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Correction to: The EMBO Journal (2003) 22, 5241-5250. doi:10.1093/emboj/cdg501 [ABSTRACT FROM AUTHOR]

The Small Ubiquitin-like Modifier (SUMO) is a crucial post-translational modifier of proteins, playing a key role in various cellular functions. All SUMOs are synthesized as precursor proteins that must be proteolytically processed. However, the maturation process of cleaving the extending C-terminal tail, preceding SUMOylation of substrates, remains poorly understood, especially within cellular environments. Chemical protein synthesis coupled with cell delivery offers great opportunities to prepare SUMO analogues to investigate this process in vitro and in live cells. Applying this unique combination we show that SUMO2 analogues containing the native tail undergo rapid cleavage and nuclear localisation, while a Gly93Ala mutation impairs cleavage and alters localisation. Tail mutations (Val94Glu, Tyr95Ala) affected cleavage rates, highlighting roles in SUMO-SENP protease interactions. In cells, SUMO2 analogues containing tail mutations underwent cleavage and subsequently incorporated into promyelocytic leukemia nuclear bodies (PML-NBs). These findings advance our understanding of SUMO2 maturation and provide a foundation for future studies of this process for different SUMO paralogues in various cell lines and tissues., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Small ubiquitin-related modifiers (SUMOs) function as post-translational protein modifications and regulate nearly every aspect of cellular function. While a single ubiquitin protein is expressed across eukaryotic organisms, multiple SUMO paralogues with distinct biomolecular properties have been identified in plants and vertebrates. Five SUMO paralogues have been characterized in humans, with SUMO1, SUMO2 and SUMO3 being the best studied. SUMO2 and SUMO3 share 97% protein sequence homology (and are thus referred to as SUMO2/3) but only 47% homology with SUMO1. To date, thousands of putative sumoylation substrates have been identified thanks to advanced proteomic techniques, but the identification of SUMO1- and SUMO2/3-specific modifications and their unique functions in physiology and pathology are not well understood. The SUMO2/3 paralogues play an important role in proteostasis, converging with ubiquitylation to mediate protein degradation. This function is achieved primarily through SUMO-targeted ubiquitin ligases (STUbLs), which preferentially bind and ubiquitylate poly-SUMO2/3 modified proteins. Effects of the SUMO1 paralogue on protein solubility and aggregation independent of STUbLs and proteasomal degradation have also been reported. Consistent with these functions, sumoylation is implicated in multiple human diseases associated with disturbed proteostasis, and a broad range of pathogenic proteins have been identified as SUMO1 and SUMO2/3 substrates. A better understanding of paralogue-specific functions of SUMO1 and SUMO2/3 in cellular protein quality control may therefore provide novel insights into disease pathogenesis and therapeutic innovation. This review summarizes current understandings of the roles of sumoylation in protein quality control and associated diseases, with a focus on the specific effects of SUMO1 and SUMO2/3 paralogues.

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ellis, Zhu, Yagle, Yang, Huang, Kwako, Seidman and Matunis.)

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

Keratin 17 ( KRT17 ; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 -null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2β (an isoform encoded by TMPO ) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Sumoylation is an important post-translational modification that involves the conjugation of the Small Ubiquitin-related Modifier (SUMO) onto target proteins. This modification is reversed through the catalytic activity of SUMO isopeptidases, known as SENPs. One of these SENPs, SENP1, was reported to be overexpressed in human pancreatic cancer cells and patient tissues. Since elevated SENP1 expression levels can be used as a prognostic marker for a subset of cancers, we set out to further explore the overexpression of SENP1 in pancreatic cancer. We found that SENP1 protein levels were not significantly different between pancreatic cancer and normal pancreas-derived cell lines. To evaluate SENP1 expression in patient samples, we analyzed large publicly available datasets and found that SENP1 mRNA levels were significantly lower in pancreatic cancer tissue as compared to normal pancreas tissue samples. Furthermore, we found that the SENP1 gene is amplified in less than 1% of sequenced pancreatic cancer patient samples and that expression levels have no association with patient survival. Based on our analysis, we conclude that SENP1 is not overexpressed in pancreatic cancer and is therefore not likely to be an effective biomarker for this disease. Through this work, we also outline a simple but powerful bioinformatics workflow for the assessment of mRNA expression levels, genomic alterations and survival analysis for putative biomarkers for common human cancers., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Journal of Gastrointestinal Oncology. All rights reserved.)

The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification in vitro . Here, we describe a sensitive method for detecting SUMO modification of target human proteins using an in vitro transcription and translation system derived from rabbit reticulocyte and radiolabeled amino acids., Competing Interests: Competing interests Authors do not declare any conflicts of interest or competing interests.

Sumoylation regulates a wide range of essential cellular functions, many of which are associated with activities in the nucleus. Although there is also emerging evidence for the involvement of the small ubiquitin-related modifier (SUMO) at intracellular membranes, the mechanisms by which sumoylation is regulated at membranes is largely unexplored. In this study, we report that the SUMO-specific isopeptidase, SENP2, uniquely associates with intracellular membranes. Using in vivo analyses and in vitro binding assays, we show that SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic α-helix that promotes direct membrane binding. Furthermore, we demonstrate that SENP2 binding to intracellular membranes is regulated by interactions with the nuclear import receptor karyopherin-α. Consistent with membrane association, biotin identification (BioID) revealed interactions between SENP2 and endoplasmic reticulum, Golgi, and inner nuclear membrane-associated proteins. Collectively, our findings indicate that SENP2 binds to intracellular membranes where it interacts with membrane-associated proteins and has the potential to regulate their sumoylation and membrane-associated functions.

The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit., Competing Interests: CL, BL, HY, MM No competing interests declared, (© 2018, Lee et al.)

A decade has passed since the first reported connection between RAP80 and BRCA1 in DNA double-strand break repair. Despite the initial identification of RAP80 as a factor localizing BRCA1 to DNA double-strand breaks and potentially promoting homologous recombination, there is increasing evidence that RAP80 instead suppresses homologous recombination to fine-tune the balance of competing DNA repair processes during the S/G 2 phase of the cell cycle. RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex. Ubiquitin and SUMO modifications of chromatin at DNA double-strand breaks recruit RAP80, which contains distinct sequence motifs that recognize ubiquitin and SUMO. Here, we review RAP80's role in repressing homologous recombination at DNA double-strand breaks and how this role is facilitated by its ability to bind ubiquitin and SUMO modifications.

SUMOylation is a critical regulator of a broad range of cellular processes, and is thought to do so in part by modulation of protein interaction. To comprehensively identify human proteins whose interaction is modulated by SUMOylation, we developed an in vitro binding assay using human proteome microarrays to identify targets of SUMO1 and SUMO2. We then integrated these results with protein SUMOylation and protein-protein interaction data to perform network motif analysis. We focused on a single network motif we termed a SUMOmodPPI (SUMO-modulated Protein-Protein Interaction) that included the INO80 chromatin remodeling complex subunits TFPT and INO80E. We validated the SUMO-binding activity of INO80E, and showed that TFPT is a SUMO substrate both in vitro and in vivo We then demonstrated a key role for SUMOylation in mediating the interaction between these two proteins, both in vitro and in vivo By demonstrating a key role for SUMOylation in regulating the INO80 chromatin remodeling complex, this work illustrates the power of bioinformatics analysis of large data sets in predicting novel biological phenomena., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways.

Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein modification by small ubiquitin-related modifiers (SUMOs) is essential and conserved in the malaria parasite, Plasmodium falciparum. We have previously shown that interactions between the SUMO E1-activating and E2-conjugating enzyme in P. falciparum are distinct compared with human, suggesting a potential target for development of parasite-specific inhibitors of SUMOylation. The parasite asexual trophozoite stage is susceptible to iron-induced oxidative stress and is subsequently a target for many of the current anti-malarial drugs. Here, we provide evidence that SUMOylation plays a role in the parasite response to oxidative stress during red blood cell stages, indicative of a protective role seen in other organisms. Using x-ray crystallography, we solved the structure of the human SUMO E1 ubiquitin fold domain in complex with the E2, Ubc9. The interface defined in this structure guided in silico modeling, mutagenesis, and in vitro biochemical studies of the P. falciparum SUMO E1 and E2 enzymes, resulting in the identification of surface residues that explain species-specific interactions. Our findings suggest that parasite-specific inhibitors of SUMOylation could be developed and used in combination therapies with drugs that induce oxidative stress., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Reversible protein modification by small ubiquitin-related modifiers (SUMOs) regulates many cellular processes, including transcription, protein quality control, cell division, and oxidative stress. SUMOylation is therefore essential for normal cell function and represents a potentially valuable target for the development of inhibitors of pathogenic eukaryotic organisms, including the malaria parasite, Plasmodium falciparum (Pf). The specific and essential functions of SUMOylation in Pf, however, remain largely uncharacterized. The further development of antimalarial drugs targeting SUMOylation would benefit significantly from a more detailed understanding of its functions and regulation during the parasite life cycle. The recent development of antibodies specific for Pf SUMO provides a valuable tool to study the functions and regulation of SUMOylation. In preliminary studies, we have used immunoblot analysis to demonstrate that SUMOylation levels vary significantly in parasites during different stages of the red blood cell cycle and also in response to oxidative stress. Owing to the dynamic nature of SUMOylation and to the robust activity of SUMO isopeptidases, analysis of SUMOylation in cultured Pf parasites requires a number of precautions during parasite purification and lysis. Here, we outline methods for preserving SUMO conjugates during isolation of Pf parasites from human red blood cell cultures, and for their detection by immunoblot analysis using PfSUMO-specific antibodies.

Protein modification by the small ubiquitin-related modifier (SUMO) was simultaneously discovered by several groups at the middle of the 1990s. Although distinct names were proposed including Sentrin, GMP1, PIC1, or SMT3, SUMO became the most popular. Early studies on the functions of SUMOylation focused on activities in the nucleus, including transcription activation, chromatin structure, and DNA repair. However, it is now recognized that SUMOylation affects a large diversity of cellular processes both in the nucleus and the cytoplasm and functions of SUMOylation appear to have undefined limits. SUMO-conjugating enzymes and specific proteases actively regulate the modification status of target proteins. The recent discoveries of ubiquitin-SUMO hybrid chains, multiple SUMO-interacting motifs, and macromolecular complexes regulated by SUMOylation underscore the high complexity of this dynamic reversible system. New conceptual frameworks suggested by these findings have motivated the development of new methodologies to study pre- and post-SUMOylation events in vitro and in vivo, using distinct model organisms. Here we summarize some of the new developments and methodologies in the field, particularly those that will be further elaborated on in the chapters integrating this book.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 (small ubiquitin-related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods, we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere- and kinetochore-associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (kobs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of kobs on TDG concentration gives kmax = 1.6 min(-1) and K1/2 = 0.55 μM, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (Kd ∼50 nM). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sumoylation of centromere, kinetochore, and other mitotic chromosome-associated proteins is essential for chromosome segregation. The mechanisms regulating spatial and temporal sumoylation of proteins in mitosis, however, are not well understood. Here we show that the small ubiquitin-related modifier (SUMO)-specific isopeptidases SENP1 and SENP2 are targeted to kinetochores in mitosis. SENP2 targeting occurs through a mechanism dependent on the Nup107-160 subcomplex of the nuclear pore complex and is modulated through interactions with karyopherin α. Overexpression of SENP2, but not other SUMO-specific isopeptidases, causes a defect in chromosome congression that depends on its precise kinetochore targeting. By altering SENP1 kinetochore associations, however, this effect on chromosome congression could be phenocopied. In contrast, RNA interference-mediated knockdown of SENP1 delays sister chromatid separation at metaphase, whereas SENP2 knockdown produces no detectable phenotypes. Our findings indicate that chromosome segregation depends on precise spatial and temporal control of sumoylation in mitosis and that SENP1 and SENP2 are important mediators of this control.

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

Polymerase stalling results in uncoupling of DNA polymerase and the replicative helicase, which generates single-stranded DNA (ssDNA). After stalling, RAD51 accumulates at stalled replication forks to stabilize the fork and to repair by homologous recombination (HR) double-strand breaks (DSBs) that accumulate there. We showed recently that SUMO modification of the BLM helicase is required in order for RAD51 to accumulate at stalled forks. In order to investigate how BLM SUMOylation controls RAD51 accumulation, we characterized the function of HR proteins and ssDNA-binding protein RPA in cells that stably expressed either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM). In HU-treated SM-BLM cells, mediators BRCA2 and RAD52, which normally substitute RAD51 for RPA on ssDNA, failed to accumulate normally at stalled forks; instead, excess RPA accumulated. SM-BLM cells also exhibited higher levels of HU-induced chromatin-bound RPA than BLM+ cells did. The excess RPA did not result from excessive intrinsic BLM helicase activity, because in vitro SUMOylated BLM unwound similar amounts of replication-fork substrate as unSUMOylated BLM. Nor did BLM SUMOylation inhibit binding of RPA to BLM in vitro; however, in immunoprecipitation experiments, more BLM-RPA complex formed in HU-treated SM-BLM cells, indicating that BLM SUMOylation controls the amount of BLM-RPA complex normally formed at stalled forks. Together, these results showed that BLM SUMOylation regulates the amount of ssDNA that accumulates during polymerase stalling. We conclude that BLM SUMOylation functions as a licensing mechanism that permits and regulates HR at damaged replication forks.

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

A major challenge in nuclear organization is the packaging of DNA into dynamic chromatin structures that can respond to changes in the transcriptional requirements of the cell. Posttranslational protein modifications, of histones and other chromatin-associated factors, are essential regulators of chromatin dynamics. In this Review, we summarize studies demonstrating that posttranslational modification of proteins by small ubiquitin-related modifiers (SUMOs) regulates chromatin structure and function at multiple levels and through a variety of mechanisms to influence gene expression and maintain genome integrity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified in 1997 as a tumor suppressor gene through mapping of homozygous mutations occurring in multiple sporadic tumor types and in patients with cancer predisposition syndromes including Cowden disease (Song et al., 2012). Since that time, PTEN has emerged as one of the most frequently mutated or deleted genes in human cancers, including human skin cancers. In particular, loss of PTEN function through mutation or deletion has been observed in up to 70% of melanoma cell lines, and epigenetic silencing of PTEN has been observed in 30-40% of malignant melanomas (Mehnert and Kluger, 2012)., (Copyright © 2012 John Wiley & Sons A/S.)

An Epstein-Barr virus (EBV) protein microarray was used to screen for proteins binding noncovalently to the small ubiquitin-like modifier SUMO2. Among the 11 SUMO binding proteins identified was the conserved protein kinase BGLF4. The mutation of potential SUMO interaction motifs (SIMs) in BGLF4 identified N- and C-terminal SIMs. The mutation of both SIMs changed the intracellular localization of BGLF4 from nuclear to cytoplasmic, while BGLF4 mutated in the N-terminal SIM remained predominantly nuclear. The mutation of the C-terminal SIM yielded an intermediate phenotype with nuclear and cytoplasmic staining. The transfer of BGLF4 amino acids 342 to 359 to a nuclear green fluorescent protein (GFP)-tagged reporter protein led to the relocalization of the reporter to the cytoplasm. Thus, the C-terminal SIM lies adjacent to a nuclear export signal, and coordinated SUMO binding by the N- and C-terminal SIMs blocks export and allows the nuclear accumulation of BGLF4. The mutation of either SIM prevented SUMO binding in vitro. The ability of BGLF4 to abolish the SUMOylation of the EBV lytic cycle transactivator ZTA was dependent on both BGLF4 SUMO binding and BGLF4 kinase activity. The global profile of SUMOylated cell proteins was also suppressed by BGLF4 but not by the SIM or kinase-dead BGLF4 mutant. The effective BGLF4-mediated dispersion of promyelocytic leukemia (PML) bodies was dependent on SUMO binding. The SUMO binding function of BGLF4 was also required to induce the cellular DNA damage response and to enhance the production of extracellular virus during EBV lytic replication. Thus, SUMO binding by BGLF4 modulates BGLF4 function and affects the efficiency of lytic EBV replication.

The association of small, ubiquitin-related modifier-specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat-containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway.

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.

An intimate relationship exists between the transcriptional coregulator Daxx, SUMO, and PML nuclear bodies. In this issue, Chang et al. (2011) provide structural insights into how phosphorylation of Daxx increases its affinity toward SUMOs and functional insights into how enhanced SUMO binding affects Daxx-PML interactions, PML nuclear body localization, and Daxx-mediated repression of genes encoding for antiapoptotic factors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased gamma-H2AX foci. Because the increased gamma-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess gamma-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function., Competing Interests: The authors have declared that no competing interests exist.

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys(63)-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13 approximately Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys(63) of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.

Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.

Vertebrates express three small ubiquitin-related modifiers (SUMO-1, SUMO-2, and SUMO-3) that are conjugated in part to unique subsets of proteins and, thereby, regulate distinct cellular processes. Mechanisms regulating paralog-selective sumoylation, however, remain poorly understood. Despite being equally well modified by SUMO-1 and SUMO-2 in vitro, RanGAP1 is selectively modified by SUMO-1 in vivo. We have found that this paralog-selective modification is determined at the level of deconjugation by isopeptidases. Our findings indicate that, relative to SUMO-2-modified RanGAP1, SUMO-1-modified RanGAP1 forms a more stable, higher affinity complex with the nucleoporin Nup358/RanBP2 that preferentially protects it from isopeptidases. By swapping residues in SUMO-1 and SUMO-2 responsible for Nup358/RanBP2 binding, or by manipulating isopeptidase expression levels, paralog-selective modification of RanGAP1 could be affected both in vitro and in vivo. Thus, protection from isopeptidases, through interactions with SUMO-binding proteins, represents an important mechanism defining paralog-selective sumoylation.

Post-translational modification of proteins, such as phosphorylation, ubiquitination, and SUMO modification, is an important means of regulating a variety of cellular activities. SUMOs (Small Ubiquitin related Modifiers) are covalently conjugated to lysine residues of many proteins by a mechanism that parallels ubiquitination (1). The effects of sumoylation, however, are distinct from ubiquitination. Sumoylation does not directly control protein stability, but regulates proteins through various mechanisms that include modulation of protein-protein interactions, protein-nucleic acid interactions, subcellular protein localization, and enzymatic activity (1-4). There are many examples, however, where the molecular bases for the effects of sumoylation on protein function and on cellular processes remain unclear. Here, we outline the use of an inducible and reversible sumoylation system, based on rapamycin heterodimerization, as a novel tool to characterize the functions of sumoylation in mammalian cells.

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are approximately 96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralog-selective modification.

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.

SUMOylation is essential for cell-cycle regulation in invertebrates; however, its functions during the mammalian cell cycle are largely uncharacterized. Mammals express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are 96% identical and referred to as SUMO-2/3). We found that SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. Consistent with this, global inhibition of SUMOylation caused a prometaphase arrest due to defects in targeting the microtubule motor protein CENP-E to kinetochores. CENP-E was found to be modified specifically by SUMO-2/3 and to possess SUMO-2/3 polymeric chain-binding activity essential for kinetochore localization. Our findings indicate that SUMOylation is a key regulator of the mammalian cell cycle, with SUMO-1 and SUMO-2/3 modification of different proteins regulating distinct processes.

Two articles in a recent issue of Molecular Cell (Shen et al., 2006; Lin et al., 2006) demonstrate that noncovalent interactions between the SUMO moieties of SUMO-modified PML, and SUMO binding motifs on PML and other PML nuclear-body-associated proteins, affect the assembly of PML nuclear bodies and the recruitment of proteins in and out of these subnuclear structures.

The nuclear envelope is a double lipid bilayer that physically separates the functions of the nucleus and the cytoplasm of eukaryotic cells. Regulated transport of molecules between the nucleus and the cytoplasm is essential for normal cell metabolism and is mediated by large protein complexes, termed nuclear pore complexes (NPCs), which span the inner and outer membranes of the nuclear envelope. Significant progress has been made in the past 10 years in identifying the protein composition of NPCs and the basic molecular mechanisms by which these complexes facilitate the selective exchange of molecules between the nucleus and the cytoplasm. However, many fundamentally important questions about the functions of NPCs, the specific functions of individual NPC-associated proteins, and the assembly and disassembly of NPCs, remain unanswered. This review describes approaches for isolating and characterizing nuclear envelopes and NPC-associated proteins from mammalian cells. It is anticipated that these procedures can be used as a starting point for further molecular and biochemical analysis of the mammalian nuclear envelope, NPCs, and NPC-associated proteins.