Institut Gustave Roussy; Villejuif, France
| |
Collapse
|
|
23
|
Felix C, Kaplan Türköz B, Ranaldi S, Koelblen T, Terradot L, O'Callaghan D, Vergunst AC. The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation. Cell Commun Signal 2014; 12:53. [PMID: 25304327 PMCID: PMC4203976 DOI: 10.1186/s12964-014-0053-y] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2014] [Accepted: 08/27/2014] [Indexed: 01/12/2023] Open
Abstract
Background The TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton. Results Unlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules. Conclusions Our results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways. Electronic supplementary material The online version of this article (doi:10.1186/s12964-014-0053-y) contains supplementary material, which is available to authorized users.
Collapse
|
|
24
|
Argyriou AA, Cavaletti G, Bruna J, Kyritsis AP, Kalofonos HP. Bortezomib-induced peripheral neurotoxicity: an update. Arch Toxicol 2014; 88:1669-79. [DOI: 10.1007/s00204-014-1316-5] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2014] [Accepted: 07/17/2014] [Indexed: 12/31/2022]
|
|
25
|
Deschênes-Simard X, Lessard F, Gaumont-Leclerc MF, Bardeesy N, Ferbeyre G. Cellular senescence and protein degradation: breaking down cancer. Cell Cycle 2014; 13:1840-58. [PMID: 24866342 DOI: 10.4161/cc.29335] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Autophagy and the ubiquitin-proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.
Collapse
Affiliation(s)
- Xavier Deschênes-Simard
- Department of Biochemistry and Molecular Medicine; Université de Montréal; Montréal, Québec, Canada
| | - Frédéric Lessard
- Department of Biochemistry and Molecular Medicine; Université de Montréal; Montréal, Québec, Canada
| | | | - Nabeel Bardeesy
- Massachusetts General Hospital Cancer Center; Harvard Medical School; Boston, MA USA
| | - Gerardo Ferbeyre
- Department of Biochemistry and Molecular Medicine; Université de Montréal; Montréal, Québec, Canada
| |
Collapse
|
|
26
|
Xu D, Lin F, Jiang Y, Huang X, Li J, Ling J, Hettiarachchi C, Tellgren-Roth C, Holm M, Deng XW. The RING-Finger E3 Ubiquitin Ligase COP1 SUPPRESSOR1 Negatively Regulates COP1 Abundance in Maintaining COP1 Homeostasis in Dark-Grown Arabidopsis Seedlings. THE PLANT CELL 2014; 26:1981-1991. [PMID: 24838976 PMCID: PMC4079363 DOI: 10.1105/tpc.114.124024] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/07/2014] [Revised: 04/13/2014] [Accepted: 04/28/2014] [Indexed: 05/18/2023]
Abstract
CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) functions as an E3 ubiquitin ligase in both plants and animals. In dark-grown Arabidopsis thaliana seedlings, COP1 targets photomorphogenesis-promoting factors for degradation to repress photomorphogenesis. Little is known, however, about how COP1 itself is regulated. Here, we identify COP1 SUPPRESSOR1 (CSU1), a RING-finger E3 ubiquitin ligase, as a regulator of COP1. Genetic evidence demonstrates that csu1 mutations suppress cop1-6 phenotypes completely in the dark. Furthermore, CSU1 colocalizes with COP1 in nuclear speckles and negatively regulates COP1 protein accumulation in darkness. CSU1 can ubiquitinate COP1 in vitro and is essential for COP1 ubiquitination in vivo. Therefore, we conclude that CSU1 plays a major role in maintaining COP1 homeostasis by targeting COP1 for ubiquitination and degradation in dark-grown seedlings.
Collapse
Affiliation(s)
- Dongqing Xu
- Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China Department of Biological and Environmental Sciences, Gothenburg University, SE-405 30 Gothenburg, Sweden
| | - Fang Lin
- Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China
| | - Yan Jiang
- Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China Department of Biological and Environmental Sciences, Gothenburg University, SE-405 30 Gothenburg, Sweden
| | - Xi Huang
- Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China
| | - Jigang Li
- State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Junjie Ling
- Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China
| | - Chamari Hettiarachchi
- Department of Biological and Environmental Sciences, Gothenburg University, SE-405 30 Gothenburg, Sweden
| | - Christian Tellgren-Roth
- Uppsala Genome Center, National Genomics Infrastructure-Science for Life Laboratory, Department of Immunology, Genetics, and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85 Uppsala, Sweden
| | - Magnus Holm
- Department of Biological and Environmental Sciences, Gothenburg University, SE-405 30 Gothenburg, Sweden
| | - Xing Wang Deng
- Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520
| |
Collapse
|
|
27
|
Palanca A, Casafont I, Berciano MT, Lafarga M. Proteasome inhibition induces DNA damage and reorganizes nuclear architecture and protein synthesis machinery in sensory ganglion neurons. Cell Mol Life Sci 2014; 71:1961-75. [PMID: 24061536 PMCID: PMC11113442 DOI: 10.1007/s00018-013-1474-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2013] [Revised: 09/02/2013] [Accepted: 09/10/2013] [Indexed: 11/24/2022]
Abstract
Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.
Collapse
Affiliation(s)
- Ana Palanca
- Department of Anatomy and Cell Biology, Faculty of Medicine and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), University of Cantabria-IFIMAV, Avd. Cardenal Herrera Oria s/n, 39011 Santander, Spain
| | - Iñigo Casafont
- Department of Anatomy and Cell Biology, Faculty of Medicine and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), University of Cantabria-IFIMAV, Avd. Cardenal Herrera Oria s/n, 39011 Santander, Spain
| | - María T. Berciano
- Department of Anatomy and Cell Biology, Faculty of Medicine and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), University of Cantabria-IFIMAV, Avd. Cardenal Herrera Oria s/n, 39011 Santander, Spain
| | - Miguel Lafarga
- Department of Anatomy and Cell Biology, Faculty of Medicine and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), University of Cantabria-IFIMAV, Avd. Cardenal Herrera Oria s/n, 39011 Santander, Spain
| |
Collapse
|
|
28
|
Liu Y, Liu Q, Yan Q, Shi L, Fang Y. Nucleolus-tethering system (NoTS) reveals that assembly of photobodies follows a self-organization model. Mol Biol Cell 2014; 25:1366-73. [PMID: 24554768 PMCID: PMC3983000 DOI: 10.1091/mbc.e13-09-0527] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2013] [Revised: 01/27/2014] [Accepted: 02/13/2014] [Indexed: 01/08/2023] Open
Abstract
Protein-protein interactions play essential roles in regulating many biological processes. At the cellular level, many proteins form nuclear foci known as nuclear bodies in which many components interact with each other. Photobodies are nuclear bodies containing proteins for light-signaling pathways in plants. What initiates the formation of photobodies is poorly understood. Here we develop a nucleolar marker protein nucleolin2 (Nuc2)-based method called the nucleolus-tethering system (NoTS) by artificially tethering a protein of interest to the nucleolus to analyze the initiation of photobodies. A candidate initiator is evaluated by visualizing whether a protein fused with Nuc2 forms body-like structures at the periphery of the nucleolus, and other components are recruited to the de novo-formed bodies. The interaction between two proteins can also be revealed through relocation and recruitment of interacting proteins to the nucleolus. Using the NoTS, we test the interactions among components in photobodies. In addition, we demonstrate that components of photobodies such as CONSTITUTIVELY PHOTOMORPHOGENIC 1, photoreceptors, and transcription factors tethered to the nucleolus have the capacity to form body-like structures at the periphery of the nucleolus, which contain other components of photobodies, suggesting a self-organization model for the biogenesis of photobodies.
Collapse
Affiliation(s)
- Yin Liu
- National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| | - Qi Liu
- National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| | - Qingqing Yan
- National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| | - Leilei Shi
- National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| | - Yuda Fang
- National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
| |
Collapse
|
|
29
|
Protein quality control and elimination of protein waste: The role of the ubiquitin–proteasome system. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2014; 1843:182-96. [DOI: 10.1016/j.bbamcr.2013.06.031] [Citation(s) in RCA: 292] [Impact Index Per Article: 26.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/03/2013] [Revised: 06/28/2013] [Accepted: 06/29/2013] [Indexed: 01/26/2023]
|
|
30
|
Abstract
The principles that determine the organization of the nucleus have become clearer in recent years, largely because of new insights into polymer, colloid, and soft-matter science. Macromolecules, together with the giant linear polymers that form the chromosomes, are confined at high concentrations within the nuclear envelope and their interactions are influenced strongly by short-range depletion or entropic forces which are negligible in dilute systems, in addition to the more familiar van der Waals, electrostatic, steric, hydrogen bonding, and hydrophobic forces. The studies described in this volume are consistent with the model that this complex and concentrated mixture of macromolecules is maintained in a delicate equilibrium by quite simple although unsuspected physicochemical principles. The sensitivity of this equilibrium to perturbation may underlie the controversies about the existence of a nuclear matrix or scaffold. In this volume, we underline the importance for cell biologists of being familiar with current work in colloid, polymer, soft matter, and nanoscience. This chapter presents a brief background to the aspects of the nucleus that are considered in detail in subsequent chapters.
Collapse
Affiliation(s)
- Ronald Hancock
- Laval University Cancer Research Centre, CRCHUQ-Oncology, Québec, Canada; Biosystems Group, Biotechnology Centre, Silesian University of Technology, Gliwice, Poland.
| |
Collapse
|
|
31
|
Palanca A, Casafont I, Berciano MT, Lafarga M. Reactive nucleolar and Cajal body responses to proteasome inhibition in sensory ganglion neurons. Biochim Biophys Acta Mol Basis Dis 2013; 1842:848-59. [PMID: 24269586 DOI: 10.1016/j.bbadis.2013.11.016] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Revised: 11/12/2013] [Accepted: 11/13/2013] [Indexed: 12/25/2022]
Abstract
The dysfunction of the ubiquitin proteasome system has been related to a broad array of neurodegenerative disorders in which the accumulation of misfolded protein aggregates causes proteotoxicity. The ability of proteasome inhibitors to induce cell cycle arrest and apoptosis has emerged as a powerful strategy for cancer therapy. Bortezomib is a proteasome inhibitor used as an antineoplastic drug, although its neurotoxicity frequently causes a severe sensory peripheral neuropathy. In this study we used a rat model of bortezomib treatment to study the nucleolar and Cajal body responses to the proteasome inhibition in sensory ganglion neurons that are major targets of bortezomib-induced neurotoxicity. Treatment with bortezomib induced dose-dependent dissociation of protein synthesis machinery (chromatolysis) and nuclear retention of poly(A) RNA granules resulting in neuronal dysfunction. However, as a compensatory response to the proteotoxic stress, both nucleoli and Cajal bodies exhibited reactive changes. These include an increase in the number and size of nucleoli, strong nucleolar incorporation of the RNA precursor 5'-fluorouridine, and increased expression of both 45S rRNA and genes encoding nucleolar proteins UBF, fibrillarin and B23. Taken together, these findings appear to reflect the activation of the nucleolar transcription in response to proteotoxic stress Furthermore, the number of Cajal bodies, a parameter related to transcriptional activity, increases upon proteasome inhibition. We propose that nucleoli and Cajal bodies are important targets in the signaling pathways that are activated by the proteotoxic stress response to proteasome inhibition. The coordinating activity of these two organelles in the production of snRNA, snoRNA and rRNA may contribute to neuronal survival after proteasome inhibition. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.
Collapse
Affiliation(s)
- Ana Palanca
- Department of Anatomy and Cell Biology, University of Cantabria-IFIMAV, Santander, Spain; "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", Santander, Spain
| | - Iñigo Casafont
- Department of Anatomy and Cell Biology, University of Cantabria-IFIMAV, Santander, Spain; "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", Santander, Spain
| | - María T Berciano
- Department of Anatomy and Cell Biology, University of Cantabria-IFIMAV, Santander, Spain; "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", Santander, Spain
| | - Miguel Lafarga
- Department of Anatomy and Cell Biology, University of Cantabria-IFIMAV, Santander, Spain; "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", Santander, Spain.
| |
Collapse
|
|
32
|
Schoborg T, Rickels R, Barrios J, Labrador M. Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death. ACTA ACUST UNITED AC 2013; 202:261-76. [PMID: 23878275 PMCID: PMC3718971 DOI: 10.1083/jcb.201304181] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins. Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol–p38 mitogen-activated protein kinase–independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization.
Collapse
Affiliation(s)
- Todd Schoborg
- Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA
| | | | | | | |
Collapse
|
|
33
|
Rivera-Molina YA, Martínez FP, Tang Q. Nuclear domain 10 of the viral aspect. World J Virol 2013; 2:110-122. [PMID: 24255882 PMCID: PMC3832855 DOI: 10.5501/wjv.v2.i3.110] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2013] [Revised: 05/31/2013] [Accepted: 07/11/2013] [Indexed: 02/05/2023] Open
Abstract
Nuclear domain 10 (ND10) are spherical bodies distributed throughout the nucleoplasm and measuring around 0.2-1.0 μm. First observed under an electron microscope, they were originally described as dense bodies found in the nucleus. They are known by a number of other names, including Promyelocytic Leukemia bodies (PML bodies), Kremer bodies, and PML oncogenic domains. ND10 are frequently associated with Cajal bodies and cleavage bodies. It has been suggested that they play a role in regulating gene transcription. ND10 were originally characterized using human autoantisera, which recognizes Speckled Protein of 100 kDa, from patients with primary biliary cirrhosis. At the immunohistochemical level, ND10 appear as nuclear punctate structures, with 10 indicating the approximate number of dots per nucleus observed. ND10 do not colocalize with kinetochores, centromeres, sites of mRNA processing, or chromosomes. Resistance of ND10 antigens to nuclease digestion and salt extraction suggest that ND10 are associated with the nuclear matrix. They are often identified by immunofluorescent assay using specific antibodies against PML, Death domain-associated protein, nuclear dot protein (NDP55), and so on. The role of ND10 has long been the subject of investigation, with the specific connection of ND10 and viral infection having been a particular focus for almost 20 years. This review summarizes the relationship of ND10 and viral infection. Some future study directions are also discussed.
Collapse
|
|
34
|
Plourde MB, Morchid A, Iranezereza L, Berthoux L. The Bcl-2/Bcl-xL inhibitor BH3I-2′ affects the dynamics and subcellular localization of sumoylated proteins. Int J Biochem Cell Biol 2013; 45:826-35. [DOI: 10.1016/j.biocel.2013.01.013] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2012] [Revised: 01/04/2013] [Accepted: 01/17/2013] [Indexed: 11/17/2022]
|
|
35
|
Chort A, Alves S, Marinello M, Dufresnois B, Dornbierer JG, Tesson C, Latouche M, Baker DP, Barkats M, El Hachimi KH, Ruberg M, Janer A, Stevanin G, Brice A, Sittler A. Interferon beta induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice. Brain 2013; 136:1732-45. [DOI: 10.1093/brain/awt061] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
|
|
36
|
Malinovska L, Kroschwald S, Alberti S. Protein disorder, prion propensities, and self-organizing macromolecular collectives. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2013; 1834:918-31. [PMID: 23328411 DOI: 10.1016/j.bbapap.2013.01.003] [Citation(s) in RCA: 142] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 11/16/2012] [Revised: 12/12/2012] [Accepted: 01/03/2013] [Indexed: 12/24/2022]
Abstract
Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.
Collapse
Affiliation(s)
- Liliana Malinovska
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | | | | |
Collapse
|
|
37
|
Herpes simplex virus 1 ubiquitin ligase ICP0 interacts with PML isoform I and induces its SUMO-independent degradation. J Virol 2012; 86:11209-22. [PMID: 22875967 DOI: 10.1128/jvi.01145-12] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction.
Collapse
|
|
38
|
3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography. PLoS Pathog 2012; 8:e1002740. [PMID: 22685402 PMCID: PMC3369938 DOI: 10.1371/journal.ppat.1002740] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2012] [Accepted: 04/25/2012] [Indexed: 12/21/2022] Open
Abstract
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level. Varicella-zoster virus (VZV), the cause of varicella and zoster, is a human herpesvirus that replicates in the host cell nucleus where viral genomes are packaged into virion nucleocapsids. We have recently identified antiviral PML (promyelocytic leukemia) nuclear cages that sequester VZV nucleocapsids and inhibit formation of infectious particles. Here we developed a novel three-dimensional (3D) imaging and reconstruction strategy, termed Serial Section Array-Scanning Electron Microscopy (SSA-SEM) that together with electron tomography made it possible to derive 3D reconstructions of complete herpesvirus infected host cell nuclei and of PML cages with ultrastructural precision for the first time. We determined the 3D distribution of several thousand nucleocapsids within reconstructed volumes of single host cell nuclei and in PML cages as well as their sequestration efficiency and sequestration capacity: more than 98% of nucleocapsids were entrapped within PML cages and individual PML cages could sequester nearly 3,000 nucleocapsids which were cross-linked by an irregular electron-dense meshwork within the PML cages. This 3D analysis provides a proof of concept for using SSA-SEM to investigate virion assembly at the whole cell level and further elucidates our observation that PML cages are antiviral nuclear domains which block VZV nucleocapsid egress from the infected cell nucleus.
Collapse
|
|
39
|
Stępiński D. Immunofluorescent localization of ubiquitin and proteasomes in nucleolar vacuoles of soybean root meristematic cells. Eur J Histochem 2012; 56:e13. [PMID: 22688294 PMCID: PMC3428962 DOI: 10.4081/ejh.2012.e13] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provide an additional level of regulation of intracellular proteolysis via compartment-specific activities of their components. It is suggested that the presence of the UPS solely in vacuolated nucleoli serves as a mechanism that enhances the speed of ribosome subunit production in very actively transcribing nucleoli. On the other hand, nucleolar vacuoles in a cell/nucleus could play additional roles associated with temporary sequestration or storage of some cellular factors, including components of the ubiquitin-proteasome system.
Collapse
|
|
40
|
Stępiński D. Immunofluorescent localization of ubiquitin and proteasomes in nucleolar vacuoles of soybean root meristematic cells. Eur J Histochem 2012; 56:e13. [PMID: 22688294 PMCID: PMC3428962 DOI: 10.4081/ejh.2012.13] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2011] [Revised: 01/27/2012] [Accepted: 01/27/2012] [Indexed: 01/01/2023] Open
Abstract
In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provide an additional level of regulation of intracellular proteolysis via compartment-specific activities of their components. It is suggested that the presence of the UPS solely in vacuolated nucleoli serves as a mechanism that enhances the speed of ribosome subunit production in very actively transcribing nucleoli. On the other hand, nucleolar vacuoles in a cell/nucleus could play additional roles associated with temporary sequestration or storage of some cellular factors, including components of the ubiquitin-proteasome system.
Collapse
Affiliation(s)
- D Stępiński
- Department of Cytophysiology, University of Łódź, Poland.
| |
Collapse
|
|
41
|
Transformation by E1A oncoprotein involves ubiquitin-mediated proteolysis of the neuronal and tumor repressor REST in the nucleus. J Virol 2012; 86:5594-602. [PMID: 22419809 DOI: 10.1128/jvi.06811-11] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF(β-TrCP), since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.
Collapse
|
|
42
|
Reorganization of Cajal bodies and nucleolar targeting of coilin in motor neurons of type I spinal muscular atrophy. Histochem Cell Biol 2012; 137:657-67. [DOI: 10.1007/s00418-012-0921-8] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/13/2012] [Indexed: 12/21/2022]
|
|
43
|
|
|
44
|
Van Buskirk EK, Decker PV, Chen M. Photobodies in light signaling. PLANT PHYSIOLOGY 2012; 158:52-60. [PMID: 21951469 PMCID: PMC3252093 DOI: 10.1104/pp.111.186411] [Citation(s) in RCA: 138] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/31/2011] [Accepted: 09/22/2011] [Indexed: 05/17/2023]
|
|
45
|
Anderson DD, Eom JY, Stover PJ. Competition between sumoylation and ubiquitination of serine hydroxymethyltransferase 1 determines its nuclear localization and its accumulation in the nucleus. J Biol Chem 2011; 287:4790-9. [PMID: 22194612 DOI: 10.1074/jbc.m111.302174] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.
Collapse
Affiliation(s)
- Donald D Anderson
- Graduate Field of Biochemistry and Molecular and Cell Biology, DK56339, USA
| | | | | |
Collapse
|
|
46
|
Noor NM, Steer DL, Wheaton BJ, Ek CJ, Truettner JS, Dietrich WD, Dziegielewska KM, Richardson SJ, Smith AI, VandeBerg JL, Saunders NR. Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica). PLoS One 2011; 6:e27465. [PMID: 22110655 PMCID: PMC3217969 DOI: 10.1371/journal.pone.0027465] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2011] [Accepted: 10/17/2011] [Indexed: 12/15/2022] Open
Abstract
Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.
Collapse
Affiliation(s)
- Natassya M. Noor
- Department of Pharmacology, the University of Melbourne, Parkville, Victoria, Australia
| | - David L. Steer
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia
| | - Benjamin J. Wheaton
- Department of Pharmacology, the University of Melbourne, Parkville, Victoria, Australia
| | - C. Joakim Ek
- Department of Pharmacology, the University of Melbourne, Parkville, Victoria, Australia
| | - Jessie S. Truettner
- The Miami Project to Cure Paralysis, University of Miami, Miller School of Medicine, Miami, Florida, United States of America
| | - W. Dalton Dietrich
- The Miami Project to Cure Paralysis, University of Miami, Miller School of Medicine, Miami, Florida, United States of America
| | | | - Samantha J. Richardson
- School of Medical Sciences and Health Innovations Research Institute, RMIT University, Bundoora, Victoria, Australia
| | - A. Ian Smith
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia
| | - John L. VandeBerg
- Texas Biomedical Research Institute, San Antonio, Texas, United States of America
| | - Norman R. Saunders
- Department of Pharmacology, the University of Melbourne, Parkville, Victoria, Australia
| |
Collapse
|
|
47
|
Kiyan Y, Limbourg A, Kiyan R, Tkachuk S, Limbourg FP, Ovsianikov A, Chichkov BN, Haller H, Dumler I. Urokinase receptor associates with myocardin to control vascular smooth muscle cells phenotype in vascular disease. Arterioscler Thromb Vasc Biol 2011; 32:110-22. [PMID: 22075245 DOI: 10.1161/atvbaha.111.234369] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
OBJECTIVE The urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) are a potent multifunctional system involved in vascular remodeling. The goal of the study was to unravel the mechanisms of uPA/uPAR-directed vascular smooth muscle cell (VSMC) differentiation. METHODS AND RESULTS Using cultured human primary VSMCs, we identified a new molecular mechanism controlling phenotypic modulation in vitro and in vivo. We found that the urokinase-type plasminogen activator receptor (uPAR) acts together with the transcriptional coactivator myocardin to regulate the VSMC phenotype. uPAR, a glycosylphosphatidylinositol-anchored cell-surface receptor family member, undergoes ligand-induced internalization and nuclear transport in VSMCs. Platelet-derived growth factor receptor β and SUMOylated RanGAP1 mediate this trafficking. Nuclear uPAR associates with myocardin, which is then recruited from the promoters of serum response factor target genes and undergoes proteasomal degradation. This chain of events initiates the synthetic VSMC phenotype. Using mouse carotid artery ligation model, we show that this mechanism contributes to adverse vascular remodeling after injury in vivo. We then cultured cells on a microstructured biomaterial and found that substrate topography induced uPAR-mediated VSMC differentiation. CONCLUSIONS These findings reveal the transcriptional activity of uPAR, controlling the differentiation of VSMCs in a vascular disease model. They also suggest a new role for uPAR as a therapeutic target and as a marker for VSMC phenotyping on prosthetic biomaterials.
Collapse
Affiliation(s)
- Yulia Kiyan
- Nephrology Department, Hannover Medical School, Carl-Neuberg Str 1, 30625 Hannover, Germany.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
48
|
Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity. Histochem Cell Biol 2011; 137:25-36. [PMID: 21993664 DOI: 10.1007/s00418-011-0869-0] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/28/2011] [Indexed: 12/12/2022]
Abstract
The bioflavonoid quercetin has long been known to exert anti-tumor effects, although the underlying mechanisms remain unknown. Investigation of the potential interference of this anti-oxidant with the efficacy of cell stress-inducing anti-cancer drugs revealed extensive intracellular vacuolation induced by quercetin in epithelial cancer cells that led to cell cycle arrest and ensuing apoptosis. Accumulation of biomarkers of autophagy, including fluorescent autophagy markers and acidotropic dyes characterized these vacuoles as phagolysosomes. Prior to the formation of autophagosomes, an immediate and pronounced inhibition of the autophagy-controlling mTOR activity in quercetin-treated cancer cells occurred, accompanied by a marked reduction in the phosphorylation of the mTOR substrates 4E-BP1 and p70S6 kinase. Assessment of cellular proteasome activity revealed an effective and immediate inhibition of the activity of the proteasome by quercetin in cancer cells. In addition to the formation of autophagosomes, accumulation of poly-ubiquitinated protein aggregates was observed. Thus, proteasome inhibition by quercetin can be regarded as a major cause of quercetin-induced cancer cell death. These results suggest potential new applications for quercetin in cancer science and identify quercetin as an easy-to-handle agent to study proteasome activity, mTOR signaling and autophagy in human cancer cells for cell biological purposes.
Collapse
|
|
49
|
Huang H, Wang H, Voehler MW, Kozekova A, Rizzo CJ, McCullough AK, Lloyd RS, Stone MP. γ-Hydroxy-1,N2-propano-2'-deoxyguanosine DNA adduct conjugates the N-terminal amine of the KWKK peptide via a carbinolamine linkage. Chem Res Toxicol 2011; 24:1123-33. [PMID: 21561113 PMCID: PMC3138414 DOI: 10.1021/tx200113n] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5'-d(GCTAGCXAGTCC)-3'·5'-d(GGACTCGCTAGC)-3' (X = γ-OH-PdG). In the presence of excess peptide KWKK, (13)C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA-KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the N(2)-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited (15)N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH(3), suggesting that they existed, in part, as imine (Schiff base) linkages. However, (13)C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA-KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [ Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. ( 2010 ) Biochemistry , 49 , 6155 -6164 ]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA-KWKK conjugates thermally stabilized duplex DNA. However, the DNA-KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5'-CpX-3' sequence, the DNA-KWKK conjugates slowly converted to interstrand N(2)-dG:N(2)-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks.
Collapse
Affiliation(s)
- Hai Huang
- Department of Chemistry, Center in Molecular Toxicology, Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37235, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
50
|
Reichelt M, Wang L, Sommer M, Perrino J, Nour AM, Sen N, Baiker A, Zerboni L, Arvin AM. Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus. PLoS Pathog 2011; 7:e1001266. [PMID: 21304940 PMCID: PMC3033373 DOI: 10.1371/journal.ppat.1001266] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2010] [Accepted: 12/30/2010] [Indexed: 12/24/2022] Open
Abstract
The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.
Collapse
Affiliation(s)
- Mike Reichelt
- Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
| | | | | | | | | | | | | | | | | |
Collapse
|