Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.

The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.

A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation.

Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold-type E3 ligase mechanism that activates the UFM1-conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.

A Chain of Events: Regulating Target Proteins by SUMO Polymers.

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.

DNA asymmetry promotes SUMO modification of the single-stranded DNA-binding protein RPA.

Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA-RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.

Stress - Regulation of SUMO conjugation and of other Ubiquitin-Like Modifiers.

Stress is unavoidable and essential to cellular and organismal evolution and failure to adapt or restore homeostasis can lead to severe diseases or even death. At the cellular level, stress drives a plethora of molecular changes, of which variations in the profile of protein post-translational modifications plays a key role in mediating the adaptative response of the genome and proteome to stress. In this context, post-translational modification of proteins by ubiquitin-like modifiers, (Ubl), notably SUMO, is an essential stress response mechanism. In this review, aiming to draw universal concepts of the Ubls stress response, we will decipher how stress alters the expression level, activity, specificity and/or localization of the proteins involved in the conjugation pathways of the various type-I Ubls, and how this result in the modification of particular Ubl targets that will translate an adaptive physiological stress response and allow cells to restore homeostasis.

SUMOylation indirectly suppresses activity of the HIF-1α pathway in intestinal epithelial cells.

The hypoxia-inducible factor (HIF) is a master regulator of the cellular transcriptional response to hypoxia. While the oxygen-sensitive regulation of HIF-1α subunit stability via the ubiquitin-proteasome pathway has been well described, less is known about how other oxygen-independent post-translational modifications impact the HIF pathway. SUMOylation, the attachment of SUMO (small ubiquitin-like modifier) proteins to a target protein, regulates the HIF pathway, although the impact of SUMO on HIF activity remains controversial. Here, we examined the effects of SUMOylation on the expression pattern of HIF-1α in response to pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) in intestinal epithelial cells. We evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression and inhibition of SUMOylation using a novel selective inhibitor of the SUMO pathway, TAK-981, on the sensitivity of HIF-1α in Caco-2 intestinal epithelial cells. Our findings demonstrate that treatment with TAK-981 decreases global SUMO-1 and SUMO-2/3 modification and enhances HIF-1α protein levels, whereas SUMO-1 and SUMO-2/3 overexpression results in decreased HIF-1α protein levels in response to DMOG. Reporter assay analysis demonstrates reduced HIF-1α transcriptional activity in cells overexpressing SUMO-1 and SUMO-2/3, whereas pretreatment with TAK-981 increased HIF-1α transcriptional activity in response to DMOG. In addition, HIF-1α nuclear accumulation was decreased in cells overexpressing SUMO-1. Importantly, we showed that HIF-1α is not directly SUMOylated, but that SUMOylation affects HIF-1α stability and activity indirectly. Taken together, our results indicate that SUMOylation indirectly suppresses HIF-1α protein stability, transcriptional activity, and nuclear accumulation in intestinal epithelial cells.

Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control.

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.

SUMO-3 promotes the ubiquitin-dependent turnover of TRIM55.

Human muscle-specific RING fingers (MURFs) are members of the tripartite motif (TRIM) family of proteins characterized by their C-terminal subgroup one signature domain. MURFs play a role in sarcomere formation and microtubule dynamics. It was previously established that some TRIMs undergo post-translational modification by small ubiquitin-like modifier (SUMO). In this study, we explored the putative SUMOylation of MURF proteins as well as their interactions with SUMO. MURF proteins (TRIM54, TRIM55, and TRIM63) were not found to be SUMOylated. However, TRIM55 turnover by proteasomal and lysosomal degradation was higher upon overexpression of SUMO-3 but not of SUMO-1. Furthermore, it is predicted that TRIM55 contains two potential SUMO-interacting motifs (SIMs). We found that SIM1- and SIM2-mutated TRIM55 were more stable than the wild-type (WT) protein partly due to decreased degradation. Consistently, SIM-mutated TRIM55 was less polyubiquitinated than the WT protein, despite similar monoubiquitination levels. Using IF microscopy, we observed that SIM motifs influenced TRIM55 subcellular localization. In conclusion, our results suggest that SUMO-3 or SUMO-3-modified proteins modulate the localization, stability, and RING ubiquitin ligase activity of TRIM55.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

IMPORTANCE: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Targeting SUMOylation with an injectable nanocomposite hydrogel to optimize radiofrequency ablation therapy for hepatocellular carcinoma.

BACKGROUND: Incomplete radiofrequency ablation (iRFA) in hepatocellular carcinoma (HCC) often leads to local recurrence and distant metastasis of the residual tumor. This is closely linked to the development of a tumor immunosuppressive environment (TIME). In this study, underlying mechanisms and potential therapeutic targets involved in the formation of TIME in residual tumors following iRFA were explored. Then, TAK-981-loaded nanocomposite hydrogel was constructed, and its therapeutic effects on residual tumors were investigated. RESULTS: This study reveals that the upregulation of small ubiquitin-like modifier 2 (Sumo2) and activated SUMOylation is intricately tied to immunosuppression in residual tumors post-iRFA. Both knockdown of Sumo2 and inhibiting SUMOylation with TAK-981 activate IFN-1 signaling in HCC cells, thereby promoting dendritic cell maturation. Herein, we propose an injectable PDLLA-PEG-PDLLA (PLEL) nanocomposite hydrogel which incorporates self-assembled TAK-981 and BSA nanoparticles for complementary localized treatment of residual tumor after iRFA. The sustained release of TAK-981 from this hydrogel curbs the expansion of residual tumors and notably stimulates the dendritic cell and cytotoxic lymphocyte-mediated antitumor immune response in residual tumors while maintaining biosafety. Furthermore, the treatment with TAK-981 nanocomposite hydrogel resulted in a widespread elevation in PD-L1 levels. Combining TAK-981 nanocomposite hydrogel with PD-L1 blockade therapy synergistically eradicates residual tumors and suppresses distant tumors. CONCLUSIONS: These findings underscore the potential of the TAK-981-based strategy as an effective therapy to enhance RFA therapy for HCC.

SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation.

During active DNA demethylation, 5-methylcytosine (5mC) is oxidized by TET proteins to 5-formyl-/5-carboxylcytosine (5fC/5caC) for replacement by unmethylated C by TDG-initiated DNA base excision repair (BER). Base excision generates fragile abasic sites (AP-sites) in DNA and has to be coordinated with subsequent repair steps to limit accumulation of genome destabilizing secondary DNA lesions. Here, we show that 5fC/5caC is generated at a high rate in genomes of differentiating mouse embryonic stem cells and that SUMOylation and the BER protein XRCC1 play critical roles in orchestrating TDG-initiated BER of these lesions. SUMOylation of XRCC1 facilitates physical interaction with TDG and promotes the assembly of a TDG-BER core complex. Within this TDG-BERosome, SUMO is transferred from XRCC1 and coupled to the SUMO acceptor lysine in TDG, promoting its dissociation while assuring the engagement of the BER machinery to complete demethylation. Although well-studied, the biological importance of TDG SUMOylation has remained obscure. Here, we demonstrate that SUMOylation of TDG suppresses DNA strand-break accumulation and toxicity to PARP inhibition in differentiating mESCs and is essential for neural lineage commitment.

Simplified cloning and isolation of peptides from "sandwiched" SUMO-peptide-intein fusion proteins.

BACKGROUND: Some peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a "sandwiched" SUMO-peptide-intein (SPI) fusion protein, which protects the target peptide sequence from truncation and improves yield. This initial system required cloning with two commercially available vectors. It used an N-terminal polyhistidine tagged small ubiquitin-like modifier (SUMO) protein and a C-terminal engineered Mycobacterium xenopii DNA Gyrase A intein with an inserted chitin binding domain (CBD) to create "sandwiched" fusion proteins of the form: His6-SUMO-peptide-intein-CBD. However, the major drawback of this previously reported fusion protein "sandwich" approach is the increased time and number of steps required to complete the cloning and isolation procedures, relative to the simple procedures to produce recombinant peptides in E. coli from a single (non-"sandwiched") fusion protein system. RESULTS: In this work we generate the plasmid pSPIH6, which improves upon the previous system by encoding both the SUMO and intein proteins and allows facile construction of a SPI protein in a single cloning step. Additionally, the Mxe GyrA intein encoded in pSPIH6 contains a C-terminal polyhistidine tag, resulting in SPI fusion proteins of the form: His6-SUMO-peptide-intein-CBD-His6. The dual polyhistidine tags greatly simplify isolation procedures compared to the original SPI system, which we have here demonstrated with two linear bacteriocin peptides: leucocin A and lactococcin A. The yields obtained for both peptides after purification were also improved compared to the previous SPI system as a result of this streamlined protocol. CONCLUSIONS: This modified SPI system and its simplified cloning and purification procedures described here may be generally useful as a heterologous E. coli expression system to obtain pure peptides in high yield, especially when degradation of the target peptide is an issue.

Conserved E1B-55K SUMOylation in Different Human Adenovirus Species Is a Potent Regulator of Intracellular Localization.

Over the past decades, studies on the biology of human adenoviruses (HAdVs) mainly focused on the HAdV prototype species C type 5 (HAdV-C5) and revealed fundamental molecular insights into mechanisms of viral replication and viral cell transformation. Recently, other HAdV species are gaining more and more attention in the field. Reports on large E1B proteins (E1B-55K) from different HAdV species showed that these multifactorial proteins possess strikingly different features along with highly conserved functions. In this work, we identified potential SUMO-conjugation motifs (SCMs) in E1B-55K proteins from HAdV species A to F. Mutational inactivation of these SCMs demonstrated that HAdV E1B-55K proteins are SUMOylated at a single lysine residue that is highly conserved among HAdV species B to E. Moreover, we provide evidence that E1B-55K SUMOylation is a potent regulator of intracellular localization and p53-mediated transcription in most HAdV species. We also identified a lysine residue at position 101 (K101), which is unique to HAdV-C5 E1B-55K and specifically regulates its SUMOylation and nucleo-cytoplasmic shuttling. Our findings reveal important new aspects on HAdV E1B-55K proteins and suggest that different E1B-55K species possess conserved SCMs while their SUMOylation has divergent cellular effects during infection. IMPORTANCE E1B-55K is a multifunctional adenoviral protein and its functions are highly regulated by SUMOylation. Although functional consequences of SUMOylated HAdV-C5 E1B-55K are well studied, we lack information on the effects of SUMOylation on homologous E1B-55K proteins from other HAdV species. Here, we show that SUMOylation is a conserved posttranslational modification in most of the E1B-55K proteins, similar to what we know about HAdV-C5 E1B-55K. Moreover, we identify subcellular localization and regulation of p53-dependent transcription as highly conserved SUMOylation-regulated E1B-55K functions. Thus, our results highlight how HAdV proteins might have evolved in different HAdV species with conserved domains involved in virus replication and differing alternative functions and interactions with the host cell machinery. Future research will link these differences and similarities to the diverse pathogenicity and organ tropism of the different HAdV species.

Anti-SAE autoantibody in dermatomyositis: original comparative study and review of the literature.

OBJECTIVE: Among specific autoantibodies in DM, the anti-small ubiquitin-like modifier activating enzyme (SAE) antibody is rare. We aim to describe the clinical characteristics, cancer prevalence, and muscle pathology of anti-SAE-positive DM. METHODS: Patients with a diagnosis of DM and sera positive for the anti-SAE antibody were recruited from 19 centres in this retrospective observational study. The available muscular biopsies were reviewed. We conducted a comparison with anti-SAE-negative DM and a review of the literature. RESULTS: Of the patients in the study (n = 49), 84% were women. Skin involvement was typical in 96% of patients, with 10% having calcinosis, 18% ulceration and 12% necrosis; 35% presented with a widespread skin rash. Muscular disease affected 84% of patients, with mild weakness [Medical Research Council (MRC) scale 4 (3, 5)], although 39% of patients had dysphagia. Muscular biopsies showed typical DM lesions. Interstitial lung disease was found in 21% of patients, mainly with organizing pneumonia pattern, and 26% of patients showed dyspnoea. Cancer-associated myositis was diagnosed in 16% of patients and was responsible for the majority of deaths, its prevalence being five times that of the general population. IVIG therapy was administered to 51% of the patients during the course of the disease. Comparison with anti-SAE-negative DM (n = 85) showed less and milder muscle weakness (P = 0.02 and P = 0.006, respectively), lower creatinine kinase levels (P < 0.0001) and less dyspnoea (P = 0.003). CONCLUSION: Anti-SAE positive DM is a rare subgroup associated with typical skin features but a potentially diffuse rash, a mild myopathy. Interstitial lung disease defines an organizing pneumonia pattern. Cancer associated DM prevalence is five times that of the general population. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov, NCT04637672.

Robust production of active Ulp1 (SUMO protease) from inclusion bodies.

High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.

Mass spectrometric assays monitoring the deubiquitinase activity of the SARS-CoV-2 papain-like protease inform on the basis of substrate selectivity and have utility for substrate identification.

The SARS-CoV-2 papain-like protease (PLpro) and main protease (Mpro) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with Mpro, PLpro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PLpro using synthetic Nε-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PLpro. They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PLpro catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S' sites. Importantly, the results reflect the reported cellular substrate selectivity of PLpro, i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PLpro catalysis is affected not only by the identity of the substrate residues binding in the S and S' sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PLpro:substrate complex. Nε-Lysine-branched oligopeptides thus have potential to help the identification of PLpro substrates. More generally, the results imply that MS-based assays with Nε-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.

SUMO1 and Defective Spermatozoa Correlate with Endogenous Hydrogen Peroxide and Live Birth Outcome in Intrauterine Insemination Cycles for Unexplained Infertility.

This study aimed to investigate the correlation between hydrogen peroxide (H2O2), small ubiquitin-like modifier molecules (SUMO), and pregnancy outcomes in couples with unexplained infertility (UI) undergoing intrauterine insemination (IUI) treatment. We prospectively collected semen samples from 56 couples with UI and divided the spermatozoa into motile and immotile fractions by density gradient centrifugation (DSC). Immunofluorescence staining was used to examine the immunostaining and localization of nuclear pore complex (NPC), SUMO1, and SUMO2/3 in spermatozoa. We detected H2O2 levels by chemiluminescence methods. We found that H2O2 levels correlated with NPC (neck) (r = 0.400) and NPC (tail) (r = 0.473) in motile sperm fractions. In immotile fractions, H2O2 positively correlated with NPC (tail) (r = 0.431) and SUMO1 (neck) (r = 0.282). Furthermore, the positive NPC (tail) group had a significantly lower live birth rate than the negative NPC group (17.9% = 5/28 vs. 42.9% = 12/28). In conclusion, H2O2 positively correlated with SUMO1 (neck) and NPC (tail) in human spermatozoa. The DSC may partially eliminate defective spermatozoa (positive NPC staining); however, if defective spermatozoa remain in the motile fraction, this scenario is associated with a low live birth rate following IUI treatment.

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Dermatomyositis (DM) is an autoimmune disease often complicated with malignant tumors. More than 50% of DM patients have myositis specific autoantibodies in their bodies. DM specific autoantibodies [including anti-migration inhibitory factor (Mi)-2 antibody, anti-nuclear matrix protein (NXP)-2 antibody, anti-transcription intermediary factor (TIF) 1-γ antibody, and anti-small ubiquitin like modifier activating enzyme (SAE) antibody] play important roles in the pathogenesis of malignancy associated DM. Revealing the role of DM specific autoantibodies in the development of malignant tumors in DM patients can provide important evidence for accurately assessing the risk of developing malignant tumors in DM patients, and also provide new ideas for clinical diagnosis of DM and precise treatment.

Heat shock transcription factor 1 is SUMOylated in the activated trimeric state.

The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response.