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Nagai R, Milam O, Niwa T, Howell W, Best J, Yoshida H, Freeburg C, Koomen J, Fujii K. Ribosomal expansion segment contributes to translation fidelity via N-terminal processing of ribosomal proteins. Nucleic Acids Res 2025; 53:gkaf448. [PMID: 40433980 PMCID: PMC12117404 DOI: 10.1093/nar/gkaf448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2024] [Revised: 04/30/2025] [Accepted: 05/14/2025] [Indexed: 05/29/2025] Open
Abstract
Eukaryotic ribosomes exhibit higher mRNA translation fidelity than prokaryotic ribosomes, partly due to eukaryote-specific ribosomal RNA (rRNA) insertions. Among these, expansion segment 27L (ES27L) on the 60S subunit enhances fidelity by anchoring methionine aminopeptidase (MetAP) at the nascent protein exit tunnel, accelerating co-translational N-terminal initiator methionine (iMet) processing. However, the mechanisms by which iMet processing influences translation fidelity remain unknown. Using yeast in vitro translation (IVT) systems, we found that inhibiting co-translational iMet processing does not impact ribosome decoding of ongoing peptide synthesis. Instead, our novel method to monitor iMet processing in vivo revealed that ribosomes purified from strains lacking MetAP ribosomal association (ES27L Δb1-4) or major yeast MetAP (Δmap1) increase iMet retention on ribosomal proteins (RPs). Given the densely packed structure of ribosomes, iMet retention on RPs may distort ribosomal structure and impair its function. Indeed, reconstituted IVT systems containing iMet-retaining ribosome subunits from ES27L Δb1-4 strain, combined with translation factors from wild-type strains, elucidated that iMet retention on the 40S ribosomal subunit causes translation errors. Our study demonstrated the critical role of ES27L in adjusting ribosome association of universally conserved MetAP enzyme to fine-tune iMet processing of key RPs, thereby ensuring the structural integrity and functional accuracy of eukaryotic ribosomes.
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Affiliation(s)
- Riku Nagai
- Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, United States
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
| | - Olivia L Milam
- Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, United States
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
| | - Tatsuya Niwa
- Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Kanagawa 226-8503, Japan
| | - William J Howell
- Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, United States
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
| | - Jacob A Best
- Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, United States
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
| | - Hideji Yoshida
- Department of Physics, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-8686, Japan
| | - Carver D Freeburg
- Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, United States
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
| | - John M Koomen
- Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL 33612, United States
| | - Kotaro Fujii
- Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, United States
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
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Øye H, Lundekvam M, Caiella A, Hellesvik M, Arnesen T. Protein N-terminal modifications: molecular machineries and biological implications. Trends Biochem Sci 2025; 50:290-310. [PMID: 39837675 DOI: 10.1016/j.tibs.2024.12.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2024] [Revised: 12/15/2024] [Accepted: 12/20/2024] [Indexed: 01/23/2025]
Abstract
The majority of eukaryotic proteins undergo N-terminal (Nt) modifications facilitated by various enzymes. These enzymes, which target the initial amino acid of a polypeptide in a sequence-dependent manner, encompass peptidases, transferases, cysteine oxygenases, and ligases. Nt modifications - such as acetylation, fatty acylations, methylation, arginylation, and oxidation - enhance proteome complexity and regulate protein targeting, stability, and complex formation. Modifications at protein N termini are thereby core components of a large number of biological processes, including cell signaling and motility, autophagy regulation, and plant and animal oxygen sensing. Dysregulation of Nt-modifying enzymes is implicated in several human diseases. In this feature review we provide an overview of the various protein Nt modifications occurring either co- or post-translationally, the enzymes involved, and the biological impact.
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Affiliation(s)
- Hanne Øye
- Department of Biomedicine, University of Bergen, Bergen, Norway.
| | - Malin Lundekvam
- Department of Biomedicine, University of Bergen, Bergen, Norway
| | - Alessia Caiella
- Department of Biomedicine, University of Bergen, Bergen, Norway
| | | | - Thomas Arnesen
- Department of Biomedicine, University of Bergen, Bergen, Norway; Department of Surgery, Haukeland University Hospital, Bergen, Norway.
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Osana S, Tsai CT, Suzuki N, Murayama K, Kaneko M, Hata K, Takada H, Kano Y, Nagatomi R. Inhibition of methionine aminopeptidase in C2C12 myoblasts disrupts cell integrity via increasing endoplasmic reticulum stress. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2025; 1872:119901. [PMID: 39814187 DOI: 10.1016/j.bbamcr.2025.119901] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/24/2024] [Revised: 12/29/2024] [Accepted: 01/08/2025] [Indexed: 01/18/2025]
Abstract
Proteasome-dependent protein degradation and the digestion of peptides by aminopeptidases are essential for myogenesis. Methionine aminopeptidases (MetAPs) are uniquely involved in, both, the proteasomal degradation of proteins and in the regulation of translation (via involvement in post-translational modification). Suppressing MetAP1 and MetAP2 expression inhibits the myogenic differentiation of C2C12 myoblasts. However, the molecular mechanism by which inhibiting MetAPs impairs cellular function remains to be elucidated. Here, we provide evidence for our hypothesis that MetAPs regulate proteostasis and that their inhibition increases ER stress by disrupting the post-translational modification, and thereby compromises cell integrity. Thus, using C2C12 myoblasts, we investigate the effect of inhibiting MetAPs on cell proliferation and the molecular mechanisms underpinning its effects. We found that exposure to bengamide B (a MetAP inhibitor) caused C2C12 myoblasts to lose their proliferative abilities via cell cycle arrest. The underlying mechanism involved the accumulation of abnormal proteins (due to the decrease in the N-terminal methionine removal function) which led to increased endoplasmic reticulum stress, decreased protein synthesis, and a protective activation of the autophagy pathway. To identify the MetAP involved in these effects, we use siRNAs to specifically knockdown MetAP1 and MetAP2 expressions. We found that only MetAP2 knockdown mimicked the effects seen with bengamide B treatment. Thus, we suggest that MetAP2, rather than MetAP1, is involved in maintaining the integrity of C2C12 myoblasts. Our results are useful in understanding muscle regeneration, obesity, and overeating disorders. It will help guide new treatment strategies for these disorders.
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Affiliation(s)
- Shion Osana
- Department of Sports and Medical Science, Graduate School of Emergency Medical System, Kokushikan University, Tokyo 206-8515, Japan; Center for Neuroscience and Biomedical Engineering, University of Electro-Communications, Tokyo 182-8585, Japan.
| | - Cheng-Ta Tsai
- The Institute of Physical Education, Kokushikan University, Tokyo 206-8515, Japan
| | - Naoki Suzuki
- Department of Rehabilitation Medicine, Graduate School of Medicine, Tohoku University, Miyagi 980-8575, Japan
| | - Kazutaka Murayama
- Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, Miyagi 980-8575, Japan
| | - Masaki Kaneko
- The Institute of Physical Education, Kokushikan University, Tokyo 206-8515, Japan
| | - Katsuhiko Hata
- Department of Sports and Medical Science, Graduate School of Emergency Medical System, Kokushikan University, Tokyo 206-8515, Japan
| | - Hiroaki Takada
- Designing Future Health Initiative, Center for Promotion of Innovation Strategy, Head Office of Enterprise Partnerships, Tohoku University, Miyagi 980-8579, Japan
| | - Yutaka Kano
- Center for Neuroscience and Biomedical Engineering, University of Electro-Communications, Tokyo 182-8585, Japan; Department of Engineering Science, Graduate School of Informatics and Engineering, University of Electro-Communications, Tokyo 182-8585, Japan
| | - Ryoichi Nagatomi
- Designing Future Health Initiative, Center for Promotion of Innovation Strategy, Head Office of Enterprise Partnerships, Tohoku University, Miyagi 980-8579, Japan.
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Lentzsch AM, Lee JH, Shan SO. Mechanistic Insights into Protein Biogenesis and Maturation on the Ribosome. J Mol Biol 2025:169056. [PMID: 40024436 DOI: 10.1016/j.jmb.2025.169056] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2025] [Revised: 02/19/2025] [Accepted: 02/25/2025] [Indexed: 03/04/2025]
Abstract
The ribosome is a major cellular machine that converts genetic information into biological function. Emerging data show that the ribosome is not only a protein synthesis machine, but also participates in the maturation of the nascent protein into properly folded and active molecules. The ribosome surface near the opening of the polypeptide exit tunnel can interact directly with the newly synthesized proteins and, more importantly, provides a platform where numerous protein biogenesis factors assemble, gain access to the nascent chain, and direct them into diverse biogenesis pathways. In this article, we review the current understanding of cotranslational protein maturation pathways, with an emphasis on systems in which biochemical studies provided a high-resolution molecular understanding and yielded generalizable mechanistic principles.
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Affiliation(s)
- Alfred M Lentzsch
- Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States
| | - Jae Ho Lee
- Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, United States
| | - Shu-Ou Shan
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, United States.
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Ranji P, Pairet E, Helaers R, Bayet B, Gerdom A, Gil-da-Silva-Lopes VL, Revencu N, Vikkula M. Four putative pathogenic ARHGAP29 variants in patients with non-syndromic orofacial clefts (NsOFC). Eur J Hum Genet 2025; 33:38-43. [PMID: 39506048 PMCID: PMC11711499 DOI: 10.1038/s41431-024-01727-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2024] [Revised: 10/11/2024] [Accepted: 10/21/2024] [Indexed: 11/08/2024] Open
Abstract
The pathophysiological basis of non-syndromic orofacial cleft (NsOFC) is still largely unclear. However, exome sequencing (ES) has led to identify several causative genes, often with reduced penetrance. Among these, the Rho GTPase activating protein 29 (ARHGAP29) has been previously implicated in 7 families with NsOFC. We investigated a cohort of 224 NsOFCs for which no genetic pathogenic variant had been identified by diagnostic testing. We used ES and bioinformatic variant filtering and identified four novel putative pathogenic variants in ARHGAP29 in four families. One was a missense variant leading to the substitution of the first methionine with threonine, two were heterozygous frameshift variants leading to a premature termination codon, and one was a nonsense variant. All variants were predicted to result in loss of function, either through mRNA decay, truncated ARHGAP29, or abnormal N-terminal initiation of translation of ARHGAP29. The truncated ARHGAP29 proteins would lack the important RhoGAP domain. The variants were either absent or rare in the control population databases, and the loss of intolerance score (pLI) of ARHGAP29 is 1.0, suggesting that ARHGAP29 haploinsufficiency is not tolerated. Phenotypes ranged from microform cleft lip (CL) to complete bilateral cleft lip and palate (CLP), with one unaffected mutation carrier. These results extend the mutational spectrum of ARHGAP29 and show that it is an important gene underlying variable NsOFC phenotypes. ARHGAP29 should be included in diagnostic genetic testing for NsOFC, especially familial cases, as it may be mutated in ∼4% of them (4/97 in our cohort) with high penetrance (89%).
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Affiliation(s)
- Peyman Ranji
- Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium
| | - Eleonore Pairet
- Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium
| | - Raphael Helaers
- Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium
| | - Bénédicte Bayet
- Centre Labio-Palatin, Division of Plastic Surgery, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium
| | - Alexander Gerdom
- Centre Labio-Palatin, Division of Plastic Surgery, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium
| | - Vera Lúcia Gil-da-Silva-Lopes
- Department of Translational Medicine, Area of Medical Genetics and Genomic Medicine, University of Campinas (UNICAMP), Campinas, SP, Brazil
| | - Nicole Revencu
- Center for Human Genetics, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium
| | - Miikka Vikkula
- Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium.
- WELBIO Department, WEL Research Institute, avenue Pasteur, 6, 1300, Wavre, Belgium.
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Lao Y, Jin Y, Wu S, Fang T, Wang Q, Sun L, Sun B. Deciphering a profiling based on multiple post-translational modifications functionally associated regulatory patterns and therapeutic opportunities in human hepatocellular carcinoma. Mol Cancer 2024; 23:283. [PMID: 39732660 PMCID: PMC11681642 DOI: 10.1186/s12943-024-02199-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2024] [Accepted: 12/11/2024] [Indexed: 12/30/2024] Open
Abstract
BACKGROUND Posttranslational modifications (PTMs) play critical roles in hepatocellular carcinoma (HCC). However, the locations of PTM-modified sites across protein secondary structures and regulatory patterns in HCC remain largely uncharacterized. METHODS Total proteome and nine PTMs (phosphorylation, acetylation, crotonylation, ubiquitination, lactylation, N-glycosylation, succinylation, malonylation, and β-hydroxybutyrylation) in tumor sections and paired normal adjacent tissues derived from 18 HCC patients were systematically profiled by 4D-Label free proteomics analysis combined with PTM-based peptide enrichment. RESULTS We detected robust preferences in locations of intrinsically disordered protein regions (IDRs) with phosphorylated sites and other site biases to locate in folded regions. Integrative analyses revealed that phosphorylated and multiple acylated-modified sites are enriched in proteins containing RRM1 domain, and RNA splicing is the key feature of this subset of proteins, as indicated by phosphorylation and acylation of splicing factor NCL at multiple residues. We confirmed that NCL-S67, K398, and K646 cooperate to regulate RNA processing. CONCLUSION Together, this proteome profiling represents a comprehensive study detailing regulatory patterns based on multiple PTMs of HCC.
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Affiliation(s)
- Yuanxiang Lao
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
- Anhui Province Key Laboratory of Tumor Immune Microenvironment and Immunotherapy, Innovative Institute of Tumor Immunity and Medicine (ITIM), Hefei, Anhui, China
- Anhui Provincial Innovation Institute for Pharmaceutical Basic Research, Hefei, Anhui, China
| | - Yirong Jin
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
- Anhui Province Key Laboratory of Tumor Immune Microenvironment and Immunotherapy, Innovative Institute of Tumor Immunity and Medicine (ITIM), Hefei, Anhui, China
- Anhui Provincial Innovation Institute for Pharmaceutical Basic Research, Hefei, Anhui, China
| | - Songfeng Wu
- Beijing Qinglian Biotech Co., Ltd, Beijing, China
| | - Ting Fang
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
- Anhui Province Key Laboratory of Tumor Immune Microenvironment and Immunotherapy, Innovative Institute of Tumor Immunity and Medicine (ITIM), Hefei, Anhui, China
- Anhui Provincial Innovation Institute for Pharmaceutical Basic Research, Hefei, Anhui, China
| | - Qiang Wang
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
- Anhui Province Key Laboratory of Tumor Immune Microenvironment and Immunotherapy, Innovative Institute of Tumor Immunity and Medicine (ITIM), Hefei, Anhui, China
- Anhui Provincial Innovation Institute for Pharmaceutical Basic Research, Hefei, Anhui, China
| | - Longqin Sun
- Beijing Qinglian Biotech Co., Ltd, Beijing, China
| | - Beicheng Sun
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
- Anhui Province Key Laboratory of Tumor Immune Microenvironment and Immunotherapy, Innovative Institute of Tumor Immunity and Medicine (ITIM), Hefei, Anhui, China.
- Anhui Provincial Innovation Institute for Pharmaceutical Basic Research, Hefei, Anhui, China.
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Brünje A, Füßl M, Eirich J, Boyer JB, Heinkow P, Neumann U, Konert M, Ivanauskaite A, Seidel J, Ozawa SI, Sakamoto W, Meinnel T, Schwarzer D, Mulo P, Giglione C, Finkemeier I. The Plastidial Protein Acetyltransferase GNAT1 Forms a Complex With GNAT2, yet Their Interaction Is Dispensable for State Transitions. Mol Cell Proteomics 2024; 23:100850. [PMID: 39349166 PMCID: PMC11585782 DOI: 10.1016/j.mcpro.2024.100850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Revised: 07/12/2024] [Accepted: 08/18/2024] [Indexed: 10/02/2024] Open
Abstract
Protein N-acetylation is one of the most abundant co- and post-translational modifications in eukaryotes, extending its occurrence to chloroplasts within vascular plants. Recently, a novel plastidial enzyme family comprising eight acetyltransferases that exhibit dual lysine and N-terminus acetylation activities was unveiled in Arabidopsis. Among these, GNAT1, GNAT2, and GNAT3 reveal notable phylogenetic proximity, forming a subgroup termed NAA90. Our study focused on characterizing GNAT1, closely related to the state transition acetyltransferase GNAT2. In contrast to GNAT2, GNAT1 did not prove essential for state transitions and displayed no discernible phenotypic difference compared to the wild type under high light conditions, while gnat2 mutants were severely affected. However, gnat1 mutants exhibited a tighter packing of the thylakoid membranes akin to gnat2 mutants. In vitro studies with recombinant GNAT1 demonstrated robust N-terminus acetylation activity on synthetic substrate peptides. This activity was confirmed in vivo through N-terminal acetylome profiling in two independent gnat1 knockout lines. This attributed several acetylation sites on plastidial proteins to GNAT1, reflecting a subset of GNAT2's substrate spectrum. Moreover, co-immunoprecipitation coupled with mass spectrometry revealed a robust interaction between GNAT1 and GNAT2, as well as a significant association of GNAT2 with GNAT3 - the third acetyltransferase within the NAA90 subfamily. This study unveils the existence of at least two acetyltransferase complexes within chloroplasts, whereby complex formation might have a critical effect on the fine-tuning of the overall acetyltransferase activities. These findings introduce a novel layer of regulation in acetylation-dependent adjustments in plastidial metabolism.
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Affiliation(s)
- Annika Brünje
- Plant Physiology, Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Münster, Germany
| | - Magdalena Füßl
- Plant Physiology, Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Münster, Germany
| | - Jürgen Eirich
- Plant Physiology, Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Münster, Germany
| | - Jean-Baptiste Boyer
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Paulina Heinkow
- Plant Physiology, Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Münster, Germany
| | - Ulla Neumann
- Central Microscopy, Max Planck Institute for Plant Breeding Research, Köln, Germany
| | - Minna Konert
- Department of Life Technologies, Molecular Plant Biology, University of Turku, Turku, Finland
| | - Aiste Ivanauskaite
- Department of Life Technologies, Molecular Plant Biology, University of Turku, Turku, Finland
| | - Julian Seidel
- Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
| | - Shin-Ichiro Ozawa
- Institute of Plant Science and Resources (IPSR) Okayama University, Kurashiki, Okayama, Japan
| | - Wataru Sakamoto
- Institute of Plant Science and Resources (IPSR) Okayama University, Kurashiki, Okayama, Japan
| | - Thierry Meinnel
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Dirk Schwarzer
- Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
| | - Paula Mulo
- Department of Life Technologies, Molecular Plant Biology, University of Turku, Turku, Finland
| | - Carmela Giglione
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Iris Finkemeier
- Plant Physiology, Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Münster, Germany.
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Das B, Chokkalingam P, Shareef MA, Shukla S, Das S, Saito M, Weiss LM. Methionine aminopeptidases: Potential therapeutic target for microsporidia and other microbes. J Eukaryot Microbiol 2024; 71:e13036. [PMID: 39036929 PMCID: PMC11576263 DOI: 10.1111/jeu.13036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Revised: 05/06/2024] [Accepted: 05/16/2024] [Indexed: 07/23/2024]
Abstract
Methionine aminopeptidases (MetAPs) have emerged as a target for medicinal chemists in the quest for novel therapeutic agents for treating cancer, obesity, and other disorders. Methionine aminopeptidase is a metalloenzyme with two structurally distinct forms in humans, MetAP-1 and MetAP-2. The MetAP2 inhibitor fumagillin, which was used as an amebicide in the 1950s, has been used for the successful treatment of microsporidiosis in humans; however, it is no longer commercially available. Despite significant efforts and investments by many pharmaceutical companies, no new MetAP inhibitors have been approved for the clinic. Several lead compounds have been designed and synthesized by researchers as potential inhibitors of MetAP and evaluated for their potential activity in a wide range of diseases. MetAP inhibitors such as fumagillin, TNP-470, beloranib, and reversible inhibitors and their analogs guide new prospects for MetAP inhibitor development in the ongoing quest for new pharmacological indications. This perspective provides insights into recent advances related to MetAP, as a potential therapeutic target in drug discovery, bioactive small molecule MetAP2 inhibitors, and data on the role of MetAP-2 as a therapeutic target for microsporidiosis.
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Affiliation(s)
- Bhaskar Das
- Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY, USA
- Department of Medicine and Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Parthiban Chokkalingam
- Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY, USA
| | - Mohammed Adil Shareef
- Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY, USA
| | - Srushti Shukla
- Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY, USA
| | - Sasmita Das
- Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY, USA
| | - Mariko Saito
- Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA
| | - Louis M. Weiss
- Department of Pathology and Medicine (Infectious Diseases) Albert Einstein College of Medicine, Bronx, NY-10461, USA
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Guedes JP, Boyer JB, Elurbide J, Carte B, Redeker V, Sago L, Meinnel T, Côrte-Real M, Giglione C, Aldabe R. NatB Protects Procaspase-8 from UBR4-Mediated Degradation and Is Required for Full Induction of the Extrinsic Apoptosis Pathway. Mol Cell Biol 2024; 44:358-371. [PMID: 39099191 PMCID: PMC11376409 DOI: 10.1080/10985549.2024.2382453] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Revised: 07/10/2024] [Accepted: 07/10/2024] [Indexed: 08/06/2024] Open
Abstract
N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.
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Affiliation(s)
- Joana P. Guedes
- CBMA/UM – Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal
- Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Braga, Portugal
- CIMA/UNAV – Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain
| | - Jean Baptiste Boyer
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Jasmine Elurbide
- CIMA/UNAV – Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain
| | - Beatriz Carte
- CIMA/UNAV – Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain
| | - Virginie Redeker
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Laila Sago
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Thierry Meinnel
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Manuela Côrte-Real
- CBMA/UM – Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal
- Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Braga, Portugal
| | - Carmela Giglione
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France
| | - Rafael Aldabe
- CIMA/UNAV – Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain
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10
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Schlößer M, Moseler A, Bodnar Y, Homagk M, Wagner S, Pedroletti L, Gellert M, Ugalde JM, Lillig CH, Meyer AJ. Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2024; 118:1455-1474. [PMID: 38394181 DOI: 10.1111/tpj.16687] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Revised: 01/31/2024] [Accepted: 02/07/2024] [Indexed: 02/25/2024]
Abstract
Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.
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Affiliation(s)
- Michelle Schlößer
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Anna Moseler
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Yana Bodnar
- Institute for Medical Biochemistry and Molecular Biology, University Medicine, University of Greifswald, Ferdinand-Sauerbruch-Straße, D-17475, Greifswald, Germany
| | - Maria Homagk
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Stephan Wagner
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Luca Pedroletti
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Manuela Gellert
- Institute for Medical Biochemistry and Molecular Biology, University Medicine, University of Greifswald, Ferdinand-Sauerbruch-Straße, D-17475, Greifswald, Germany
| | - José M Ugalde
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Christopher H Lillig
- Institute for Medical Biochemistry and Molecular Biology, University Medicine, University of Greifswald, Ferdinand-Sauerbruch-Straße, D-17475, Greifswald, Germany
| | - Andreas J Meyer
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
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11
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Venezian J, Bar-Yosef H, Ben-Arie Zilberman H, Cohen N, Kleifeld O, Fernandez-Recio J, Glaser F, Shiber A. Diverging co-translational protein complex assembly pathways are governed by interface energy distribution. Nat Commun 2024; 15:2638. [PMID: 38528060 DOI: 10.1038/s41467-024-46881-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Accepted: 03/12/2024] [Indexed: 03/27/2024] Open
Abstract
Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find that only a few residues serve as "hotspots," initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes' complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly.
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Affiliation(s)
- Johannes Venezian
- Faculty of Biology, Technion Israel institute of Technology, Haifa, Israel
| | - Hagit Bar-Yosef
- Faculty of Biology, Technion Israel institute of Technology, Haifa, Israel
| | | | - Noam Cohen
- Faculty of Biology, Technion Israel institute of Technology, Haifa, Israel
| | - Oded Kleifeld
- Faculty of Biology, Technion Israel institute of Technology, Haifa, Israel
| | - Juan Fernandez-Recio
- Instituto de Ciencias de la Vid y del Vino (ICVV), CSIC-Universidad de La Rioja-Gobierno de La Rioja, Logroño, Spain
| | - Fabian Glaser
- Lorry I. Lokey Interdisciplinary Center for Life Sciences & Engineering, Haifa, Israel
| | - Ayala Shiber
- Faculty of Biology, Technion Israel institute of Technology, Haifa, Israel.
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12
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Gamerdinger M, Deuerling E. Cotranslational sorting and processing of newly synthesized proteins in eukaryotes. Trends Biochem Sci 2024; 49:105-118. [PMID: 37919225 DOI: 10.1016/j.tibs.2023.10.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2023] [Revised: 09/28/2023] [Accepted: 10/06/2023] [Indexed: 11/04/2023]
Abstract
Ribosomes interact with a variety of different protein biogenesis factors that guide newly synthesized proteins to their native 3D shapes and cellular localization. Depending on the type of translated substrate, a distinct set of cotranslational factors must interact with the ribosome in a timely and coordinated manner to ensure proper protein biogenesis. While cytonuclear proteins require cotranslational maturation and folding factors, secretory proteins must be maintained in an unfolded state and processed cotranslationally by transport and membrane translocation factors. Here we explore the specific cotranslational processing steps for cytonuclear, secretory, and membrane proteins in eukaryotes and then discuss how the nascent polypeptide-associated complex (NAC) cotranslationally sorts these proteins into the correct protein biogenesis pathway.
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Affiliation(s)
- Martin Gamerdinger
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany.
| | - Elke Deuerling
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany.
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13
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Lee Y, Kim H, Lee E, Hahn H, Heo Y, Jang DM, Kwak K, Kim HJ, Kim HS. Structural insights into N-terminal methionine cleavage by the human mitochondrial methionine aminopeptidase, MetAP1D. Sci Rep 2023; 13:22326. [PMID: 38102161 PMCID: PMC10724148 DOI: 10.1038/s41598-023-49332-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Accepted: 12/07/2023] [Indexed: 12/17/2023] Open
Abstract
Isozymes are enzymes that catalyze identical biological reactions, yet exhibit slight variations in structures and catalytic efficiency, which enables the precise adjustment of metabolism to fulfill the specific requirements of a particular tissue or stage of development. Methionine aminopeptidase (MetAP) isozymes function a critical role in cleaving N-terminal methionine from nascent proteins to generate functional proteins. In humans, two distinct MetAP types I and II have been identified, with type I further categorized into cytosolic (MetAP1) and mitochondrial (MetAP1D) variants. However, despite extensive structural studies on both bacterial and human cytosolic MetAPs, the structural information remains unavailable for human mitochondrial MetAP. This study was aimed to elucidate the high-resolution structures of human mitochondrial MetAP1D in its apo-, cobalt-, and methionine-bound states. Through a comprehensive analysis of the determined structures and a docking simulation model with mitochondrial substrate peptides, we present mechanistic insights into the cleavage process of the initiator methionine from mitochondrial proteins. Notably, despite the shared features at the active site between the cytosolic and mitochondrial MetAP type I isozymes, we identified distinct structural disparities within the active-site pocket primarily contributed by two specific loops that could play a role in accommodating specific substrates. These structural insights offer a basis for the further exploration of MetAP isozymes as critical players in cellular processes and potential therapeutic applications.
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Affiliation(s)
- Yeon Lee
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
| | - Hayoung Kim
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
- Division of Medical Sciences, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea
| | - Eunji Lee
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
| | - Hyunggu Hahn
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
| | - Yoonyoung Heo
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
| | - Dong Man Jang
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
| | - Kihyuck Kwak
- Division of Medical Sciences, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea
| | - Hyo Jung Kim
- College of Pharmacy, Woosuk University, Wanju, 55338, Republic of Korea.
| | - Hyoun Sook Kim
- Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea.
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14
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Höpfler M, Hegde RS. Control of mRNA fate by its encoded nascent polypeptide. Mol Cell 2023; 83:2840-2855. [PMID: 37595554 PMCID: PMC10501990 DOI: 10.1016/j.molcel.2023.07.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Revised: 07/03/2023] [Accepted: 07/11/2023] [Indexed: 08/20/2023]
Abstract
Cells tightly regulate mRNA processing, localization, and stability to ensure accurate gene expression in diverse cellular states and conditions. Most of these regulatory steps have traditionally been thought to occur before translation by the action of RNA-binding proteins. Several recent discoveries highlight multiple co-translational mechanisms that modulate mRNA translation, localization, processing, and stability. These mechanisms operate by recognition of the nascent protein, which is necessarily coupled to its encoding mRNA during translation. Hence, the distinctive sequence or structure of a particular nascent chain can recruit recognition factors with privileged access to the corresponding mRNA in an otherwise crowded cellular environment. Here, we draw on both well-established and recent examples to provide a conceptual framework for how cells exploit nascent protein recognition to direct mRNA fate. These mechanisms allow cells to dynamically and specifically regulate their transcriptomes in response to changes in cellular states to maintain protein homeostasis.
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15
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Messana I, Manconi B, Cabras T, Boroumand M, Sanna MT, Iavarone F, Olianas A, Desiderio C, Rossetti DV, Vincenzoni F, Contini C, Guadalupi G, Fiorita A, Faa G, Castagnola M. The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms. Int J Mol Sci 2023; 24:12776. [PMID: 37628956 PMCID: PMC10454625 DOI: 10.3390/ijms241612776] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Revised: 08/05/2023] [Accepted: 08/08/2023] [Indexed: 08/27/2023] Open
Abstract
In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.
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Affiliation(s)
- Irene Messana
- Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Consiglio Nazionale delle Ricerche, 00168 Rome, Italy; (I.M.); (C.D.); (D.V.R.)
| | - Barbara Manconi
- Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; (B.M.); (M.T.S.); (A.O.); (C.C.); (G.G.)
| | - Tiziana Cabras
- Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; (B.M.); (M.T.S.); (A.O.); (C.C.); (G.G.)
| | | | - Maria Teresa Sanna
- Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; (B.M.); (M.T.S.); (A.O.); (C.C.); (G.G.)
| | - Federica Iavarone
- Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (F.I.); (F.V.)
- Fondazione Policlinico Universitario A. Gemelli Fondazione IRCCS, 00168 Rome, Italy;
| | - Alessandra Olianas
- Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; (B.M.); (M.T.S.); (A.O.); (C.C.); (G.G.)
| | - Claudia Desiderio
- Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Consiglio Nazionale delle Ricerche, 00168 Rome, Italy; (I.M.); (C.D.); (D.V.R.)
| | - Diana Valeria Rossetti
- Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Consiglio Nazionale delle Ricerche, 00168 Rome, Italy; (I.M.); (C.D.); (D.V.R.)
| | - Federica Vincenzoni
- Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (F.I.); (F.V.)
- Fondazione Policlinico Universitario A. Gemelli Fondazione IRCCS, 00168 Rome, Italy;
| | - Cristina Contini
- Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; (B.M.); (M.T.S.); (A.O.); (C.C.); (G.G.)
| | - Giulia Guadalupi
- Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; (B.M.); (M.T.S.); (A.O.); (C.C.); (G.G.)
| | - Antonella Fiorita
- Fondazione Policlinico Universitario A. Gemelli Fondazione IRCCS, 00168 Rome, Italy;
- Dipartimento di Scienze dell’Invecchiamento, Neurologiche, Ortopediche e della Testa e del Collo, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
| | - Gavino Faa
- Unit of Pathology, Department of Medical Sciences and Public Health, University of Cagliari, 09124 Cagliari, Italy;
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA
| | - Massimo Castagnola
- Proteomics Laboratory, European Center for Brain Research, (IRCCS) Santa Lucia Foundation, 00168 Rome, Italy;
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16
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Briney CA, Rissland OS. Planes, trains, and automobiles: How cells localize their molecules. Mol Cell 2023; 83:2618-2620. [PMID: 37541217 DOI: 10.1016/j.molcel.2023.07.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Revised: 07/11/2023] [Accepted: 07/11/2023] [Indexed: 08/06/2023]
Abstract
In this issue of Molecular Cell, Gasparski et al.1 and Loedige et al.2 reshape our understanding of subcellular gene product localization by highlighting the importance of messenger RNA (mRNA) stability and co-translational mechanisms in mRNA and protein localization.
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Affiliation(s)
- Chloe A Briney
- Department of Biochemistry & Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA; RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, USA
| | - Olivia S Rissland
- Department of Biochemistry & Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA; RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, USA.
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17
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Nashed S, El Barbry H, Benchouaia M, Dijoux-Maréchal A, Delaveau T, Ruiz-Gutierrez N, Gaulier L, Tribouillard-Tanvier D, Chevreux G, Le Crom S, Palancade B, Devaux F, Laine E, Garcia M. Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import. PLoS Genet 2023; 19:e1010848. [PMID: 37585488 PMCID: PMC10482271 DOI: 10.1371/journal.pgen.1010848] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 09/06/2023] [Accepted: 06/29/2023] [Indexed: 08/18/2023] Open
Abstract
N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.
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Affiliation(s)
- Salomé Nashed
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Houssam El Barbry
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Médine Benchouaia
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Angélie Dijoux-Maréchal
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Thierry Delaveau
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Nadia Ruiz-Gutierrez
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Lucie Gaulier
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | | | | | - Stéphane Le Crom
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | | | - Frédéric Devaux
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Elodie Laine
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
| | - Mathilde Garcia
- Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, UMR 7238, Laboratoire de Biologie Computationnelle et Quantitative, Paris, France
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18
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Chang YH. Impact of Protein N α-Modifications on Cellular Functions and Human Health. Life (Basel) 2023; 13:1613. [PMID: 37511988 PMCID: PMC10381334 DOI: 10.3390/life13071613] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Revised: 07/14/2023] [Accepted: 07/17/2023] [Indexed: 07/30/2023] Open
Abstract
Most human proteins are modified by enzymes that act on the α-amino group of a newly synthesized polypeptide. Methionine aminopeptidases can remove the initiator methionine and expose the second amino acid for further modification by enzymes responsible for myristoylation, acetylation, methylation, or other chemical reactions. Specific acetyltransferases can also modify the initiator methionine and sometimes the acetylated methionine can be removed, followed by further modifications. These modifications at the protein N-termini play critical roles in cellular protein localization, protein-protein interaction, protein-DNA interaction, and protein stability. Consequently, the dysregulation of these modifications could significantly change the development and progression status of certain human diseases. The focus of this review is to highlight recent progress in our understanding of the roles of these modifications in regulating protein functions and how these enzymes have been used as potential novel therapeutic targets for various human diseases.
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Affiliation(s)
- Yie-Hwa Chang
- Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Medical School, Saint Louis, MO 63104, USA
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19
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Gamerdinger M, Jia M, Schloemer R, Rabl L, Jaskolowski M, Khakzar KM, Ulusoy Z, Wallisch A, Jomaa A, Hunaeus G, Scaiola A, Diederichs K, Ban N, Deuerling E. NAC controls cotranslational N-terminal methionine excision in eukaryotes. Science 2023; 380:1238-1243. [PMID: 37347872 DOI: 10.1126/science.adg3297] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Accepted: 05/18/2023] [Indexed: 06/24/2023]
Abstract
N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.
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Affiliation(s)
- Martin Gamerdinger
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Min Jia
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
| | - Renate Schloemer
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Laurenz Rabl
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Mateusz Jaskolowski
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
| | - Katrin M Khakzar
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Zeynel Ulusoy
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Annalena Wallisch
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Ahmad Jomaa
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
| | - Gundula Hunaeus
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
| | - Alain Scaiola
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
| | - Kay Diederichs
- Department of Biology, Molecular Bioinformatics, University of Konstanz, 78457 Konstanz, Germany
| | - Nenad Ban
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland
| | - Elke Deuerling
- Department of Biology, Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany
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20
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Hedtke B, Strätker SM, Pulido ACC, Grimm B. Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes. PLANT PHYSIOLOGY 2023; 192:871-885. [PMID: 36806676 PMCID: PMC10231370 DOI: 10.1093/plphys/kiad107] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Revised: 12/22/2022] [Accepted: 01/24/2023] [Indexed: 06/01/2023]
Abstract
All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified 2 ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the 2 isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. Mitochondrial localization of PPO2 in Arabidopsis was ruled out. Since Arabidopsis PPO2 does not encode a cleavable transit peptide, integration of the protein into the chloroplast envelope must make use of a noncanonical import route. However, when a chloroplast transit peptide was fused to the N-terminus of PPO2, the enzyme was detected predominantly in thylakoid membranes and was able to fully complement ppo1. Thus, the 2 PPO isoforms in Arabidopsis are functionally equivalent but spatially separated. Their distinctive localizations within plastids thus enable the synthesis of discrete subpools of the PPO product protoporphyrin IX, which may serve different cellular needs.
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Affiliation(s)
- Boris Hedtke
- Humboldt-Universität zu Berlin, Institute of Biology/Plant Physiology, Philippstraße 13 (Building 12), Berlin 10115, Germany
| | - Sarah Melissa Strätker
- Humboldt-Universität zu Berlin, Institute of Biology/Plant Physiology, Philippstraße 13 (Building 12), Berlin 10115, Germany
| | - Andrea C Chiappe Pulido
- Humboldt-Universität zu Berlin, Institute of Biology/Plant Physiology, Philippstraße 13 (Building 12), Berlin 10115, Germany
| | - Bernhard Grimm
- Humboldt-Universität zu Berlin, Institute of Biology/Plant Physiology, Philippstraße 13 (Building 12), Berlin 10115, Germany
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21
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Hanna R, Rozenberg A, Saied L, Ben-Yosef D, Lavy T, Kleifeld O. In-Depth Characterization of Apoptosis N-terminome Reveals a Link Between Caspase-3 Cleavage and Post-Translational N-terminal Acetylation. Mol Cell Proteomics 2023:100584. [PMID: 37236440 PMCID: PMC10362333 DOI: 10.1016/j.mcpro.2023.100584] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Revised: 05/16/2023] [Accepted: 05/22/2023] [Indexed: 05/28/2023] Open
Abstract
The N-termini of proteins contain information about their biochemical properties and functions. These N-termini can be processed by proteases, and can undergo other co- or post-translational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-terminomic method to study caspase-3 mediated proteolysis both in vitro and during apoptosis in cells. This has enabled us to identify many unreported caspase-3 cleavages, some of which cannot be identified by other methods. Moreover, we have found direct evidence that neo-N-termini generated by caspase-3 cleavage can be further modified by Nt-acetylation. Some of these neo-Nt-acetylation events occur in the early phase of the apoptotic process and may have a role in translation inhibition. This has provided a comprehensive overview of the caspase-3 degradome and has uncovered previously unrecognized crosstalk between post-translational Nt-acetylation and caspase proteolytic pathways.
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Affiliation(s)
- Rawad Hanna
- Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 3200003, Israel
| | - Andrey Rozenberg
- Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 3200003, Israel
| | - Layla Saied
- Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 3200003, Israel
| | - Daniel Ben-Yosef
- Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 3200003, Israel
| | - Tali Lavy
- Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 3200003, Israel
| | - Oded Kleifeld
- Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 3200003, Israel.
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22
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Knorr AG, Mackens-Kiani T, Musial J, Berninghausen O, Becker T, Beatrix B, Beckmann R. The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains. PLoS Biol 2023; 21:e3001995. [PMID: 37079644 PMCID: PMC10118133 DOI: 10.1371/journal.pbio.3001995] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 01/10/2023] [Indexed: 04/21/2023] Open
Abstract
Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel.
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Affiliation(s)
- Alexandra G. Knorr
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
| | - Timur Mackens-Kiani
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
| | - Joanna Musial
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
| | - Otto Berninghausen
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
| | - Thomas Becker
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
| | - Birgitta Beatrix
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
| | - Roland Beckmann
- Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, University of Munich, Munich, Germany
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23
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Müller F, Bange T. Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry. Methods Enzymol 2023; 686:67-97. [PMID: 37532409 DOI: 10.1016/bs.mie.2023.02.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/04/2023]
Abstract
Regulated protein degradation controls protein levels of all short-lived proteins to ensure cellular homeostasis and also protects cells from misfolded or other abnormal proteins. The most important players in the degradation system are E3 ubiquitin ligases which recognize exposed sequence motifs, so-called degrons, of target proteins and mark them through the attachment of ubiquitin for degradation. N-terminal (Nt) sequences are extensively used as degrons (N-degrons) and all 20 amino acids are able to feed proteins in 1 of the 5 known N-degron pathways. Studies have mainly focused on characterizing systematically the role of the starting amino acid on protein stability and less on the identification of the E3 ligases involved. Recent data from our lab and literature suggest that there is an extensive interplay of N-recognins and Nt-modifying enzymes like Nt-acetyltransferases (NATs) or N-myristoyltransferases which only starts to be elucidated. It suggests that improperly modified or unexpectedly unmodified proteins become rapidly removed after synthesis ensuring protein maturation and quality control of specific subsets of proteins. Here, we describe a peptide pull-down and down-stream bioinformatics workflow conducted in the MaxQuant and Perseus computational environment to identify N-recognin candidates in an unbiased way using quantitative mass spectrometry (MS)-based proteomics. Our workflow allows the identification of N-recognin candidates for specific N-degrons, to determine their sequence specificity and it can be applied as well more general to identify binding partners of N-terminal modifications. This method paves the way to identify pathways involved in protein quality control and stability acting at the N-terminus.
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Affiliation(s)
- Franziska Müller
- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
| | - Tanja Bange
- Institute of Medical Psychology, Faculty of Medicine, Ludwig-Maximilians University, Munich, Germany.
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24
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A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin. BIOCHIMICA ET BIOPHYSICA ACTA. PROTEINS AND PROTEOMICS 2023; 1871:140881. [PMID: 36396098 DOI: 10.1016/j.bbapap.2022.140881] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Revised: 11/01/2022] [Accepted: 11/11/2022] [Indexed: 11/16/2022]
Abstract
In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.
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25
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Bögeholz LA, Mercier E, Wintermeyer W, Rodnina MV. Deformylation of nascent peptide chains on the ribosome. Methods Enzymol 2023; 684:39-70. [DOI: 10.1016/bs.mie.2023.02.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/12/2023]
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26
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Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W. Nat Struct Mol Biol 2023; 30:62-71. [PMID: 36593310 DOI: 10.1038/s41594-022-00866-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Accepted: 10/10/2022] [Indexed: 01/03/2023]
Abstract
Protein post-translational modification (PTM) regulates nearly every aspect of cellular processes in eukaryotes. However, the identification of new protein PTMs is very challenging. Here, using genetically encoded unnatural amino acids as chemical probes, we report the identification and validation of a previously unreported form of protein PTM, aminoacylated lysine ubiquitination, in which the modification occurs on the α-amine group of aminoacylated lysine. We identify more than 2,000 ubiquitination sites on all 20 aminoacylated lysines in two human cell lines. The modifications can mediate rapid protein degradation, complementing the canonical lysine ubiquitination-mediated proteome degradation. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme UBE2W acts as a writer of aminoacylated lysine ubiquitination and facilitates the ubiquitination event on proteins. More broadly, the discovery and validation of aminoacylated lysine ubiquitination paves the way for the identification and verification of new protein PTMs with the genetic code expansion strategy.
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27
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Meinnel T, Boyer JB, Giglione C. The Global Acetylation Profiling Pipeline for Quick Assessment of Protein N-Acetyltransferase Specificity In Cellulo. Methods Mol Biol 2023; 2718:137-150. [PMID: 37665458 DOI: 10.1007/978-1-0716-3457-8_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Global acetylation profiling (GAP) consists of heterologous expression of a given N-acetyltransferase (NAT) in Escherichia coli to assess its specificity. The remarkable sensitivity and robustness of the GAP pipeline relies on the very low frequency of known N-terminal acetylated proteins in E. coli, including their degree of N-terminal acetylation. Using the SILProNAQ mass spectrometry strategy on bacterial protein extracts, GAP permits easy acquisition of both qualitative and quantitative data to decipher the impact of any putative NAT of interest on the N-termini of newly acetylated proteins. This strategy allows rapid determination of the substrate specificity of any NAT.
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Affiliation(s)
- Thierry Meinnel
- Université Paris Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.
| | - Jean-Baptiste Boyer
- Université Paris Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Carmela Giglione
- Université Paris Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.
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28
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Ku YS, Cheng SS, Cheung MY, Law CH, Lam HM. The Re-Localization of Proteins to or Away from Membranes as an Effective Strategy for Regulating Stress Tolerance in Plants. MEMBRANES 2022; 12:membranes12121261. [PMID: 36557168 PMCID: PMC9788111 DOI: 10.3390/membranes12121261] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 12/08/2022] [Accepted: 12/09/2022] [Indexed: 06/12/2023]
Abstract
The membranes of plant cells are dynamic structures composed of phospholipids and proteins. Proteins harboring phospholipid-binding domains or lipid ligands can localize to membranes. Stress perception can alter the subcellular localization of these proteins dynamically, causing them to either associate with or detach from membranes. The mechanisms behind the re-localization involve changes in the lipidation state of the proteins and interactions with membrane-associated biomolecules. The functional significance of such re-localization includes the regulation of molecular transport, cell integrity, protein folding, signaling, and gene expression. In this review, proteins that re-localize to or away from membranes upon abiotic and biotic stresses will be discussed in terms of the mechanisms involved and the functional significance of their re-localization. Knowledge of the re-localization mechanisms will facilitate research on increasing plant stress adaptability, while the study on re-localization of proteins upon stresses will further our understanding of stress adaptation strategies in plants.
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29
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McGrath H, Černeková M, Kolář MH. Binding of the peptide deformylase on the ribosome surface modulates the exit tunnel interior. Biophys J 2022; 121:4443-4451. [PMID: 36335428 PMCID: PMC9748369 DOI: 10.1016/j.bpj.2022.11.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 08/26/2022] [Accepted: 11/01/2022] [Indexed: 11/06/2022] Open
Abstract
Proteosynthesis on ribosomes is regulated at many levels. Conformational changes of the ribosome, possibly induced by external factors, may transfer over large distances and contribute to the regulation. The molecular principles of this long-distance allostery within the ribosome remain poorly understood. Here, we use structural analysis and atomistic molecular dynamics simulations to investigate peptide deformylase (PDF), an enzyme that binds to the ribosome surface near the ribosomal protein uL22 during translation and chemically modifies the emerging nascent peptide. Our simulations of the entire ribosome-PDF complex reveal that the PDF undergoes a swaying motion on the ribosome surface at the submicrosecond timescale. We show that the PDF affects the conformational dynamics of parts of the ribosome over distances of more than 5 nm. Using a supervised-learning algorithm, we demonstrate that the exit tunnel is influenced by the presence or absence of PDF. Our findings suggest a possible effect of the PDF on the nascent peptide translocation through the ribosome exit tunnel.
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Affiliation(s)
- Hugo McGrath
- Department of Physical Chemistry, University of Chemistry and Technology, Prague, Czech Republic
| | - Michaela Černeková
- Department of Physical Chemistry, University of Chemistry and Technology, Prague, Czech Republic
| | - Michal H Kolář
- Department of Physical Chemistry, University of Chemistry and Technology, Prague, Czech Republic.
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30
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Pożoga M, Armbruster L, Wirtz M. From Nucleus to Membrane: A Subcellular Map of the N-Acetylation Machinery in Plants. Int J Mol Sci 2022; 23:ijms232214492. [PMID: 36430970 PMCID: PMC9692967 DOI: 10.3390/ijms232214492] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Revised: 11/14/2022] [Accepted: 11/17/2022] [Indexed: 11/23/2022] Open
Abstract
N-terminal acetylation (NTA) is an ancient protein modification conserved throughout all domains of life. N-terminally acetylated proteins are present in the cytosol, the nucleus, the plastids, mitochondria and the plasma membrane of plants. The frequency of NTA differs greatly between these subcellular compartments. While up to 80% of cytosolic and 20-30% of plastidic proteins are subject to NTA, NTA of mitochondrial proteins is rare. NTA alters key characteristics of proteins such as their three-dimensional structure, binding properties and lifetime. Since the majority of proteins is acetylated by five ribosome-bound N-terminal acetyltransferases (Nats) in yeast and humans, NTA was long perceived as an exclusively co-translational process in eukaryotes. The recent characterization of post-translationally acting plant Nats, which localize to the plasma membrane and the plastids, has challenged this view. Moreover, findings in humans, yeast, green algae and higher plants uncover differences in the cytosolic Nat machinery of photosynthetic and non-photosynthetic eukaryotes. These distinctive features of the plant Nat machinery might constitute adaptations to the sessile lifestyle of plants. This review sheds light on the unique role of plant N-acetyltransferases in development and stress responses as well as their evolution-driven adaptation to function in different cellular compartments.
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31
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Regioselectivity in inhibition of peptide deformylase from Haemophilus influenzae by 4- vs 5-azaindole hydroxamic acid derivatives: Biochemical, structural and antimicrobial studies. Bioorg Chem 2022; 128:106095. [DOI: 10.1016/j.bioorg.2022.106095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2022] [Revised: 08/04/2022] [Accepted: 08/09/2022] [Indexed: 11/20/2022]
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32
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Meinnel T, Giglione C. N-terminal modifications, the associated processing machinery, and their evolution in plastid-containing organisms. JOURNAL OF EXPERIMENTAL BOTANY 2022; 73:6013-6033. [PMID: 35768189 DOI: 10.1093/jxb/erac290] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Accepted: 06/28/2022] [Indexed: 06/15/2023]
Abstract
The N-terminus is a frequent site of protein modifications. Referring primarily to knowledge gained from land plants, here we review the modifications that change protein N-terminal residues and provide updated information about the associated machinery, including that in Archaeplastida. These N-terminal modifications include many proteolytic events as well as small group additions such as acylation or arginylation and oxidation. Compared with that of the mitochondrion, the plastid-dedicated N-terminal modification landscape is far more complex. In parallel, we extend this review to plastid-containing Chromalveolata including Stramenopiles, Apicomplexa, and Rhizaria. We report a well-conserved machinery, especially in the plastid. Consideration of the two most abundant proteins on Earth-Rubisco and actin-reveals the complexity of N-terminal modification processes. The progressive gene transfer from the plastid to the nuclear genome during evolution is exemplified by the N-terminus modification machinery, which appears to be one of the latest to have been transferred to the nuclear genome together with crucial major photosynthetic landmarks. This is evidenced by the greater number of plastid genes in Paulinellidae and red algae, the most recent and fossil recipients of primary endosymbiosis.
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Affiliation(s)
- Thierry Meinnel
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Carmela Giglione
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
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33
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Structural and large-scale analysis unveil the intertwined paths promoting NMT-catalyzed lysine and glycine myristoylation. J Mol Biol 2022; 434:167843. [PMID: 36181773 DOI: 10.1016/j.jmb.2022.167843] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Revised: 09/22/2022] [Accepted: 09/25/2022] [Indexed: 11/20/2022]
Abstract
N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate's reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.
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34
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Yang CI, Zhu Z, Jones JJ, Lomenick B, Chou TF, Shan SO. System-wide analyses reveal essential roles of N-terminal protein modification in bacterial membrane integrity. iScience 2022; 25:104756. [PMID: 35942092 PMCID: PMC9356101 DOI: 10.1016/j.isci.2022.104756] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Revised: 06/20/2022] [Accepted: 07/07/2022] [Indexed: 11/18/2022] Open
Abstract
The removal of the N-terminal formyl group on nascent proteins by peptide deformylase (PDF) is the most prevalent protein modification in bacteria. PDF is a critical target of antibiotic development; however, its role in bacterial physiology remains a long-standing question. This work used the time-resolved analyses of the Escherichia coli translatome and proteome to investigate the consequences of PDF inhibition. Loss of PDF activity rapidly induces cellular stress responses, especially those associated with protein misfolding and membrane defects, followed by a global down-regulation of metabolic pathways. Rapid membrane hyperpolarization and impaired membrane integrity were observed shortly after PDF inhibition, suggesting that the plasma membrane disruption is the most immediate and primary consequence of formyl group retention on nascent proteins. This work resolves the physiological function of a ubiquitous protein modification and uncovers its crucial role in maintaining the structure and function of the bacterial membrane.
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Affiliation(s)
- Chien-I Yang
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA
| | - Zikun Zhu
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA
| | - Jeffrey J. Jones
- Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, USA
| | - Brett Lomenick
- Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, USA
| | - Tsui-Fen Chou
- Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, USA
| | - Shu-ou Shan
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA
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35
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Sindlinger J, Schön S, Eirich J, Kirchgäßner S, Finkemeier I, Schwarzer D. Investigating peptide-Coenzyme A-conjugates as chemical probes for proteomic profiling of N-terminal and lysine acetyltransferases. Chembiochem 2022; 23:e202200255. [PMID: 35776679 PMCID: PMC9541820 DOI: 10.1002/cbic.202200255] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Revised: 07/01/2022] [Indexed: 11/18/2022]
Abstract
Acetyl groups are transferred from acetyl‐coenzyme A (Ac‐CoA) to protein N‐termini and lysine side chains by N‐terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs), respectively. Building on lysine‐CoA conjugates as KAT probes, we have synthesized peptide probes with CoA conjugated to N‐terminal alanine (α‐Ala‐CoA), proline (α‐Pro‐CoA) or tri‐glutamic acid (α‐3Glu‐CoA) units for interactome profiling of NAT complexes. The α‐Ala‐CoA probe enriched the majority of NAT catalytic and auxiliary subunits, while a lysine CoA‐conjugate bound only a subset of endogenous KATs. Interactome profiling with the α‐Pro‐CoA probe showed reduced NAT recruitment in favor of metabolic CoA binding proteins and α‐3Glu‐CoA steered the interactome towards NAA80 and NatB. These findings agreed with the inherent substrate specificities of the target proteins and showed that N‐terminal CoA‐conjugated peptides are versatile probes for NAT complex profiling in lysates of physiological and pathological backgrounds.
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Affiliation(s)
- Julia Sindlinger
- Eberhard Karls Universitat Tubingen Mathematisch-Naturwissenschaftliche Fakultat, Interfaculty Institute of Biochemistry, GERMANY
| | - Stefan Schön
- Eberhard Karls Universitat Tubingen Mathematisch-Naturwissenschaftliche Fakultat, Interfakultäres Institut für Biochemie, GERMANY
| | - Jürgen Eirich
- WWU Münster FB 13 Biologie: Westfalische Wilhelms-Universitat Munster Fachbereich 13 Biologie, Institute of Plant Biology and Biotechnology, GERMANY
| | - Sören Kirchgäßner
- Eberhard Karls Universität Tübingen: Eberhard Karls Universitat Tubingen, Interfakultäres Institut für Biochemie, GERMANY
| | - Iris Finkemeier
- WWU Münster FB 13 Biologie: Westfalische Wilhelms-Universitat Munster Fachbereich 13 Biologie, Institute of Plant Biology and Biotechnology, GERMANY
| | - Dirk Schwarzer
- Interfakultäres Institut für Biochemie Eberhard Karls Universität Tübingen, Chemical Biology, Hoppe-Seyler-Str. 4, 72076, Tübingen, GERMANY
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Meinnel T. Comment on “Binding Affinity Determines Substrate Specificity and Enables Discovery of Substrates for N-Myristoyltransferases”. ACS Catal 2022. [DOI: 10.1021/acscatal.2c01060] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Thierry Meinnel
- Université Paris Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette cedex, France
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37
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Nascimento JM, Gouvêa-Junqueira D, Zuccoli GS, Pedrosa CDSG, Brandão-Teles C, Crunfli F, Antunes ASLM, Cassoli JS, Karmirian K, Salerno JA, de Souza GF, Muraro SP, Proenca-Módena JL, Higa LM, Tanuri A, Garcez PP, Rehen SK, Martins-de-Souza D. Zika Virus Strains and Dengue Virus Induce Distinct Proteomic Changes in Neural Stem Cells and Neurospheres. Mol Neurobiol 2022; 59:5549-5563. [PMID: 35732867 DOI: 10.1007/s12035-022-02922-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 06/05/2022] [Indexed: 11/30/2022]
Abstract
Brain abnormalities and congenital malformations have been linked to the circulating strain of Zika virus (ZIKV) in Brazil since 2016 during the microcephaly outbreak; however, the molecular mechanisms behind several of these alterations and differential viral molecular targets have not been fully elucidated. Here we explore the proteomic alterations induced by ZIKV by comparing the Brazilian (Br ZIKV) and the African (MR766) viral strains, in addition to comparing them to the molecular responses to the Dengue virus type 2 (DENV). Neural stem cells (NSCs) derived from induced pluripotent stem (iPSCs) were cultured both as monolayers and in suspension (resulting in neurospheres), which were then infected with ZIKV (Br ZIKV or ZIKV MR766) or DENV to assess alterations within neural cells. Large-scale proteomic analyses allowed the comparison not only between viral strains but also regarding the two- and three-dimensional cellular models of neural cells derived from iPSCs, and the effects on their interaction. Altered pathways and biological processes were observed related to cell death, cell cycle dysregulation, and neurogenesis. These results reinforce already published data and provide further information regarding the biological alterations induced by ZIKV and DENV in neural cells.
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Affiliation(s)
- Juliana Minardi Nascimento
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil.,D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro, RJ, 22281-100, Brazil.,Department of Biosciences, Federal University of São Paulo, Santos, Brazil
| | - Danielle Gouvêa-Junqueira
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil
| | - Giuliana S Zuccoli
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil
| | | | - Caroline Brandão-Teles
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil
| | - Fernanda Crunfli
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil
| | - André S L M Antunes
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil
| | - Juliana S Cassoli
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil.,Institute of Biological Sciences, Federal University of Pará (UFPA), Belém, Brazil
| | - Karina Karmirian
- D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro, RJ, 22281-100, Brazil
| | - José Alexandre Salerno
- D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro, RJ, 22281-100, Brazil
| | - Gabriela Fabiano de Souza
- Laboratory of Emerging Viruses, Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (UNICAMP), São Paulo, Brazil
| | - Stéfanie Primon Muraro
- Laboratory of Emerging Viruses, Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (UNICAMP), São Paulo, Brazil
| | - Jose Luiz Proenca-Módena
- Laboratory of Emerging Viruses, Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (UNICAMP), São Paulo, Brazil
| | - Luiza M Higa
- Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
| | - Amilcar Tanuri
- Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
| | - Patricia P Garcez
- D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro, RJ, 22281-100, Brazil.,Institute of Biomedical Sciences, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
| | - Stevens K Rehen
- D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro, RJ, 22281-100, Brazil. .,Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
| | - Daniel Martins-de-Souza
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Rua Monteiro Lobato, Campinas, SP, 255, 13083-862, Brazil. .,D'Or Institute for Research and Education (IDOR), Rua Diniz Cordeiro, 30, Rio de Janeiro, RJ, 22281-100, Brazil. .,Experimental Medicine Research Cluster (EMRC), University of Campinas, Campinas, Brazil. .,Instituto Nacional de Biomarcadores Em Neuropsiquiatria (INBION), Conselho Nacional de Desenvolvimento Científico E Tecnológico, São Paulo, Brazil.
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38
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Nyonda MA, Boyer JB, Belmudes L, Krishnan A, Pino P, Couté Y, Brochet M, Meinnel T, Soldati-Favre D, Giglione C. N-Acetylation of secreted proteins is widespread in Apicomplexa and independent of acetyl-CoA ER-transporter AT1. J Cell Sci 2022; 135:275539. [PMID: 35621049 DOI: 10.1242/jcs.259811] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2022] [Accepted: 05/05/2022] [Indexed: 11/20/2022] Open
Abstract
Acetyl-CoA participates in post-translational modification of proteins, central carbon and lipid metabolism in several cell compartments. In mammals, the acetyl-CoA transporter 1 (AT1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with dedicated acetyltransferases including NAT8. However, the implication of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. We identify homologues of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei. Proteome-wide analyses revealed widespread N-terminal acetylation marks of secreted proteins in both parasites. Such acetylation profile of N-terminally processed proteins was never observed so far in any other organisms. AT1 deletion resulted in a considerable reduction of parasite fitness. In P. berghei, AT1 is important for growth of asexual blood stages and production of female gametocytes and male gametocytogenesis impaling its requirement for transmission. In the absence of AT1, the lysine and N-terminal acetylation sites remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.
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Affiliation(s)
- Mary Akinyi Nyonda
- Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland
| | - Jean-Baptiste Boyer
- Université Paris-Saclay, CEA, CNRS, Institute for Intergrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - Lucid Belmudes
- Université Grenoble Alpes, INSERM, CEA, UMR BioSanté U1292, CNRS, CEA, FR2048, 38000 Grenoble, France
| | - Aarti Krishnan
- Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland
| | - Paco Pino
- Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.,ExcellGene SA, CH1870 Monthey, Switzerland
| | - Yohann Couté
- Université Grenoble Alpes, INSERM, CEA, UMR BioSanté U1292, CNRS, CEA, FR2048, 38000 Grenoble, France
| | - Mathieu Brochet
- Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland
| | - Thierry Meinnel
- Université Paris-Saclay, CEA, CNRS, Institute for Intergrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
| | - Dominique Soldati-Favre
- Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland
| | - Carmela Giglione
- Université Paris-Saclay, CEA, CNRS, Institute for Intergrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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39
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Oberleitner L, Perrar A, Macorano L, Huesgen PF, Nowack ECM. A bipartite chromatophore transit peptide and N-terminal protein processing in the Paulinella chromatophore. PLANT PHYSIOLOGY 2022; 189:152-164. [PMID: 35043947 PMCID: PMC9070848 DOI: 10.1093/plphys/kiac012] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/19/2021] [Accepted: 12/06/2021] [Indexed: 05/19/2023]
Abstract
The amoeba Paulinella chromatophora contains photosynthetic organelles, termed chromatophores, which evolved independently from plastids in plants and algae. At least one-third of the chromatophore proteome consists of nucleus-encoded (NE) proteins that are imported across the chromatophore double envelope membranes. Chromatophore-targeted proteins exceeding 250 amino acids (aa) carry a conserved N-terminal extension presumably involved in protein targeting, termed the chromatophore transit peptide (crTP). Short imported proteins do not carry discernable targeting signals. To explore whether the import of proteins is accompanied by their N-terminal processing, here we identified N-termini of 208 chromatophore-localized proteins by a mass spectrometry-based approach. Our study revealed extensive N-terminal acetylation and proteolytic processing in both NE and chromatophore-encoded (CE) fractions of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Surprisingly, only the N-terminal ∼50 aa (part 1) become cleaved upon import. This part contains a conserved adaptor protein-1 complex-binding motif known to mediate protein sorting at the trans-Golgi network followed by a predicted transmembrane helix, implying that part 1 anchors the protein co-translationally in the endoplasmic reticulum and mediates trafficking to the chromatophore via the Golgi. The C-terminal part 2 contains conserved secondary structural elements, remains attached to the mature proteins, and might mediate translocation across the chromatophore inner membrane. Short imported proteins remain largely unprocessed. Finally, this work illuminates N-terminal processing of proteins encoded in an evolutionary-early-stage organelle and suggests host-derived posttranslationally acting factors involved in regulation of the CE chromatophore proteome.
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Affiliation(s)
- Linda Oberleitner
- Department of Biology, Institute of Microbial Cell Biology, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany
| | - Andreas Perrar
- Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, 52425 Jülich, Germany
- Cologne Excellence Cluster on Stress Responses in Ageing-Associated Diseases, CECAD, Medical Faculty and University Hospital, University of Cologne, 50931 Cologne, Germany
| | - Luis Macorano
- Department of Biology, Institute of Microbial Cell Biology, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany
| | - Pitter F Huesgen
- Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, 52425 Jülich, Germany
- Cologne Excellence Cluster on Stress Responses in Ageing-Associated Diseases, CECAD, Medical Faculty and University Hospital, University of Cologne, 50931 Cologne, Germany
- Department of Chemistry, Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany
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40
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Mercier E, Wang X, Bögeholz LAK, Wintermeyer W, Rodnina MV. Cotranslational Biogenesis of Membrane Proteins in Bacteria. Front Mol Biosci 2022; 9:871121. [PMID: 35573737 PMCID: PMC9099147 DOI: 10.3389/fmolb.2022.871121] [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: 02/07/2022] [Accepted: 04/12/2022] [Indexed: 12/26/2022] Open
Abstract
Nascent polypeptides emerging from the ribosome during translation are rapidly scanned and processed by ribosome-associated protein biogenesis factors (RPBs). RPBs cleave the N-terminal formyl and methionine groups, assist cotranslational protein folding, and sort the proteins according to their cellular destination. Ribosomes translating inner-membrane proteins are recognized and targeted to the translocon with the help of the signal recognition particle, SRP, and SRP receptor, FtsY. The growing nascent peptide is then inserted into the phospholipid bilayer at the translocon, an inner-membrane protein complex consisting of SecY, SecE, and SecG. Folding of membrane proteins requires that transmembrane helices (TMs) attain their correct topology, the soluble domains are inserted at the correct (cytoplasmic or periplasmic) side of the membrane, and – for polytopic membrane proteins – the TMs find their interaction partner TMs in the phospholipid bilayer. This review describes the recent progress in understanding how growing nascent peptides are processed and how inner-membrane proteins are targeted to the translocon and find their correct orientation at the membrane, with the focus on biophysical approaches revealing the dynamics of the process. We describe how spontaneous fluctuations of the translocon allow diffusion of TMs into the phospholipid bilayer and argue that the ribosome orchestrates cotranslational targeting not only by providing the binding platform for the RPBs or the translocon, but also by helping the nascent chains to find their correct orientation in the membrane. Finally, we present the auxiliary role of YidC as a chaperone for inner-membrane proteins. We show how biophysical approaches provide new insights into the dynamics of membrane protein biogenesis and raise new questions as to how translation modulates protein folding.
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41
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Gong X, Huang Y, Liang Y, Yuan Y, Liu Y, Han T, Li S, Gao H, Lv B, Huang X, Linster E, Wang Y, Wirtz M, Wang Y. OsHYPK-mediated protein N-terminal acetylation coordinates plant development and abiotic stress responses in rice. MOLECULAR PLANT 2022; 15:740-754. [PMID: 35381198 DOI: 10.1016/j.molp.2022.03.001] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 02/08/2022] [Accepted: 02/28/2022] [Indexed: 06/14/2023]
Abstract
N-terminal acetylation is one of the most common protein modifications in eukaryotes, and approximately 40% of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A (NatA) in a co-translational manner. However, the in vivo regulatory mechanism of NatA and the global impact of NatA-mediated N-terminal acetylation on protein fate remain unclear. Here, we identify Huntingtin Yeast partner K (HYPK), an evolutionarily conserved chaperone-like protein, as a positive regulator of NatA activity in rice. We found that loss of OsHYPK function leads to developmental defects in rice plant architecture but increased resistance to abiotic stresses, attributable to perturbation of the N-terminal acetylome and accelerated global protein turnover. Furthermore, we demonstrated that OsHYPK is also a substrate of NatA and that N-terminal acetylation of OsHYPK promotes its own degradation, probably through the Ac/N-degron pathway, which could be induced by abiotic stresses. Taken together, our findings suggest that the OsHYPK-NatA complex plays a critical role in coordinating plant development and stress responses by dynamically regulating NatA-mediated N-terminal acetylation and global protein turnover, which are essential for maintaining adaptive phenotypic plasticity in rice.
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Affiliation(s)
- Xiaodi Gong
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yaqian Huang
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yan Liang
- State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China
| | - Yundong Yuan
- State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China
| | - Yuhao Liu
- State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China
| | - Tongwen Han
- State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China
| | - Shujia Li
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Hengbin Gao
- State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China
| | - Bo Lv
- State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China
| | - Xiahe Huang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Eric Linster
- Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany
| | - Yingchun Wang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Markus Wirtz
- Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany
| | - Yonghong Wang
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agriculture University, Taian, Shandong 271018, China.
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42
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Yang CI, Kim J, Shan SO. Ribosome-nascent chain interaction regulates N-terminal protein modification. J Mol Biol 2022; 434:167535. [PMID: 35278477 PMCID: PMC9126151 DOI: 10.1016/j.jmb.2022.167535] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 03/01/2022] [Accepted: 03/04/2022] [Indexed: 01/02/2023]
Abstract
Numerous proteins initiate their folding, localization, and modifications early during translation, and emerging data show that the ribosome actively participates in diverse protein biogenesis pathways. Here we show that the ribosome imposes an additional layer of substrate selection during N-terminal methionine excision (NME), an essential protein modification in bacteria. Biochemical analyses show that cotranslational NME is exquisitely sensitive to a hydrophobic signal sequence or transmembrane domain near the N terminus of the nascent polypeptide. The ability of the nascent chain to access the active site of NME enzymes dictates NME efficiency, which is inhibited by confinement of the nascent chain on the ribosome surface and exacerbated by signal recognition particle. In vivo measurements corroborate the inhibition of NME by an N-terminal hydrophobic sequence, suggesting the retention of formylmethionine on a substantial fraction of the secretory and membrane proteome. Our work demonstrates how molecular features of a protein regulate its cotranslational modification and highlights the active participation of the ribosome in protein biogenesis pathways via interactions of the ribosome surface with the nascent protein.
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43
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(De)Activation (Ir)Reversibly or Degradation: Dynamics of Post-Translational Protein Modifications in Plants. Life (Basel) 2022; 12:life12020324. [PMID: 35207610 PMCID: PMC8874572 DOI: 10.3390/life12020324] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 02/11/2022] [Accepted: 02/16/2022] [Indexed: 11/22/2022] Open
Abstract
The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants.
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44
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Abstract
Protein N-termini provide unique and distinguishing information on proteolytically processed or N-terminally modified proteoforms. Also splicing, use of alternative translation initiation sites, and a variety of co- and post-translational N-terminal modifications generate distinct proteoforms that are unambiguously identified by their N-termini. However, N-terminal peptides are only a small fraction among all peptides generated in a shotgun proteome digest, are often of low stoichiometric abundance, and therefore require enrichment. Various protocols for enrichment of N-terminal peptides have been established and successfully been used for protease substrate discovery and profiling of N-terminal modification, but often require large amounts of proteome. We have recently established the High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) as a fast and sensitive method to enable enrichment of protein N-termini from limited sample sources with as little as a few microgram proteome. Here we present our current HUNTER protocol for sensitive plant N-terminome profiling, including sample preparation, enrichment of N-terminal peptides, and mass spectrometry data analysis.
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Affiliation(s)
- Fatih Demir
- Central Institute for Engineering, Electronics and Analytics, Forschungszentrum Jülich, Jülich, Germany
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Andreas Perrar
- Central Institute for Engineering, Electronics and Analytics, Forschungszentrum Jülich, Jülich, Germany
- Cologne Cluster of Excellence on Aging-related Disorders, CECAD, Medical Faculty and University Hospital, University of Cologne, Cologne, Germany
| | - Melissa Mantz
- Central Institute for Engineering, Electronics and Analytics, Forschungszentrum Jülich, Jülich, Germany
- Cologne Cluster of Excellence on Aging-related Disorders, CECAD, Medical Faculty and University Hospital, University of Cologne, Cologne, Germany
| | - Pitter F Huesgen
- Central Institute for Engineering, Electronics and Analytics, Forschungszentrum Jülich, Jülich, Germany.
- Cologne Cluster of Excellence on Aging-related Disorders, CECAD, Medical Faculty and University Hospital, University of Cologne, Cologne, Germany.
- Institute of Biochemistry, Department for Chemistry , University of Cologne, Cologne, Germany.
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45
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Bögeholz LAK, Mercier E, Wintermeyer W, Rodnina MV. Kinetic control of nascent protein biogenesis by peptide deformylase. Sci Rep 2021; 11:24457. [PMID: 34961771 PMCID: PMC8712518 DOI: 10.1038/s41598-021-03969-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2021] [Accepted: 12/13/2021] [Indexed: 12/05/2022] Open
Abstract
Synthesis of bacterial proteins on the ribosome starts with a formylated methionine. Removal of the N-terminal formyl group is essential and is carried out by peptide deformylase (PDF). Deformylation occurs co-translationally, shortly after the nascent-chain emerges from the ribosomal exit tunnel, and is necessary to allow for further N-terminal processing. Here we describe the kinetic mechanism of deformylation by PDF of ribosome-bound nascent-chains and show that PDF binding to and dissociation from ribosomes is rapid, allowing for efficient scanning of formylated substrates in the cell. The rate-limiting step in the PDF mechanism is a conformational rearrangement of the nascent-chain that takes place after cleavage of the formyl group. Under conditions of ongoing translation, the nascent-chain is deformylated rapidly as soon as it becomes accessible to PDF. Following deformylation, the enzyme is slow in releasing the deformylated nascent-chain, thereby delaying further processing and potentially acting as an early chaperone that protects short nascent chains before they reach a length sufficient to recruit other protein biogenesis factors.
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Affiliation(s)
- Lena A K Bögeholz
- Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany
| | - Evan Mercier
- Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany
| | - Wolfgang Wintermeyer
- Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany
| | - Marina V Rodnina
- Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
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46
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Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes. Genomics 2021; 114:292-304. [PMID: 34915127 DOI: 10.1016/j.ygeno.2021.12.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Revised: 11/28/2021] [Accepted: 12/09/2021] [Indexed: 11/24/2022]
Abstract
Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5' end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes. Totally, 1047 proteins were identified with 2058 TMPP labeled N-terminal peptides from all the 2625 mass spectrometer (MS) sequenced proteins. Comparative genomics analysis allowed the re-annotation of 43 proteins' N-termini in H37Rv and 762 proteins in Mycobacteriaceae. All revised N-termini start sites were distributed in 5'-UTR of annotated genes due to over-annotation of previous N-terminal initiation codon, especially the ATG. In addition, we identified and verified a novel gene Rv1078A in +3 frame different from the annotated gene Rv1078 in +2 frame. Altogether, our findings contribute to the better understanding of N-terminal of H37Rv and other species from Mycobacteriaceae that can assist future studies on biological study.
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Giglione C, Meinnel T. Mapping the myristoylome through a complete understanding of protein myristoylation biochemistry. Prog Lipid Res 2021; 85:101139. [PMID: 34793862 DOI: 10.1016/j.plipres.2021.101139] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Revised: 11/04/2021] [Accepted: 11/06/2021] [Indexed: 12/22/2022]
Abstract
Protein myristoylation is a C14 fatty acid modification found in all living organisms. Myristoylation tags either the N-terminal alpha groups of cysteine or glycine residues through amide bonds or lysine and cysteine side chains directly or indirectly via glycerol thioester and ester linkages. Before transfer to proteins, myristate must be activated into myristoyl coenzyme A in eukaryotes or, in bacteria, to derivatives like phosphatidylethanolamine. Myristate originates through de novo biosynthesis (e.g., plants), from external uptake (e.g., human tissues), or from mixed origins (e.g., unicellular organisms). Myristate usually serves as a molecular anchor, allowing tagged proteins to be targeted to membranes and travel across endomembrane networks in eukaryotes. In this review, we describe and discuss the metabolic origins of protein-bound myristate. We review strategies for in vivo protein labeling that take advantage of click-chemistry with reactive analogs, and we discuss new approaches to the proteome-wide discovery of myristate-containing proteins. The machineries of myristoylation are described, along with how protein targets can be generated directly from translating precursors or from processed proteins. Few myristoylation catalysts are currently described, with only N-myristoyltransferase described to date in eukaryotes. Finally, we describe how viruses and bacteria hijack and exploit myristoylation for their pathogenicity.
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Affiliation(s)
- Carmela Giglione
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France.
| | - Thierry Meinnel
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France.
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Metal utilization in genome-reduced bacteria: Do human mycoplasmas rely on iron? Comput Struct Biotechnol J 2021; 19:5752-5761. [PMID: 34765092 PMCID: PMC8566771 DOI: 10.1016/j.csbj.2021.10.022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 10/08/2021] [Accepted: 10/12/2021] [Indexed: 12/04/2022] Open
Abstract
Mycoplasmas are parasitic bacteria with streamlined genomes and complex nutritional requirements. Although iron is vital for almost all organisms, its utilization by mycoplasmas is controversial. Despite its minimalist nature, mycoplasmas can survive and persist within the host, where iron availability is rigorously restricted through nutritional immunity. In this review, we describe the putative iron-enzymes, transporters, and metalloregulators of four relevant human mycoplasmas. This work brings in light critical differences in the mycoplasma-iron interplay. Mycoplasma penetrans, the species with the largest genome (1.36 Mb), shows a more classic repertoire of iron-related proteins, including different enzymes using iron-sulfur clusters as well as iron storage and transport systems. In contrast, the iron requirement is less apparent in the three species with markedly reduced genomes, Mycoplasma genitalium (0.58 Mb), Mycoplasma hominis (0.67 Mb) and Mycoplasma pneumoniae (0.82 Mb), as they exhibit only a few proteins possibly involved in iron homeostasis. The multiple facets of iron metabolism in mycoplasmas illustrate the remarkable evolutive potential of these minimal organisms when facing nutritional immunity and question the dependence of several human-infecting species for iron. Collectively, our data contribute to better understand the unique biology and infective strategies of these successful pathogens.
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Key Words
- ABC, ATP-binding cassette
- ECF transporter
- ECF, energy-coupling factor
- Fur, ferric uptake regulator
- Hrl, histidine-rich lipoprotein
- Iron homeostasis
- Metal acquisition
- Metalloenzyme
- Mge, Mycoplasma genitalium
- Mho, Mycoplasma hominis
- Mollicutes
- Mpe, Mycoplasma penetrans
- Mpn, Mycoplasma pneumonia
- Mycoplasmas
- PDB, protein data bank
- RNR, ribonucleotide reductase
- XRF, X-ray fluorescence
- ZIP, zinc-iron permease
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Li X, Jiang Q, Yang X. Discovery of Inhibitors for Mycobacterium Tuberculosis Peptide Deformylase Based on Virtual Screening in Silico. Mol Inform 2021; 41:e2100002. [PMID: 34708566 DOI: 10.1002/minf.202100002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 08/21/2021] [Indexed: 01/02/2023]
Abstract
Tuberculosis has been the serious disease threatening human health and public safety due to the emergence of MDR and XDR-TB. Mycobacterium tuberculosis peptide deformylase (MtPDF) is a valuable target for antituberculotics. In order to discover new potential inhibitor candidates of MtPDF as leads for antituberculotics, Discovery Studio (DS) 2019 was used to perform molecular docking for virtual screening in silico with the bioactive compound library-I (L1700) against MtPDF. Six compounds with high docking scores and favourable ligand-protein interactions by LibDock and CDOCKER were selected for the evaluation of the inhibition potencies against MtPDF and Mycobacterium smegmatis. GST-6×His tagged MtPDF was recombinant expressed and purified firstly by Glutathione Sepharose 4B, and secondly by Ni Sepharose 6 FF after the cleavage of human rhinovirus 3C protease. These compounds showed IC50 values from 0.5 μmol/L to 112 μmol/L against MtPDF, among which CUDC-101 bearing hydroxamic acid exhibited IC50 of 0.5 μmol/L on MtPDF and MIC against Mycobacterium smegmatis of 32 μg/mL, and Ixazomib Citrate with IC50 of 63 μmol/L and MIC of 16 μg/mL. CUDC-101 and Ixazomib Citrate are promising as the potential leads for antituberculotics.
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Affiliation(s)
- Xinpeng Li
- Key Laboratory of Medical Laboratory Diagnostics of the, Ministry of Education of China, Chongqing Medical University, Chongqing, China
| | - Qihua Jiang
- College of pharmacy, Chongqing Medical University, Chongqing, China
| | - Xiaolan Yang
- Key Laboratory of Medical Laboratory Diagnostics of the, Ministry of Education of China, Chongqing Medical University, Chongqing, China
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Schlott AC, Knuepfer E, Green JL, Hobson P, Borg AJ, Morales-Sanfrutos J, Perrin AJ, Maclachlan C, Collinson LM, Snijders AP, Tate EW, Holder AA. Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion. PLoS Biol 2021; 19:e3001408. [PMID: 34695132 PMCID: PMC8544853 DOI: 10.1371/journal.pbio.3001408] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Accepted: 09/07/2021] [Indexed: 11/29/2022] Open
Abstract
We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition. Understanding the essential factors needed for malaria parasite development could help us find new therapeutic targets. This study reveals that N-myristoylation is a posttranslational modification of proteins essential for the parasites’ growth and their invasion of red blood cells.
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Affiliation(s)
- Anja C. Schlott
- Malaria Parasitology Laboratory, Francis Crick Institute, London, United Kingdom
- Molecular Sciences Research Hub, Imperial College, London, United Kingdom
| | - Ellen Knuepfer
- Malaria Parasitology Laboratory, Francis Crick Institute, London, United Kingdom
- Department of Pathobiology and Population Sciences, The Royal Veterinary College, Hatfield, United Kingdom
| | - Judith L. Green
- Malaria Parasitology Laboratory, Francis Crick Institute, London, United Kingdom
| | - Philip Hobson
- Flow Cytometry Science Technology Platform, Francis Crick Institute, London, United Kingdom
| | - Aaron J. Borg
- Mass Spectrometry Proteomics Science Technology Platform, Francis Crick Institute, London, United Kingdom
| | | | - Abigail J. Perrin
- Malaria Biochemistry Laboratory, Francis Crick Institute, London, United Kingdom
| | - Catherine Maclachlan
- Electron Microscopy Science Technology Platform, Francis Crick Institute, London, United Kingdom
| | - Lucy M. Collinson
- Electron Microscopy Science Technology Platform, Francis Crick Institute, London, United Kingdom
| | - Ambrosius P. Snijders
- Mass Spectrometry Proteomics Science Technology Platform, Francis Crick Institute, London, United Kingdom
| | - Edward W. Tate
- Molecular Sciences Research Hub, Imperial College, London, United Kingdom
- Francis Crick Institute, London, United Kingdom
- * E-mail: (EWT); (AAH)
| | - Anthony A. Holder
- Malaria Parasitology Laboratory, Francis Crick Institute, London, United Kingdom
- * E-mail: (EWT); (AAH)
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