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1
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Tröster V, Wong RP, Börgel A, Cakilkaya B, Renz C, Möckel MM, Eifler-Olivi K, Marinho J, Reinberg T, Furler S, Schaefer JV, Plückthun A, Wolf E, Ulrich HD. Custom affinity probes reveal DNA-damage-induced, ssDNA-independent chromatin SUMOylation in budding yeast. Cell Rep 2025; 44:115353. [PMID: 40019834 DOI: 10.1016/j.celrep.2025.115353] [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: 08/01/2024] [Revised: 12/17/2024] [Accepted: 02/05/2025] [Indexed: 03/29/2025] Open
Abstract
The small ubiquitin-related modifier SUMO regulates cellular processes in eukaryotes either by modulating individual protein-protein interactions or with relaxed substrate selectivity by group modification. Here, we report the isolation and characterization of designed ankyrin repeat protein (DARPin)-based affinity probes directed against budding yeast SUMO (Smt3). We validate selected DARPins as compartment-specific inhibitors or neutral detection agents. Structural characterization reveals a recognition mode distinct from that of natural SUMO interactors. In vivo application pinpoints Smt3's essential function to the nucleus and demonstrates DARPin-mediated sensitization toward various stress conditions. A subset of selected clones is validated as SUMOylation reporters in cells. In this manner, we identify a DNA-damage-induced nuclear SUMOylation response that-in contrast to previously reported chromatin group SUMOylation-is independent of single-stranded DNA and the SUMO-E3 Siz2 but depends on Mms21 and likely reflects late intermediates of homologous recombination. Thus, Smt3-specific DARPins can provide insight into the dynamics of SUMOylation in defined subcellular structures.
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Affiliation(s)
- Vera Tröster
- Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany
| | - Ronald P Wong
- Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany
| | - Arne Börgel
- Institute of Molecular Physiology, Johannes Gutenberg University, 55128 Mainz, Germany
| | - Baris Cakilkaya
- Institute of Molecular Physiology, Johannes Gutenberg University, 55128 Mainz, Germany
| | - Christian Renz
- Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany
| | - Martin M Möckel
- Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany
| | | | - Joana Marinho
- Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland
| | - Thomas Reinberg
- Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland
| | - Sven Furler
- Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland
| | - Jonas V Schaefer
- Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland
| | - Andreas Plückthun
- Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland
| | - Eva Wolf
- Institute of Molecular Physiology, Johannes Gutenberg University, 55128 Mainz, Germany
| | - Helle D Ulrich
- Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany.
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2
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Tharuka MDN, Courelli AS, Chen Y. Immune regulation by the SUMO family. Nat Rev Immunol 2025:10.1038/s41577-025-01155-4. [PMID: 40108400 DOI: 10.1038/s41577-025-01155-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/20/2025] [Indexed: 03/22/2025]
Abstract
Post-translational protein modifications by the small ubiquitin-like modifier (SUMO) family have been shown to regulate immune cells in the context of infection, autoimmunity and, more recently, cancer. Recent clinical trials investigating sumoylation inhibition as a therapeutic approach for cancer have established that sumoylation has important immune modulatory effects. Sumoylation suppresses transcription factors in innate immune cells and in cytotoxic T cells through the direct modification of these factors, which leads to the recruitment of transcriptional repressor complexes containing histone deacetylases. By contrast, in regulatory T cells and T helper 17 cells, sumoylation of transcription factors can enhance transcriptional activity by recruiting transcriptional coactivators. Sumoylation is also involved in the repression of IFNB1 and endogenous retroviruses and is therefore important for regulating interferon expression. A central theme from literature is that the sumoylation of a group of proteins, instead of a single target, collectively contributes to the regulation of various immune processes. In this Review, we consider how these studies provide scientific basis for future exploration of SUMO-mediated immune modulation for the treatment of cancers and autoimmune disorders.
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Affiliation(s)
- Mohottige D Neranjan Tharuka
- Division of Surgical Sciences, Department of Surgery, School of Medicine, University of California San Diego, La Jolla, CA, USA
- Moores Cancer Center, University of California San Diego, La Jolla, CA, USA
| | - Asimina S Courelli
- Division of Surgical Sciences, Department of Surgery, School of Medicine, University of California San Diego, La Jolla, CA, USA
- Moores Cancer Center, University of California San Diego, La Jolla, CA, USA
| | - Yuan Chen
- Division of Surgical Sciences, Department of Surgery, School of Medicine, University of California San Diego, La Jolla, CA, USA.
- Moores Cancer Center, University of California San Diego, La Jolla, CA, USA.
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3
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Chakraborty S, Strachan J, Schirmeisen K, Besse L, Mercier E, Fréon K, Zhang H, Zhao N, Bayne EH, Lambert SAE. The fission yeast SUMO-targeted ubiquitin ligase Slx8 functionally associates with clustered centromeres and the silent mating-type region at the nuclear periphery. Biol Open 2024; 13:bio061746. [PMID: 39786922 PMCID: PMC11708773 DOI: 10.1242/bio.061746] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2024] [Accepted: 11/22/2024] [Indexed: 01/12/2025] Open
Abstract
The SUMO-targeted ubiquitin ligase (STUbL) family is involved in multiple cellular processes via a wide range of mechanisms to maintain genome stability. One of the evolutionarily conserved functions of STUbL is to promote changes in the nuclear positioning of DNA lesions, targeting them to the nuclear periphery. In Schizossacharomyces pombe, the STUbL Slx8 is a regulator of SUMOylated proteins and promotes replication stress tolerance by counteracting the toxicity of SUMO conjugates. In order to study the dynamic dialectic between ubiquitinylation and SUMOylation in the nuclear space of the S. pombe genome, we analyzed Slx8 localization. Unexpectedly, we did not detect replication stress-induced Slx8 foci. However, we discovered that Slx8 forms a single nuclear focus, enriched at the nuclear periphery, which marks both clustered centromeres at the spindle pole body and the silent mating-type region. The formation of this single Slx8 focus requires the E3 SUMO ligase Pli1, poly-SUMOylation and the histone methyl transferase Clr4 that is responsible for the heterochromatin histone mark H3-K9 methylation. Finally, we established that Slx8 promotes centromere clustering and gene silencing at heterochromatin domains. Altogether, our data highlight evolutionarily conserved and functional relationships between STUbL and heterochromatin domains to promote gene silencing and nuclear organization.
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Affiliation(s)
- Shrena Chakraborty
- Institut Curie, Université PSL, CNRS UMR3348, 91400 Orsay, France
- Université Paris-Saclay, CNRS UMR3348, 91400 Orsay, France
| | - Joanna Strachan
- Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK
| | - Kamila Schirmeisen
- Institut Curie, Université PSL, CNRS UMR3348, 91400 Orsay, France
- Université Paris-Saclay, CNRS UMR3348, 91400 Orsay, France
| | - Laetitia Besse
- Institut Curie, Université PSL, CNRS UAR2016, Inserm US43, Université Paris-Saclay, Multimodal Imaging Center, 91400 Orsay, France
| | - Eve Mercier
- Institut Curie, Université PSL, CNRS UMR3348, 91400 Orsay, France
- Université Paris-Saclay, CNRS UMR3348, 91400 Orsay, France
| | - Karine Fréon
- Institut Curie, Université PSL, CNRS UMR3348, 91400 Orsay, France
- Université Paris-Saclay, CNRS UMR3348, 91400 Orsay, France
| | - Haidao Zhang
- Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK
| | - Ning Zhao
- Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK
| | - Elizabeth H. Bayne
- Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK
| | - Sarah A. E. Lambert
- Institut Curie, Université PSL, CNRS UMR3348, 91400 Orsay, France
- Université Paris-Saclay, CNRS UMR3348, 91400 Orsay, France
- Equipe Labélisée Ligue Nationale Contre le Cancer, 91400 Orsay, France
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4
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Gutierrez-Morton E, Haluska C, Collins L, Rizkallah R, Tomko RJ, Wang Y. The polySUMOylation axis promotes nucleolar release of Tof2 for mitotic exit. Cell Rep 2024; 43:114492. [PMID: 39002125 PMCID: PMC11298248 DOI: 10.1016/j.celrep.2024.114492] [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: 12/22/2023] [Revised: 05/02/2024] [Accepted: 06/26/2024] [Indexed: 07/15/2024] Open
Abstract
In budding yeast, the nucleolus serves as the site to sequester Cdc14, a phosphatase essential for mitotic exit. Nucleolar proteins Tof2, Net1, and Fob1 are required for this sequestration. Although it is known that these nucleolar proteins are SUMOylated, how SUMOylation regulates their activity remains unknown. Here, we show that Tof2 exhibits cell-cycle-regulated nucleolar delocalization and turnover. Depletion of the nuclear small ubiquitin-like modifier (SUMO) protease Ulp2 not only causes Tof2 polySUMOylation, nucleolar delocalization, and degradation but also leads to Cdc14 nucleolar release and activation. This outcome depends on polySUMOylation and the activity of downstream enzymes, including SUMO-targeted ubiquitin ligase and Cdc48/p97 segregase. We further developed a system to tether SUMO machinery to Tof2 and generated a SUMO-deficient tof2 mutant, and the results indicate that Tof2 polySUMOylation is necessary and sufficient for its nucleolar delocalization and degradation. Together, our work reveals a polySUMO-dependent mechanism that delocalizes Tof2 from the nucleolus to facilitate mitotic exit.
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Affiliation(s)
- Emily Gutierrez-Morton
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
| | - Cory Haluska
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Liam Collins
- College of Arts and Sciences, Florida State University, Tallahassee, FL 32306, USA
| | - Raed Rizkallah
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
| | - Robert J Tomko
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
| | - Yanchang Wang
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA.
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5
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Cho T, Hoeg L, Setiaputra D, Durocher D. NFATC2IP is a mediator of SUMO-dependent genome integrity. Genes Dev 2024; 38:233-252. [PMID: 38503515 PMCID: PMC11065178 DOI: 10.1101/gad.350914.123] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 03/04/2024] [Indexed: 03/21/2024]
Abstract
The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to genome duplication and repair. To investigate the mechanisms underpinning the essential function of SUMO, we undertook a genome-scale CRISPR/Cas9 screen probing the response to SUMOylation inhibition. This effort identified 130 genes whose disruption reduces or enhances the toxicity of TAK-981, a clinical-stage inhibitor of the SUMO E1-activating enzyme. Among the strongest hits, we validated and characterized NFATC2IP, an evolutionarily conserved protein related to the fungal Esc2 and Rad60 proteins that harbors tandem SUMO-like domains. Cells lacking NFATC2IP are viable but are hypersensitive to SUMO E1 inhibition, likely due to the accumulation of mitotic chromosome bridges and micronuclei. NFATC2IP primarily acts in interphase and associates with nascent DNA, suggesting a role in the postreplicative resolution of replication or recombination intermediates. Mechanistically, NFATC2IP interacts with the SMC5/6 complex and UBC9, the SUMO E2, via its first and second SUMO-like domains, respectively. AlphaFold-Multimer modeling suggests that NFATC2IP positions and activates the UBC9-NSMCE2 complex, the SUMO E3 ligase associated with SMC5/SMC6. We conclude that NFATC2IP is a key mediator of SUMO-dependent genomic integrity that collaborates with the SMC5/6 complex.
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Affiliation(s)
- Tiffany Cho
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
| | - Lisa Hoeg
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
| | - Dheva Setiaputra
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
| | - Daniel Durocher
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada;
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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6
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Han J, Mu Y, Huang J. Preserving genome integrity: The vital role of SUMO-targeted ubiquitin ligases. CELL INSIGHT 2023; 2:100128. [PMID: 38047137 PMCID: PMC10692494 DOI: 10.1016/j.cellin.2023.100128] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Revised: 10/09/2023] [Accepted: 10/09/2023] [Indexed: 12/05/2023]
Abstract
Various post-translational modifications (PTMs) collaboratively fine-tune protein activities. SUMO-targeted ubiquitin E3 ligases (STUbLs) emerge as specialized enzymes that recognize SUMO-modified substrates through SUMO-interaction motifs and subsequently ubiquitinate them via the RING domain, thereby bridging the SUMO and ubiquitin signaling pathways. STUbLs participate in a wide array of molecular processes, including cell cycle regulation, DNA repair, replication, and mitosis, operating under both normal conditions and in response to challenges such as genotoxic stress. Their ability to catalyze various types of ubiquitin chains results in diverse proteolytic and non-proteolytic outcomes for target substrates. Importantly, STUbLs are strategically positioned in close proximity to SUMO proteases and deubiquitinases (DUBs), ensuring precise and dynamic control over their target proteins. In this review, we provide insights into the unique properties and indispensable roles of STUbLs, with a particular emphasis on their significance in preserving genome integrity in humans.
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Affiliation(s)
- Jinhua Han
- Institute of Geriatrics, Affiliated Zhejiang Hospital, Zhejiang University School of Medicine, Hangzhou, 310030, Zhejiang, China
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, Zhejiang, China
| | - Yanhua Mu
- National-Local Joint Engineering Research Center of Biodiagnosis & Biotherapy, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710004, Shaanxi, China
| | - Jun Huang
- Institute of Geriatrics, Affiliated Zhejiang Hospital, Zhejiang University School of Medicine, Hangzhou, 310030, Zhejiang, China
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, Zhejiang, China
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7
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Liu S, Atkinson E, Paulucci-Holthauzen A, Wang B. A CK2 and SUMO-dependent, PML NB-involved regulatory mechanism controlling BLM ubiquitination and G-quadruplex resolution. Nat Commun 2023; 14:6111. [PMID: 37777511 PMCID: PMC10542384 DOI: 10.1038/s41467-023-41705-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Accepted: 09/14/2023] [Indexed: 10/02/2023] Open
Abstract
The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex.
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Affiliation(s)
- Shichang Liu
- Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA
| | - Erin Atkinson
- Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA
- Genetics and Epigenetics Program, The MD Anderson Cancer Center and UT Health Graduate School of Biomedical Sciences, Houston, TX, 77030, USA
| | | | - Bin Wang
- Department of Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA.
- Genetics and Epigenetics Program, The MD Anderson Cancer Center and UT Health Graduate School of Biomedical Sciences, Houston, TX, 77030, USA.
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8
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Cheng X, Yang W, Lin W, Mei F. Paradoxes of Cellular SUMOylation Regulation: A Role of Biomolecular Condensates? Pharmacol Rev 2023; 75:979-1006. [PMID: 37137717 PMCID: PMC10441629 DOI: 10.1124/pharmrev.122.000784] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 04/20/2023] [Accepted: 04/27/2023] [Indexed: 05/05/2023] Open
Abstract
Protein SUMOylation is a major post-translational modification essential for maintaining cellular homeostasis. SUMOylation has long been associated with stress responses as a diverse array of cellular stress signals are known to trigger rapid alternations in global protein SUMOylation. In addition, while there are large families of ubiquitination enzymes, all small ubiquitin-like modifiers (SUMOs) are conjugated by a set of enzymatic machinery comprising one heterodimeric SUMO-activating enzyme, a single SUMO-conjugating enzyme, and a small number of SUMO protein ligases and SUMO-specific proteases. How a few SUMOylation enzymes specifically modify thousands of functional targets in response to diverse cellular stresses remains an enigma. Here we review recent progress toward understanding the mechanisms of SUMO regulation, particularly the potential roles of liquid-liquid phase separation/biomolecular condensates in regulating cellular SUMOylation during cellular stresses. In addition, we discuss the role of protein SUMOylation in pathogenesis and the development of novel therapeutics targeting SUMOylation. SIGNIFICANCE STATEMENT: Protein SUMOylation is one of the most prevalent post-translational modifications and plays a vital role in maintaining cellular homeostasis in response to stresses. Protein SUMOylation has been implicated in human pathogenesis, such as cancer, cardiovascular diseases, neurodegeneration, and infection. After more than a quarter century of extensive research, intriguing enigmas remain regarding the mechanism of cellular SUMOylation regulation and the therapeutic potential of targeting SUMOylation.
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Affiliation(s)
- Xiaodong Cheng
- Department of Integrative Biology & Pharmacology and Texas Therapeutics Institute, Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas
| | - Wenli Yang
- Department of Integrative Biology & Pharmacology and Texas Therapeutics Institute, Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas
| | - Wei Lin
- Department of Integrative Biology & Pharmacology and Texas Therapeutics Institute, Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas
| | - Fang Mei
- Department of Integrative Biology & Pharmacology and Texas Therapeutics Institute, Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas
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9
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Chang YC, Lin K, Baxley RM, Durrett W, Wang L, Stojkova O, Billmann M, Ward H, Myers CL, Bielinsky AK. RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication. Open Biol 2023; 13:230068. [PMID: 37607592 PMCID: PMC10444366 DOI: 10.1098/rsob.230068] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 07/27/2023] [Indexed: 08/24/2023] Open
Abstract
DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.
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Affiliation(s)
- Ya-Chu Chang
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Kevin Lin
- Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Ryan M. Baxley
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Wesley Durrett
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Liangjun Wang
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Olivera Stojkova
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Maximilian Billmann
- Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Henry Ward
- Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Chad L. Myers
- Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Anja-Katrin Bielinsky
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
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10
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van den Berg SJW, Jansen LET. SUMO control of centromere homeostasis. Front Cell Dev Biol 2023; 11:1193192. [PMID: 37181753 PMCID: PMC10172491 DOI: 10.3389/fcell.2023.1193192] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Accepted: 04/17/2023] [Indexed: 05/16/2023] Open
Abstract
Centromeres are unique chromosomal loci that form the anchorage point for the mitotic spindle during mitosis and meiosis. Their position and function are specified by a unique chromatin domain featuring the histone H3 variant CENP-A. While typically formed on centromeric satellite arrays, CENP-A nucleosomes are maintained and assembled by a strong self-templated feedback mechanism that can propagate centromeres even at non-canonical sites. Central to the epigenetic chromatin-based transmission of centromeres is the stable inheritance of CENP-A nucleosomes. While long-lived at centromeres, CENP-A can turn over rapidly at non-centromeric sites and even erode from centromeres in non-dividing cells. Recently, SUMO modification of the centromere complex has come to the forefront as a mediator of centromere complex stability, including CENP-A chromatin. We review evidence from different models and discuss the emerging view that limited SUMOylation appears to play a constructive role in centromere complex formation, while polySUMOylation drives complex turnover. The deSUMOylase SENP6/Ulp2 and the proteins segregase p97/Cdc48 constitute the dominant opposing forces that balance CENP-A chromatin stability. This balance may be key to ensuring proper kinetochore strength at the centromere while preventing ectopic centromere formation.
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Affiliation(s)
- Sebastiaan J. W. van den Berg
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
- Instituto Gulbenkian de Ciencia, Oeiras, Portugal
| | - Lars E. T. Jansen
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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11
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Alghoul E, Paloni M, Takedachi A, Urbach S, Barducci A, Gaillard PH, Basbous J, Constantinou A. Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair. Mol Cell 2023; 83:1640-1658.e9. [PMID: 37059091 DOI: 10.1016/j.molcel.2023.03.021] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Revised: 02/06/2023] [Accepted: 03/21/2023] [Indexed: 04/16/2023]
Abstract
SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.
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Affiliation(s)
- Emile Alghoul
- Institut de Génétique Humaine, Université de Montpellier, CNRS, Montpellier, France
| | - Matteo Paloni
- Centre de Biologie Structurale (CBS), Université de Montpellier, CNRS, INSERM, Montpellier, France
| | - Arato Takedachi
- Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille, France
| | - Serge Urbach
- Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, Montpellier, France; Montpellier RIO Imaging, Montpellier, France
| | - Alessandro Barducci
- Centre de Biologie Structurale (CBS), Université de Montpellier, CNRS, INSERM, Montpellier, France
| | | | - Jihane Basbous
- Institut de Génétique Humaine, Université de Montpellier, CNRS, Montpellier, France.
| | - Angelos Constantinou
- Institut de Génétique Humaine, Université de Montpellier, CNRS, Montpellier, France.
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12
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Xia J, Jiang S, Dong S, Liao Y, Zhou Y. The Role of Post-Translational Modifications in Regulation of NLRP3 Inflammasome Activation. Int J Mol Sci 2023; 24:ijms24076126. [PMID: 37047097 PMCID: PMC10093848 DOI: 10.3390/ijms24076126] [Citation(s) in RCA: 26] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Revised: 03/16/2023] [Accepted: 03/20/2023] [Indexed: 04/14/2023] Open
Abstract
Pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) induce NLRP3 inflammasome activation, and subsequent formation of active caspase-1 as well as the maturation of interleukin-1β (IL-1β) and gasdermin D (GSDMD), mediating the occurrence of pyroptosis and inflammation. Aberrant NLRP3 inflammasome activation causes a variety of diseases. Therefore, the NLRP3 inflammasome pathway is a target for prevention and treatment of relative diseases. Recent studies have suggested that NLRP3 inflammasome activity is closely associated with its post-translational modifications (PTMs). This review focuses on PTMs of the components of the NLRP3 inflammasome and the resultant effects on regulation of its activity to provide references for the exploration of the mechanisms by which the NLRP3 inflammasome is activated and controlled.
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Affiliation(s)
- Jing Xia
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China
| | - Songhong Jiang
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China
| | - Shiqi Dong
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China
| | - Yonghong Liao
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China
- National Center of Technology Innovation for Pigs, Chongqing 402460, China
| | - Yang Zhou
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China
- National Center of Technology Innovation for Pigs, Chongqing 402460, China
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13
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Wang W, Lu J, Yang WC, Spear ED, Michaelis S, Matunis MJ. Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control. J Biol Chem 2023; 299:102851. [PMID: 36587767 PMCID: PMC9898758 DOI: 10.1016/j.jbc.2022.102851] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 12/16/2022] [Accepted: 12/19/2022] [Indexed: 12/30/2022] Open
Abstract
Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMO-dependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQC-associated diseases.
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Affiliation(s)
- Wei Wang
- Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Jian Lu
- Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Wei-Chih Yang
- Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Eric D Spear
- Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA
| | - Susan Michaelis
- Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA
| | - Michael J Matunis
- Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, USA.
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14
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SUMO enhances unfolding of SUMO-polyubiquitin-modified substrates by the Ufd1/Npl4/Cdc48 complex. Proc Natl Acad Sci U S A 2023; 120:e2213703120. [PMID: 36574706 PMCID: PMC9910466 DOI: 10.1073/pnas.2213703120] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The Ufd1/Npl4/Cdc48 complex is a universal protein segregase that plays key roles in eukaryotic cellular processes. Its functions orchestrating the clearance or removal of polyubiquitylated targets are established; however, prior studies suggest that the complex also targets substrates modified by the ubiquitin-like protein SUMO. Here, we show that interactions between Ufd1 and SUMO enhance unfolding of substrates modified by SUMO-polyubiquitin hybrid chains by the budding yeast Ufd1/Npl4/Cdc48 complex compared to substrates modified by polyubiquitin chains, a difference that is accentuated when the complex has a choice between these substrates. Incubating Ufd1/Npl4/Cdc48 with a substrate modified by a SUMO-polyubiquitin hybrid chain produced a series of single-particle cryo-EM structures that reveal features of interactions between Ufd1/Npl4/Cdc48 and ubiquitin prior to and during unfolding of ubiquitin. These results are consistent with cellular functions for SUMO and ubiquitin modifications and support a physical model wherein Ufd1/Npl4/Cdc48, SUMO, and ubiquitin conjugation pathways converge to promote clearance of proteins modified with SUMO and polyubiquitin.
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15
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Lascorz J, Codina-Fabra J, Reverter D, Torres-Rosell J. SUMO-SIM interactions: From structure to biological functions. Semin Cell Dev Biol 2022; 132:193-202. [PMID: 34840078 DOI: 10.1016/j.semcdb.2021.11.007] [Citation(s) in RCA: 37] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 11/08/2021] [Accepted: 11/08/2021] [Indexed: 12/15/2022]
Abstract
Post-translational modification by Small Ubiquitin-like Modifier (SUMO) proteins regulates numerous cellular processes. This modification involves the covalent and reversible attachment of SUMO to target proteins through an isopeptide bond, using a cascade of E1, E2 and E3 SUMOylation enzymes. Most functions of SUMO depend on the establishment of non-covalent protein-protein interactions between SUMOylated substrates and their binding partners. The vast majority of these interactions involve a conserved surface in the SUMO protein and a SUMO interacting motif (SIM), a short stretch of hydrophobic amino acids and an acidic region, in the interactor protein. Despite single SUMO-SIM interactions are relatively weak, they can have a huge impact at different levels, altering the activity, localization and stability of proteins, triggering the formation of macromolecular assemblies or inducing phase separation. Moreover, SUMO-SIM interactions are ubiquitous in most enzymes of the SUMO pathway, and play essential roles in SUMO conjugation and deconjugation. Here, we analyze the role of SUMO-SIM contacts in SUMO enzymes and targets and discuss how this humble interaction participates in SUMOylation reactions and mediates the outcome of this essential post-translational modification.
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Affiliation(s)
- Jara Lascorz
- Institut de Biotecnologia i de Biomedicina (IBB) and Dept. de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
| | - Joan Codina-Fabra
- Departament de Ciencies Mediques Basiques, Institut de Recerca Biomedica de Lleida, Universitat de Lleida, 25198 Lleida, Spain
| | - David Reverter
- Institut de Biotecnologia i de Biomedicina (IBB) and Dept. de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
| | - Jordi Torres-Rosell
- Departament de Ciencies Mediques Basiques, Institut de Recerca Biomedica de Lleida, Universitat de Lleida, 25198 Lleida, Spain.
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16
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Basu-Shrivastava M, Mojsa B, Mora S, Robbins I, Bossis G, Lassot I, Desagher S. Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3. Cell Death Differ 2022; 29:2107-2122. [PMID: 35449213 PMCID: PMC9613758 DOI: 10.1038/s41418-022-01002-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Revised: 04/04/2022] [Accepted: 04/05/2022] [Indexed: 01/29/2023] Open
Abstract
NFATc3 is the predominant member of the NFAT family of transcription factors in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 binds and ubiquitinates NFATc3 in vitro and in cells where it reduces NFATc3 protein level and transcriptional activity. In contrast, silencing of endogenous Trim39 decreases NFATc3 ubiquitination and increases its activity, thereby resulting in enhanced neuronal apoptosis. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, we identify Trim39 as a new SUMO-targeted E3 ubiquitin-ligase (STUbL). Indeed, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motifs in Trim39 reduces NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. In addition, Trim39 preferentially ubiquitinates SUMOylated forms of NFATc3 in vitro. As a consequence, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity in neurons. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a STUbL for NFATc3.
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Affiliation(s)
- Meenakshi Basu-Shrivastava
- IGMM, Univ Montpellier, CNRS, Montpellier, France
- Massachusetts General Hospital Cancer Center and Department of Medicine, Harvard Medical School, Boston, MA, USA
| | - Barbara Mojsa
- IGMM, Univ Montpellier, CNRS, Montpellier, France
- Centre for Gene Regulation and Expression, School of Life Science, University of Dundee, Dundee, UK
| | - Stéphan Mora
- IGMM, Univ Montpellier, CNRS, Montpellier, France
| | - Ian Robbins
- IGMM, Univ Montpellier, CNRS, Montpellier, France
| | | | - Iréna Lassot
- IGMM, Univ Montpellier, CNRS, Montpellier, France
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17
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Leng X, Duxin JP. Targeting DNA-Protein Crosslinks via Post-Translational Modifications. Front Mol Biosci 2022; 9:944775. [PMID: 35860355 PMCID: PMC9289515 DOI: 10.3389/fmolb.2022.944775] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2022] [Accepted: 06/03/2022] [Indexed: 11/13/2022] Open
Abstract
Covalent binding of proteins to DNA forms DNA-protein crosslinks (DPCs), which represent cytotoxic DNA lesions that interfere with essential processes such as DNA replication and transcription. Cells possess different enzymatic activities to counteract DPCs. These include enzymes that degrade the adducted proteins, resolve the crosslinks, or incise the DNA to remove the crosslinked proteins. An important question is how DPCs are sensed and targeted for removal via the most suited pathway. Recent advances have shown the inherent role of DNA replication in triggering DPC removal by proteolysis. However, DPCs are also efficiently sensed and removed in the absence of DNA replication. In either scenario, post-translational modifications (PTMs) on DPCs play essential and versatile roles in orchestrating the repair routes. In this review, we summarize the current knowledge of the mechanisms that trigger DPC removal via PTMs, focusing on ubiquitylation, small ubiquitin-related modifier (SUMO) conjugation (SUMOylation), and poly (ADP-ribosyl)ation (PARylation). We also briefly discuss the current knowledge gaps and emerging hypotheses in the field.
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18
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Vertegaal ACO. Signalling mechanisms and cellular functions of SUMO. Nat Rev Mol Cell Biol 2022; 23:715-731. [PMID: 35750927 DOI: 10.1038/s41580-022-00500-y] [Citation(s) in RCA: 157] [Impact Index Per Article: 52.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/23/2022] [Indexed: 12/22/2022]
Abstract
Sumoylation is an essential post-translational modification that is catalysed by a small number of modifying enzymes but regulates thousands of target proteins in a dynamic manner. Small ubiquitin-like modifiers (SUMOs) can be attached to target proteins as one or more monomers or in the form of polymers of different types. Non-covalent readers recognize SUMO-modified proteins via SUMO interaction motifs. SUMO simultaneously modifies groups of functionally related proteins to regulate predominantly nuclear processes, including gene expression, the DNA damage response, RNA processing, cell cycle progression and proteostasis. Recent progress has increased our understanding of the cellular and pathophysiological roles of SUMO modifications, extending their functions to the regulation of immunity, pluripotency and nuclear body assembly in response to oxidative stress, which partly occurs through the recently characterized mechanism of liquid-liquid phase separation. Such progress in understanding the roles and regulation of sumoylation opens new avenues for the targeting of SUMO to treat disease, and indeed the first drug blocking sumoylation is currently under investigation in clinical trials as a possible anticancer agent.
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Affiliation(s)
- Alfred C O Vertegaal
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands.
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19
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Ding L, Luo Y, Tian T, Chen X, Yang Y, Bu M, Han J, Yang B, Yan H, Liu T, Wu M, Zhang G, Xu Y, Zhu S, Huen MSY, Mao G, Huang J. RNF4 controls the extent of replication fork reversal to preserve genome stability. Nucleic Acids Res 2022; 50:5672-5687. [PMID: 35640614 PMCID: PMC9177969 DOI: 10.1093/nar/gkac447] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Revised: 05/09/2022] [Accepted: 05/15/2022] [Indexed: 11/14/2022] Open
Abstract
Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT–TOP2A–PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.
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Affiliation(s)
- Linli Ding
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Yi Luo
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Tian Tian
- The Eighth Affiliated Hospital, Sun Yat-Sen University, Shenzhen 518033, Guangdong, China
| | - Xu Chen
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Yulan Yang
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Min Bu
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Jinhua Han
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Bing Yang
- The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
| | - Haiyan Yan
- School of Medicine, Zhejiang University City of College, Hangzhou 310015, Zhejiang, China
| | - Ting Liu
- Department of Cell Biology, and Department of General Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang, China
| | - Mengjie Wu
- The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine and Key laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou 310058, Zhejiang, China
| | - Guofei Zhang
- Department of Thoracic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang, China
| | - Yipeng Xu
- Department of Urology, Cancer Hospital of the University of Chinese Academy of Sciences, Zhejiang Cancer Hospital, Hangzhou 310058, Zhejiang, China
| | - Shaoxing Zhu
- Department of Urology, Cancer Hospital of the University of Chinese Academy of Sciences, Zhejiang Cancer Hospital, Hangzhou 310058, Zhejiang, China
| | - Michael S Y Huen
- Department of Anatomy, The University of Hong Kong, Hong Kong, China
| | - Genxiang Mao
- Zhejiang Provincial Key Lab of Geriatrics and Geriatrics Institute of Zhejiang Province, Department of Geriatrics, Zhejiang Hospital, Hangzhou 310030, Zhejiang, China
| | - Jun Huang
- Zhejiang Provincial Key Lab of Geriatrics and Geriatrics Institute of Zhejiang Province, Department of Geriatrics, Zhejiang Hospital, Hangzhou 310030, Zhejiang, China.,Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, Zhejiang, China
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20
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SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites. Genes (Basel) 2021; 12:genes12122010. [PMID: 34946958 PMCID: PMC8701742 DOI: 10.3390/genes12122010] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 12/13/2021] [Accepted: 12/13/2021] [Indexed: 12/20/2022] Open
Abstract
DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.
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21
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Ellis N, Zhu J, Yagle MK, Yang WC, Huang J, Kwako A, Seidman MM, Matunis MJ. RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse. Front Genet 2021; 12:753535. [PMID: 34868226 PMCID: PMC8633118 DOI: 10.3389/fgene.2021.753535] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Accepted: 10/25/2021] [Indexed: 12/01/2022] Open
Abstract
Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.
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Affiliation(s)
- Nathan Ellis
- University of Arizona Cancer Center, University of Arizona, Tucson, AZ, United States
| | - Jianmei Zhu
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States
| | - Mary K Yagle
- University of Arizona Cancer Center, University of Arizona, Tucson, AZ, United States
| | - Wei-Chih Yang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States
| | - Jing Huang
- Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore, MD, United States
| | - Alexander Kwako
- University of Arizona Cancer Center, University of Arizona, Tucson, AZ, United States
| | - Michael M Seidman
- Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore, MD, United States
| | - Michael J Matunis
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States
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22
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Fottner M, Weyh M, Gaussmann S, Schwarz D, Sattler M, Lang K. A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes. Nat Commun 2021; 12:6515. [PMID: 34764289 PMCID: PMC8585875 DOI: 10.1038/s41467-021-26812-9] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Accepted: 10/13/2021] [Indexed: 11/09/2022] Open
Abstract
The post-translational modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) represents one of the most important regulators in eukaryotic biology. Polymeric Ub/Ubl chains of distinct topologies control the activity, stability, interaction and localization of almost all cellular proteins and elicit a variety of biological outputs. Our ability to characterize the roles of distinct Ub/Ubl topologies and to identify enzymes and receptors that create, recognize and remove these modifications is however hampered by the difficulty to prepare them. Here we introduce a modular toolbox (Ubl-tools) that allows the stepwise assembly of Ub/Ubl chains in a flexible and user-defined manner facilitated by orthogonal sortase enzymes. We demonstrate the universality and applicability of Ubl-tools by generating distinctly linked Ub/Ubl hybrid chains, and investigate their role in DNA damage repair. Importantly, Ubl-tools guarantees straightforward access to target proteins, site-specifically modified with distinct homo- and heterotypic (including branched) Ub chains, providing a powerful approach for studying the functional impact of these complex modifications on cellular processes.
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Affiliation(s)
- Maximilian Fottner
- grid.6936.a0000000123222966Department of Chemistry, Lab for Synthetic Biochemistry, Technical University of Munich, Institute for Advanced Study, TUM-IAS, Lichtenberg Str. 4, 85748 Garching, Germany ,grid.5801.c0000 0001 2156 2780Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland
| | - Maria Weyh
- grid.6936.a0000000123222966Department of Chemistry, Lab for Synthetic Biochemistry, Technical University of Munich, Institute for Advanced Study, TUM-IAS, Lichtenberg Str. 4, 85748 Garching, Germany
| | - Stefan Gaussmann
- grid.6936.a0000000123222966Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Lichtenberg Str. 4, 85748 Garching, Germany ,grid.4567.00000 0004 0483 2525Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
| | - Dominic Schwarz
- grid.6936.a0000000123222966Department of Chemistry, Lab for Synthetic Biochemistry, Technical University of Munich, Institute for Advanced Study, TUM-IAS, Lichtenberg Str. 4, 85748 Garching, Germany
| | - Michael Sattler
- grid.6936.a0000000123222966Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Lichtenberg Str. 4, 85748 Garching, Germany ,grid.4567.00000 0004 0483 2525Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
| | - Kathrin Lang
- Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093, Zurich, Switzerland.
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23
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Franz A, Valledor P, Ubieto-Capella P, Pilger D, Galarreta A, Lafarga V, Fernández-Llorente A, de la Vega-Barranco G, den Brave F, Hoppe T, Fernandez-Capetillo O, Lecona E. USP7 and VCP FAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork. Cell Rep 2021; 37:109819. [PMID: 34644576 PMCID: PMC8527565 DOI: 10.1016/j.celrep.2021.109819] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 07/20/2021] [Accepted: 09/21/2021] [Indexed: 12/16/2022] Open
Abstract
The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.
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Affiliation(s)
- André Franz
- Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany
| | - Pablo Valledor
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
| | - Patricia Ubieto-Capella
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
| | - Domenic Pilger
- The Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK
| | - Antonio Galarreta
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
| | - Vanesa Lafarga
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
| | - Alejandro Fernández-Llorente
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain
| | - Guillermo de la Vega-Barranco
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain
| | - Fabian den Brave
- Institute of Biochemistry and Molecular Biology, University of Bonn, 53115 Bonn, Germany
| | - Thorsten Hoppe
- Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
| | - Oscar Fernandez-Capetillo
- Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain; Science for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 21 Stockholm, Sweden.
| | - Emilio Lecona
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain.
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24
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ATM controls the extent of DNA end resection by eliciting sequential posttranslational modifications of CtIP. Proc Natl Acad Sci U S A 2021; 118:2022600118. [PMID: 33723063 DOI: 10.1073/pnas.2022600118] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
DNA end resection is a critical step in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR). However, the mechanisms governing the extent of resection at DSB sites undergoing homology-directed repair remain unclear. Here, we show that, upon DSB induction, the key resection factor CtIP is modified by the ubiquitin-like protein SUMO at lysine 578 in a PIAS4-dependent manner. CtIP SUMOylation occurs on damaged chromatin and requires prior hyperphosphorylation by the ATM protein kinase. SUMO-modified hyperphosphorylated CtIP is targeted by the SUMO-dependent E3 ubiquitin ligase RNF4 for polyubiquitination and subsequent degradation. Consequently, disruption of CtIP SUMOylation results in aberrant accumulation of CtIP at DSBs, which, in turn, causes uncontrolled excessive resection, defective HR, and increased cellular sensitivity to DSB-inducing agents. These findings reveal a previously unidentified regulatory mechanism that regulates CtIP activity at DSBs and thus the extent of end resection via ATM-dependent sequential posttranslational modification of CtIP.
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25
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Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase. Nat Commun 2021; 12:4918. [PMID: 34389719 PMCID: PMC8363623 DOI: 10.1038/s41467-021-25205-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Accepted: 07/20/2021] [Indexed: 12/12/2022] Open
Abstract
Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.
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26
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Decoding post translational modification crosstalk with proteomics. Mol Cell Proteomics 2021; 20:100129. [PMID: 34339852 PMCID: PMC8430371 DOI: 10.1016/j.mcpro.2021.100129] [Citation(s) in RCA: 125] [Impact Index Per Article: 31.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2021] [Revised: 07/06/2021] [Accepted: 07/27/2021] [Indexed: 12/12/2022] Open
Abstract
Post-translational modification (PTM) of proteins allows cells to regulate protein functions, transduce signals and respond to perturbations. PTMs expand protein functionality and diversity, which leads to increased proteome complexity. PTM crosstalk describes the combinatorial action of multiple PTMs on the same or on different proteins for higher order regulation. Here we review how recent advances in proteomic technologies, mass spectrometry instrumentation, and bioinformatics spurred the proteome-wide identification of PTM crosstalk through measurements of PTM sites. We provide an overview of the basic modes of PTM crosstalk, the proteomic methods to elucidate PTM crosstalk, and approaches that can inform about the functional consequences of PTM crosstalk.
Description of basic modules and different modes of PTM crosstalk. Overview of current proteomic methods to identify and infer PTM crosstalk. Discussion of large-scale approaches to characterize functional PTM crosstalk. Future directions and potential proteomic methods for elucidating PTM crosstalk.
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27
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Juncker M, Kim C, Reed R, Haas A, Schwartzenburg J, Desai S. ISG15 attenuates post-translational modifications of mitofusins and congression of damaged mitochondria in Ataxia Telangiectasia cells. Biochim Biophys Acta Mol Basis Dis 2021; 1867:166102. [PMID: 33617986 DOI: 10.1016/j.bbadis.2021.166102] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2020] [Revised: 02/11/2021] [Accepted: 02/12/2021] [Indexed: 10/22/2022]
Abstract
Mitophagy is defective in several neurodegenerative diseases, including Ataxia Telangiectasia (A-T). However, the molecular mechanism underlying defective mitophagy in A-T is unknown. Literature indicates that damaged mitochondria are transported to the perinuclear region prior to their removal via mitophagy. Our previous work has indicated that conjugation of SUMO2 (Small Ubiquitin-like Modifier 2) to mitofusins (Mfns) may be necessary for congression of mitochondria into SUMO2-/ubiquitin-/LC3-positive compact structures resembling mito-aggresomes at the perinuclear region in CCCP-treated HEK293 cells. Here, we demonstrate that Mfns are SUMOylated, and mitochondria are transported to the perinuclear region; however, mitochondria fail to congress into mito-aggresome-like structures in CCCP-treated A-T cells. Defect in mitochondrial congression is causally related to constitutively elevated ISG15 (Interferon-Stimulated Gene 15), an antagonist of the ubiquitin pathway, in A-T cells. Suppression of the ISG15 pathway restores mitochondrial congression, reduce oxidative stress, and level of unhealthy mitochondria, which is suggestive of restoration of mitophagy in A-T cells. ISG15 is also constitutively elevated and mitophagy is defective in Amytrophic Lateral Sclerosis (ALS). The constitutively elevated ISG15 pathway therefore appears to be a common unifying biochemical mechanism underlying defective mitophagy in neurodegenerative disorders thus, implying the broader significance of our findings, and suggest the potential role of ISG15 inhibitors in their treatment.
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Affiliation(s)
- Meredith Juncker
- Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, 1901 Perdido Street, New Orleans, LA 70112, USA
| | - Catherine Kim
- Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, 1901 Perdido Street, New Orleans, LA 70112, USA
| | - Ryan Reed
- Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, 1901 Perdido Street, New Orleans, LA 70112, USA
| | - Arthur Haas
- Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, 1901 Perdido Street, New Orleans, LA 70112, USA
| | - Joshua Schwartzenburg
- Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, 1901 Perdido Street, New Orleans, LA 70112, USA
| | - Shyamal Desai
- Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, 1901 Perdido Street, New Orleans, LA 70112, USA.
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28
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Chang YC, Oram MK, Bielinsky AK. SUMO-Targeted Ubiquitin Ligases and Their Functions in Maintaining Genome Stability. Int J Mol Sci 2021; 22:ijms22105391. [PMID: 34065507 PMCID: PMC8161396 DOI: 10.3390/ijms22105391] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Revised: 05/14/2021] [Accepted: 05/16/2021] [Indexed: 02/06/2023] Open
Abstract
Small ubiquitin-like modifier (SUMO)-targeted E3 ubiquitin ligases (STUbLs) are specialized enzymes that recognize SUMOylated proteins and attach ubiquitin to them. They therefore connect the cellular SUMOylation and ubiquitination circuits. STUbLs participate in diverse molecular processes that span cell cycle regulated events, including DNA repair, replication, mitosis, and transcription. They operate during unperturbed conditions and in response to challenges, such as genotoxic stress. These E3 ubiquitin ligases modify their target substrates by catalyzing ubiquitin chains that form different linkages, resulting in proteolytic or non-proteolytic outcomes. Often, STUbLs function in compartmentalized environments, such as the nuclear envelope or kinetochore, and actively aid in nuclear relocalization of damaged DNA and stalled replication forks to promote DNA repair or fork restart. Furthermore, STUbLs reside in the same vicinity as SUMO proteases and deubiquitinases (DUBs), providing spatiotemporal control of their targets. In this review, we focus on the molecular mechanisms by which STUbLs help to maintain genome stability across different species.
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29
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Bryan L, Clynes M, Meleady P. The emerging role of cellular post-translational modifications in modulating growth and productivity of recombinant Chinese hamster ovary cells. Biotechnol Adv 2021; 49:107757. [PMID: 33895332 DOI: 10.1016/j.biotechadv.2021.107757] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Revised: 04/19/2021] [Accepted: 04/19/2021] [Indexed: 02/06/2023]
Abstract
Chinese hamster ovary (CHO) cells are one of the most commonly used host cell lines used for the production human therapeutic proteins. Much research over the past two decades has focussed on improving the growth, titre and cell specific productivity of CHO cells and in turn lowering the costs associated with production of recombinant proteins. CHO cell engineering has become of particular interest in recent years following the publication of the CHO cell genome and the availability of data relating to the proteome, transcriptome and metabolome of CHO cells. However, data relating to the cellular post-translational modification (PTMs) which can affect the functionality of CHO cellular proteins has only begun to be presented in recent years. PTMs are important to many cellular processes and can further alter proteins by increasing the complexity of proteins and their interactions. In this review, we describe the research presented from CHO cells to date related on three of the most important PTMs; glycosylation, phosphorylation and ubiquitination.
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Affiliation(s)
- Laura Bryan
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
| | - Martin Clynes
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
| | - Paula Meleady
- National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
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30
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Protein feature analysis of heat shock induced ubiquitination sites reveals preferential modification site localization. J Proteomics 2021; 239:104182. [PMID: 33705978 DOI: 10.1016/j.jprot.2021.104182] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2020] [Revised: 02/04/2021] [Accepted: 03/01/2021] [Indexed: 11/20/2022]
Abstract
Protein aggregation is indicative of failing protein quality control systems. These systems are responsible for the refolding or degradation of aberrant and misfolded proteins. Heat stress can cause proteins to misfold, triggering cellular responses including a marked increase in the ubiquitination of proteins. This response has been characterized in yeast, however more studies are needed within mammalian cells. Herein, we examine proteins that become ubiquitinated during heat shock in human tissue culture cells using diGly enrichment coupled with mass spectrometry. A majority of these proteins are localized in the nucleus or cytosol. Proteins which are conjugated under stress display longer sequence lengths, more interaction partners, and more hydrophobic patches than controls but do not show lower melting temperatures. Furthermore, heat-induced conjugation sites occur less frequently in disordered regions and are closer to hydrophobic patches than other ubiquitination sites; perhaps providing novel insight into the molecular mechanism mediating this response. Nuclear and cytosolic pools of modified proteins appear to have different protein features. Using a pulse-SILAC approach, we found that both long-lived and newly-synthesized proteins are conjugated under stress. Modified long-lived proteins are predominately nuclear and were distinct from newly-synthesized proteins, indicating that different pathways may mediate the heat-induced increase of polyubiquitination. SIGNIFICANCE: The maintenance of protein homeostasis requires a balance of protein synthesis, folding, and degradation. Under stress conditions, the cell must rapidly adapt by increasing its folding capacity to eliminate aberrant proteins. A major pathway for proteolysis is mediated by the ubiquitin proteasome system. While increased ubiquitination after heat stress was observed over 30 years ago, it remains unclear which proteins are conjugated during heat shock in mammalian cells and by what means this conjugation occurs. In this study, we combined SILAC-based mass spectrometry with computational analyses to reveal features associated to proteins ubiquitinated while under heat shock. Interestingly, we found that conjugation sites induced by the stress are less often located within disordered regions and more often located near hydrophobic patches. Our study showcases how proteomics can reveal distinct feature associated to a cohort of proteins that are modified post translationally and how the ubiquitin conjugation sites are preferably selected in these conditions. Our work opens a new path for delineating the molecular mechanisms leading to the heat stress response and the regulation of protein homeostasis.
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31
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González-Prieto R, Eifler-Olivi K, Claessens LA, Willemstein E, Xiao Z, Talavera Ormeno CMP, Ovaa H, Ulrich HD, Vertegaal ACO. Global non-covalent SUMO interaction networks reveal SUMO-dependent stabilization of the non-homologous end joining complex. Cell Rep 2021; 34:108691. [PMID: 33503430 DOI: 10.1016/j.celrep.2021.108691] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Revised: 12/11/2020] [Accepted: 01/05/2021] [Indexed: 12/17/2022] Open
Abstract
In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors.
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Affiliation(s)
- Román González-Prieto
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
| | - Karolin Eifler-Olivi
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands; Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
| | - Laura A Claessens
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Edwin Willemstein
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Zhenyu Xiao
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Cami M P Talavera Ormeno
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands; Oncode Institute, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Huib Ovaa
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands; Oncode Institute, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Helle D Ulrich
- Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
| | - Alfred C O Vertegaal
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
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32
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Effects of dietary carbohydrate sources on lipid metabolism and SUMOylation modification in the liver tissues of yellow catfish. Br J Nutr 2020; 124:1241-1250. [PMID: 32600495 DOI: 10.1017/s0007114520002408] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Dysregulation in hepatic lipid synthesis by excess dietary carbohydrate intake is often relevant with the occurrence of fatty liver; therefore, the thorough understanding of the regulation of lipid deposition and metabolism seems crucial to search for potential regulatory targets. In the present study, we examined TAG accumulation, lipid metabolism-related gene expression, the enzyme activities of lipogenesis-related enzymes, the protein levels of transcription factors or genes involving lipogenesis in the livers of yellow catfish fed five dietary carbohydrate sources, such as glucose, maize starch, sucrose, potato starch and dextrin, respectively. Generally speaking, compared with other carbohydrate sources, dietary glucose promoted TAG accumulation, up-regulated lipogenic enzyme activities and gene expressions, and down-regulated mRNA expression of genes involved in lipolysis and small ubiquitin-related modifier (SUMO) modification pathways. Further studies found that sterol regulatory element binding protein 1 (SREBP1), a key transcriptional factor relevant to lipogenic regulation, was modified by SUMO1. Mutational analyses found two important sites for SUMOylation modification (K254R and K264R) in SREBP1. Mutant SREBP lacking lysine 264 up-regulated the transactivation capacity on an SREBP-responsive promoter. Glucose reduced the SUMOylation level of SREBP1 and promoted the protein expression of SREBP1 and its target gene stearoyl-CoA desaturase 1 (SCD1), indicating that SUMOylation of SREBP1 mediated glucose-induced hepatic lipid metabolism. Our study elucidated the molecular mechanism of dietary glucose increasing hepatic lipid deposition and found that the SREBP-dependent transactivation was regulated by SUMO1 modification, which served as a new target for the transcriptional programmes governing lipid metabolism.
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33
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Double-edged role of PML nuclear bodies during human adenovirus infection. Virus Res 2020; 295:198280. [PMID: 33370557 DOI: 10.1016/j.virusres.2020.198280] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Revised: 12/21/2020] [Accepted: 12/23/2020] [Indexed: 01/31/2023]
Abstract
PML nuclear bodies are matrix-bound nuclear structures with a variety of functions in human cells. These nuclear domains are interferon regulated and play an essential role during virus infections involving accumulation of SUMO-dependent host and viral factors. PML-NBs are targeted and subsequently manipulated by adenoviral regulatory proteins, illustrating their crucial role during productive infection and virus-mediated oncogenic transformation. PML-NBs have a longstanding antiviral reputation; however, the genomes of Human Adenoviruses and initial sites of viral transcription/replication are found juxtaposed to these domains, resulting in a double-edged capacity of these nuclear multiprotein/multifunctional complexes. This enigma provides evidence that Human Adenoviruses selectively counteract antiviral responses, and simultaneously benefit from or even depend on proviral PML-NB associated components by active recruitment to PML track-like structures, that are induced during infection. Thereby, a positive microenvironment for adenoviral transcription and replication is created at these nuclear subdomains. Based on the available data, this review aims to provide a detailed overview of the current knowledge of Human Adenovirus crosstalk with nuclear PML body compartments as sites of SUMOylation processes in the host cells, evaluating the currently known principles and molecular mechanisms.
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34
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The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal. Nat Commun 2020; 11:5643. [PMID: 33159083 PMCID: PMC7648084 DOI: 10.1038/s41467-020-19516-z] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Accepted: 10/16/2020] [Indexed: 12/31/2022] Open
Abstract
Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences. In yeast, collapsed forks shift to the nuclear periphery to associate with two distinct perinuclear anchorage sites such as the nuclear pore complex. Here, the authors reveal the mechanisms engaged at nuclear pore complex facilitating fork integrity and restart via SUMO regulation.
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35
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Abstract
Sentrin/small ubiquitin-like modifier (SUMO) is protein modification pathway that regulates multiple biological processes, including cell division, DNA replication/repair, signal transduction, and cellular metabolism. In this review, we will focus on recent advances in the mechanisms of disease pathogenesis, such as cancer, diabetes, seizure, and heart failure, which have been linked to the SUMO pathway. SUMO is conjugated to lysine residues in target proteins through an isopeptide linkage catalyzed by SUMO-specific activating (E1), conjugating (E2), and ligating (E3) enzymes. In steady state, the quantity of SUMO-modified substrates is usually a small fraction of unmodified substrates due to the deconjugation activity of the family Sentrin/SUMO-specific proteases (SENPs). In contrast to the complexity of the ubiquitination/deubiquitination machinery, the biochemistry of SUMOylation and de-SUMOylation is relatively modest. Specificity of the SUMO pathway is achieved through redox regulation, acetylation, phosphorylation, or other posttranslational protein modification of the SUMOylation and de-SUMOylation enzymes. There are three major SUMOs. SUMO-1 usually modifies a substrate as a monomer; however, SUMO-2/3 can form poly-SUMO chains. The monomeric SUMO-1 or poly-SUMO chains can interact with other proteins through SUMO-interactive motif (SIM). Thus SUMO modification provides a platform to enhance protein-protein interaction. The consequence of SUMOylation includes changes in cellular localization, protein activity, or protein stability. Furthermore, SUMO may join force with ubiquitin to degrade proteins through SUMO-targeted ubiquitin ligases (STUbL). After 20 yr of research, SUMO has been shown to play critical roles in most, if not all, biological pathways. Thus the SUMO enzymes could be targets for drug development to treat human diseases.
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Affiliation(s)
- Hui-Ming Chang
- Center for Precision Medicine, Department of Medicine, University of Missouri, Columbia, Missouri
| | - Edward T H Yeh
- Center for Precision Medicine, Department of Medicine, University of Missouri, Columbia, Missouri
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36
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Jiang HK, Kurkute P, Li CL, Wang YH, Chen PJ, Lin SY, Wang YS. Revealing USP7 Deubiquitinase Substrate Specificity by Unbiased Synthesis of Ubiquitin Tagged SUMO2. Biochemistry 2020; 59:3796-3801. [PMID: 33006472 DOI: 10.1021/acs.biochem.0c00701] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Ubiquitination and SUMOylation of protein are crucial for various biological responses. The recent unraveling of cross-talk between SUMO and ubiquitin (Ub) has shown the pressing needs to develop the platform for the synthesis of Ub tagged SUMO2 dimers to decipher its biological functions. Still, the platforms for facile synthesis of dimers under native condition are less explored and remain major challenges. Here, we have developed the platform that can expeditiously synthesize all eight Ub tagged SUMO2 and SUMOylated proteins under native condition. Expanding genetic code (EGC) method was employed to incorporate Se-alkylselenocysteine at lysine positions. Oxidative selenoxide elimination generates the electrophilic center, dehydroalanine, which upon Michael addition with C-terminal modified ubiquitin, a nucleophile, yield Ub tagged SUMO2. The dimers were further interrogated with USP7, a SUMO2 deubiquitinase, which is involved in DNA repair, to understand specificity toward the Ub tagged SUMO2 dimer. Our results have shown that the C-terminal domain of USP7 is crucial for USP7 efficiency and selectivity for the Ub tagged SUMO2 dimer.
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Affiliation(s)
- Han-Kai Jiang
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.,Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.,Department of Chemistry, National Tsing Hua University, Hsinchu 30044, Taiwan
| | - Prashant Kurkute
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.,Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.,Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
| | - Chien-Lung Li
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.,Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
| | - Yi-Hui Wang
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
| | - Pei-Jung Chen
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.,Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
| | - Shu-Yu Lin
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
| | - Yane-Shih Wang
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.,Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.,Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
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37
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Jansen NS, Vertegaal ACO. A Chain of Events: Regulating Target Proteins by SUMO Polymers. Trends Biochem Sci 2020; 46:113-123. [PMID: 33008689 DOI: 10.1016/j.tibs.2020.09.002] [Citation(s) in RCA: 56] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Revised: 08/21/2020] [Accepted: 09/04/2020] [Indexed: 02/07/2023]
Abstract
Small ubiquitin-like modifiers (SUMOs) regulate virtually all nuclear processes. The fate of the target protein is determined by the architecture of the attached SUMO protein, which can be of polymeric nature. Here, we highlight the multifunctional aspects of dynamic signal transduction by SUMO polymers. The SUMO-targeted ubiquitin ligases (STUbLs) RING-finger protein 4 (RNF4) and RNF111 recognize SUMO polymers in a chain-architecture-dependent manner, leading to the formation of hybrid chains, which could enable proteasomal destruction of proteins. Recent publications have highlighted essential roles for SUMO chain disassembly by the mammalian SUMO proteases SENP6 and SENP7 and the yeast SUMO protease Ulp2. SENP6 is particularly important for centromere assembly. These recent findings demonstrate the diversity of SUMO polymer signal transduction for proteolytic and nonproteolytic purposes.
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Affiliation(s)
- Nicolette S Jansen
- Department of Cell and Chemical Biology, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands
| | - Alfred C O Vertegaal
- Department of Cell and Chemical Biology, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands.
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Du LL. Resurrection from lethal knockouts: Bypass of gene essentiality. Biochem Biophys Res Commun 2020; 528:405-412. [PMID: 32507598 DOI: 10.1016/j.bbrc.2020.05.207] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 05/27/2020] [Indexed: 01/03/2023]
Abstract
Understanding genotype-phenotype relationships is a central pursuit in biology. Gene knockout generates a complete loss-of-function genotype and is a commonly used approach for probing gene functions. The most severe phenotypic consequence of gene knockout is lethality. Genes with a lethal knockout phenotype are called essential genes. Based on genome-wide knockout analyses in yeasts, up to approximately a quarter of genes in a genome can be essential. Like other genotype-phenotype relationships, gene essentiality is subject to background effects and can vary due to gene-gene interactions. In particular, for some essential genes, lethality caused by knockout can be rescued by extragenic suppressors. Such "bypass of essentiality" (BOE) gene-gene interactions have been an understudied type of genetic suppression. A recent systematic analysis revealed that, remarkably, the essentiality of nearly 30% of essential genes in the fission yeast Schizosaccharomyces pombe can be bypassed by BOE interactions. Here, I review the history and recent progress on uncovering and understanding the bypass of gene essentiality.
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Affiliation(s)
- Li-Lin Du
- National Institute of Biological Sciences, Beijing, 102206, China; Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, 100084, China.
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39
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Heras G, Namuduri AV, Traini L, Shevchenko G, Falk A, Bergström Lind S, Jia M, Tian G, Gastaldello S. Muscle RING-finger protein-1 (MuRF1) functions and cellular localization are regulated by SUMO1 post-translational modification. J Mol Cell Biol 2020; 11:356-370. [PMID: 29868881 PMCID: PMC7727263 DOI: 10.1093/jmcb/mjy036] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2018] [Revised: 05/25/2018] [Accepted: 06/01/2018] [Indexed: 01/02/2023] Open
Abstract
The muscle RING-finger protein-1 (MuRF1) is an E3 ubiquitin ligase expressed in skeletal and cardiac muscle tissues and it plays important roles in muscle remodeling. Upregulation of MuRF1 gene transcription participates in skeletal muscle atrophy, on contrary downregulation of protein expression leads to cardiac hypertrophy. MuRF1 gene point mutations have been found to generate protein aggregate myopathies defined as muscle disorder characterized by protein accumulation in muscle fibers. We have discovered that MuRF1 turned out to be also a target for a new post-translational modification arbitrated by conjugation of SUMO1 and it is mediated by the SUMO ligases E2 UBC9 and the E3 PIASγ/4. SUMOylation takes place at lysine 238 localized at the second coiled-coil protein domain that is required for efficient substrate interaction for polyubiquitination. We provided evidence that SUMOylation is essential for MuRF1 nuclear translocation and its mitochondria accumulation is enhanced in hyperglycemic conditions delivering a stabilization of the overall SUMOylated proteins in cultured myocytes. Thus, our findings add this SUMO1 post-translational modification as a new concept to understand muscle disorders related to the defect in MuRF1 activity.
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Affiliation(s)
- Gabriel Heras
- Department of Physiology and Pharmacology, Karolinska Institutet, Solnavägen 9, Quarter B5, Stockholm, Sweden
| | - Arvind Venkat Namuduri
- Department of Physiology and Pharmacology, Karolinska Institutet, Solnavägen 9, Quarter B5, Stockholm, Sweden
| | - Leonardo Traini
- Department of Physiology and Pharmacology, Karolinska Institutet, Solnavägen 9, Quarter B5, Stockholm, Sweden
| | - Ganna Shevchenko
- Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, Uppsala, Sweden
| | - Alexander Falk
- Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, Uppsala, Sweden
| | - Sara Bergström Lind
- Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, Uppsala, Sweden
| | - Mi Jia
- Precision Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, China
| | - Geng Tian
- Precision Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, China
| | - Stefano Gastaldello
- Department of Physiology and Pharmacology, Karolinska Institutet, Solnavägen 9, Quarter B5, Stockholm, Sweden.,Precision Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, China
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40
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Zhang Y, Zeng L. Crosstalk between Ubiquitination and Other Post-translational Protein Modifications in Plant Immunity. PLANT COMMUNICATIONS 2020; 1:100041. [PMID: 33367245 PMCID: PMC7748009 DOI: 10.1016/j.xplc.2020.100041] [Citation(s) in RCA: 61] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/01/2020] [Revised: 02/07/2020] [Accepted: 03/19/2020] [Indexed: 05/05/2023]
Abstract
Post-translational modifications (PTMs) are central to the modulation of protein activity, stability, subcellular localization, and interaction with partners. They greatly expand the diversity and functionality of the proteome and have taken the center stage as key players in regulating numerous cellular and physiological processes. Increasing evidence indicates that in addition to a single regulatory PTM, many proteins are modified by multiple different types of PTMs in an orchestrated manner to collectively modulate the biological outcome. Such PTM crosstalk creates a combinatorial explosion in the number of proteoforms in a cell and greatly improves the ability of plants to rapidly mount and fine-tune responses to different external and internal cues. While PTM crosstalk has been investigated in depth in humans, animals, and yeast, the study of interplay between different PTMs in plants is still at its infant stage. In the past decade, investigations showed that PTMs are widely involved and play critical roles in the regulation of interactions between plants and pathogens. In particular, ubiquitination has emerged as a key regulator of plant immunity. This review discusses recent studies of the crosstalk between ubiquitination and six other PTMs, i.e., phosphorylation, SUMOylation, poly(ADP-ribosyl)ation, acetylation, redox modification, and glycosylation, in the regulation of plant immunity. The two basic ways by which PTMs communicate as well as the underlying mechanisms and diverse outcomes of the PTM crosstalk in plant immunity are highlighted.
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41
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Whalen JM, Freudenreich CH. Location, Location, Location: The Role of Nuclear Positioning in the Repair of Collapsed Forks and Protection of Genome Stability. Genes (Basel) 2020; 11:E635. [PMID: 32526925 PMCID: PMC7348918 DOI: 10.3390/genes11060635] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 05/29/2020] [Accepted: 06/04/2020] [Indexed: 12/14/2022] Open
Abstract
Components of the nuclear pore complex (NPC) have been shown to play a crucial role in protecting against replication stress, and recovery from some types of stalled or collapsed replication forks requires movement of the DNA to the NPC in order to maintain genome stability. The role that nuclear positioning has on DNA repair has been investigated in several systems that inhibit normal replication. These include structure forming sequences (expanded CAG repeats), protein mediated stalls (replication fork barriers (RFBs)), stalls within the telomere sequence, and the use of drugs known to stall or collapse replication forks (HU + MMS or aphidicolin). Recently, the mechanism of relocation for collapsed replication forks to the NPC has been elucidated. Here, we will review the types of replication stress that relocate to the NPC, the current models for the mechanism of relocation, and the currently known protective effects of this movement.
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Affiliation(s)
- Jenna M. Whalen
- Department of Biology, Tufts University, Medford, MA 02155, USA;
| | - Catherine H. Freudenreich
- Department of Biology, Tufts University, Medford, MA 02155, USA;
- Program in Genetics, Tufts University, Boston, MA 02111, USA
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42
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Rinfret Robert C, McManus FP, Lamoliatte F, Thibault P. Interplay of Ubiquitin-Like Modifiers Following Arsenic Trioxide Treatment. J Proteome Res 2020; 19:1999-2010. [PMID: 32223133 DOI: 10.1021/acs.jproteome.9b00807] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Arsenic trioxide (ATO) is a therapeutic agent used to treat acute promyelocytic leukemia (APL), a disease caused by a chromosomal translocation of the retinoic acid receptor α (RARα) gene that can occur reciprocally with the promyelocytic leukemia (PML) gene. The mechanisms through which ATO exerts its effects on cells are not fully characterized though they involve the SUMOylation, the ubiquitylation, and the degradation of the PML/RARα oncoprotein through the PML moiety. To better understand the mechanisms that underlie the cytotoxicity induced with increasing ATO levels, we profiled the changes in protein SUMOylation, phosphorylation, and ubiquitylation on HEK293 cells following exposure to low (1 μM) or elevated (10 μM) ATO for 4 h. Our analyses revealed that a low dose of ATO resulted in the differential modification of selected substrates including the SUMOylation (K380, K394, K490, and K497) and ubiquitylation (K337, K401) of PML. These experiments also highlighted a number of unexpected SUMOylated substrates involved in DNA damage response (e.g., PCNA, YY1, and poly[ADP-ribose] polymerase 1 (PARP1)) and messenger RNA (mRNA) splicing (e.g., ACIN1, USP39, and SART1) that were regulated at higher ATO concentrations. Interestingly, additional enzymatic assays revealed that SUMOylation of PARP1 impeded its proteolytic cleavage by caspase-3, suggesting that SUMOylation could have a protective role in delaying cell apoptosis.
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Affiliation(s)
- Clémence Rinfret Robert
- Institute for Research in Immunology and Cancer, Montreal, Québec H3T 1J4, Canada.,Department of Biochemistry, University of Montréal, Montreal, Québec H3T 1J4, Canada
| | - Francis P McManus
- Institute for Research in Immunology and Cancer, Montreal, Québec H3T 1J4, Canada
| | - Frédéric Lamoliatte
- Institute for Research in Immunology and Cancer, Montreal, Québec H3T 1J4, Canada.,Department of Chemistry, University of Montréal, P.O. Box 6128, Station Centre-Ville, Montreal, Québec H3T 1J4, Canada
| | - Pierre Thibault
- Institute for Research in Immunology and Cancer, Montreal, Québec H3T 1J4, Canada.,Department of Biochemistry, University of Montréal, Montreal, Québec H3T 1J4, Canada.,Department of Chemistry, University of Montréal, P.O. Box 6128, Station Centre-Ville, Montreal, Québec H3T 1J4, Canada
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Küçükali CI, Salman B, Yüceer H, Ulusoy C, Abacı N, Ekmekci SS, Tüzün E, Bilgiç B, Hanağası HA. Small ubiquitin-related modifier (SUMO) 3 and SUMO4 gene polymorphisms in Parkinson’s disease. Neurol Res 2020; 42:451-457. [DOI: 10.1080/01616412.2020.1724464] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Affiliation(s)
- Cem Ismail Küçükali
- Department of Neuroscience, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Burcu Salman
- Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Hande Yüceer
- Department of Neuroscience, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Canan Ulusoy
- Department of Neuroscience, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Neslihan Abacı
- Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Sema Sırma Ekmekci
- Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Erdem Tüzün
- Department of Neuroscience, Istanbul University, Aziz Sancar Institute of Experimental Medicine, Istanbul, Türkiye
| | - Başar Bilgiç
- Istanbul Faculty of Medicine, Department of Neurology, Istanbul University, Istanbul, Turkey
| | - Haşmet Ayhan Hanağası
- Istanbul Faculty of Medicine, Department of Neurology, Istanbul University, Istanbul, Turkey
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44
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RNF4-mediated SUMO-targeted ubiquitination relieves PARIS/ZNF746-mediated transcriptional repression. Biochem Biophys Res Commun 2020; 526:110-116. [PMID: 32197837 DOI: 10.1016/j.bbrc.2020.03.063] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2020] [Accepted: 03/10/2020] [Indexed: 01/28/2023]
Abstract
The transcriptional repressor PARIS, which is a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α. However, little is known about how its repression activity is regulated. We have previously shown that PARIS is SUMOylated, and this SUMOylation plays an important role in regulating its transcriptional repression activity. In this study, we demonstrated that PARIS SUMOylation induced its ubiquitination and subsequent proteasomal degradation, which was mediated by the SUMO-targeted ubiquitin ligase RNF4. Reporter gene assays revealed that co-expression of SUMO3 and RNF4 relieved PARIS-mediated transcriptional repression. Conversely, the SUMO E3 ligase PIASy inhibited the RNF4-mediated ubiquitination of PARIS and blocked the RNF4-mediated relief of PARIS-mediated transcriptional repression. These results suggest that RNF4 regulates PARIS ubiquitination to control its transcriptional repression activity.
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45
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The Viral SUMO–Targeted Ubiquitin Ligase ICP0 is Phosphorylated and Activated by Host Kinase Chk2. J Mol Biol 2020; 432:1952-1977. [DOI: 10.1016/j.jmb.2020.01.021] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Revised: 01/06/2020] [Accepted: 01/17/2020] [Indexed: 11/22/2022]
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46
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Asymmetric Processing of DNA Ends at a Double-Strand Break Leads to Unconstrained Dynamics and Ectopic Translocation. Cell Rep 2019; 24:2614-2628.e4. [PMID: 30184497 DOI: 10.1016/j.celrep.2018.07.102] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2018] [Revised: 06/07/2018] [Accepted: 07/27/2018] [Indexed: 01/10/2023] Open
Abstract
Multiple pathways regulate the repair of double-strand breaks (DSBs) to suppress potentially dangerous ectopic recombination. Both sequence and chromatin context are thought to influence pathway choice between non-homologous end-joining (NHEJ) and homology-driven recombination. To test the effect of repetitive sequences on break processing, we have inserted TG-rich repeats on one side of an inducible DSB at the budding yeast MAT locus on chromosome III. Five clustered Rap1 sites within a break-proximal TG repeat are sufficient to block Mre11-Rad50-Xrs2 recruitment, impair resection, and favor elongation by telomerase. The two sides of the break lose end-to-end tethering and show enhanced, uncoordinated movement. Only the TG-free side is resected and shifts to the nuclear periphery. In contrast to persistent DSBs without TG repeats that are repaired by imprecise NHEJ, nearly all survivors of repeat-proximal DSBs repair the break by a homology-driven, non-reciprocal translocation from ChrIII-R to ChrVII-L. This suppression of imprecise NHEJ at TG-repeat-flanked DSBs requires the Uls1 translocase activity.
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47
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Kumar R, Sabapathy K. RNF4—A Paradigm for SUMOylation‐Mediated Ubiquitination. Proteomics 2019; 19:e1900185. [DOI: 10.1002/pmic.201900185] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Revised: 09/13/2019] [Indexed: 01/08/2023]
Affiliation(s)
- Ramesh Kumar
- Cancer & Stem Cell Biology Program Duke–NUS Medical School 8 College Road Singapore 169857 Singapore
| | - Kanaga Sabapathy
- Cancer & Stem Cell Biology Program Duke–NUS Medical School 8 College Road Singapore 169857 Singapore
- Laboratory of Molecular Carcinogenesis Division of Cellular & Molecular Research Humphrey Oei Institute of Cancer Research National Cancer Centre Singapore 11 Hospital Drive Singapore 169610 Singapore
- Department of Biochemistry National University of Singapore 8 Medical Drive Singapore 117597 Singapore
- Institute of Molecular and Cellular Biology 61 Biopolis Drive Singapore 138673 Singapore
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48
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Wagner K, Kunz K, Piller T, Tascher G, Hölper S, Stehmeier P, Keiten-Schmitz J, Schick M, Keller U, Müller S. The SUMO Isopeptidase SENP6 Functions as a Rheostat of Chromatin Residency in Genome Maintenance and Chromosome Dynamics. Cell Rep 2019; 29:480-494.e5. [PMID: 31597105 DOI: 10.1016/j.celrep.2019.08.106] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Revised: 07/21/2019] [Accepted: 08/29/2019] [Indexed: 11/20/2022] Open
Abstract
Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.
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Affiliation(s)
- Kristina Wagner
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Kathrin Kunz
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Tanja Piller
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Georg Tascher
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Soraya Hölper
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Per Stehmeier
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Jan Keiten-Schmitz
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Markus Schick
- Internal Medicine III, School of Medicine, Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany; Department of Hematology, Oncology and Tumor Immunology (Campus Benjamin Franklin), Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany
| | - Ulrich Keller
- Internal Medicine III, School of Medicine, Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany; Department of Hematology, Oncology and Tumor Immunology (Campus Benjamin Franklin), Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany; German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Stefan Müller
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
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The poly-SUMO2/3 protease SENP6 enables assembly of the constitutive centromere-associated network by group deSUMOylation. Nat Commun 2019; 10:3987. [PMID: 31485003 PMCID: PMC6726658 DOI: 10.1038/s41467-019-11773-x] [Citation(s) in RCA: 44] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Accepted: 07/26/2019] [Indexed: 12/20/2022] Open
Abstract
In contrast to our extensive knowledge on ubiquitin polymer signaling, we are severely limited in our understanding of poly-SUMO signaling. We set out to identify substrates conjugated to SUMO polymers, using knockdown of the poly-SUMO2/3 protease SENP6. We identify over 180 SENP6 regulated proteins that represent highly interconnected functional groups of proteins including the constitutive centromere-associated network (CCAN), the CENP-A loading factors Mis18BP1 and Mis18A and DNA damage response factors. Our results indicate a striking protein group de-modification by SENP6. SENP6 deficient cells are severely compromised for proliferation, accumulate in G2/M and frequently form micronuclei. Accumulation of CENP-T, CENP-W and CENP-A to centromeres is impaired in the absence of SENP6. Surprisingly, the increase of SUMO chains does not lead to ubiquitin-dependent proteasomal degradation of the CCAN subunits. Our results indicate that SUMO polymers can act in a proteolysis-independent manner and consequently, have a more diverse signaling function than previously expected. While the biological roles of ubiquitin chains are well studied, little is known about the functions of SUMO polymers. Here, the authors identify poly-SUMOylation substrates and provide evidence that SUMO polymers regulate the accumulation of CCAN subunits at chromatin and centromeres.
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50
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Arkadia/RNF111 is a SUMO-targeted ubiquitin ligase with preference for substrates marked with SUMO1-capped SUMO2/3 chain. Nat Commun 2019; 10:3678. [PMID: 31417085 PMCID: PMC6695498 DOI: 10.1038/s41467-019-11549-3] [Citation(s) in RCA: 51] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Accepted: 07/22/2019] [Indexed: 11/18/2022] Open
Abstract
Modification with SUMO regulates many eukaryotic proteins. Down-regulation of sumoylated forms of proteins involves either their desumoylation, and hence recycling of the unmodified form, or their proteolytic targeting by ubiquitin ligases that recognize their SUMO modification (termed STUbL or ULS). STUbL enzymes such as Uls1 and Slx5-Slx8 in budding yeast or RNF4 and Arkadia/RNF111 in humans bear multiple SUMO interaction motifs to recognize substrates carrying poly-SUMO chains. Using yeast as experimental system and isothermal titration calorimetry, we here show that Arkadia specifically selects substrates carrying SUMO1-capped SUMO2/3 hybrid conjugates and targets them for proteasomal degradation. Our data suggest that a SUMO1-specific binding site in Arkadia with sequence similarity to a SUMO1-binding site in DPP9 is required for targeting endogenous hybrid SUMO conjugates and PML nuclear bodies in human cells. We thus characterize Arkadia as a STUbL with a preference for substrate proteins marked with distinct hybrid SUMO chains. The cellular functions of poly-SUMO chains of different compositions are not fully understood. Here, the authors characterize Arkadia/RNF111 as a SUMO-targeted ubiquitin ligase that recognizes proteins with hybrid SUMO1-capped SUMO2/3 chains and targets them for proteasomal degradation.
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