Stepwise phosphorylation and SUMOylation of PIDD1 drive PIDDosome assembly in response to DNA repair failure.

SUMOylation regulates numerous cellular stress responses, yet targets in the apoptotic machinery remain elusive. We show that a single, DNA damage-induced monoSUMOylation event controls PIDDosome (PIDD1/RAIDD/caspase-2) formation and apoptotic death in response to unresolved DNA interstrand crosslinks (ICLs). SUMO-1 conjugation occurs on conserved K879 in the PIDD1 death domain (DD); is catalyzed by PIAS1 and countered by SENP3; and is triggered by ATR phosphorylation of neighboring T788 in the PIDD1 DD, which enables PIAS1 docking. Phospho/SUMO-PIDD1 proteins are captured by nucleolar RAIDD monomers via a SUMO-interacting motif (SIM) in the RAIDD DD, thus compartmentalizing nascent PIDDosomes for caspase-2 recruitment. Denying SUMOylation or the SUMO-SIM interaction spares the onset of PIDDosome assembly but blocks its completion, thus eliminating the apoptotic response to ICL repair failure. Conversely, removal of SENP3 forces apoptosis, even in cells with tolerable ICL levels. SUMO-mediated PIDDosome control is also seen in response to DNA breaks but not supernumerary centrosomes. These results illuminate PIDDosome formation in space and time and identify a direct role for SUMOylation in the assembly of a major pro-apoptotic device.

ß-catenin-inhibited Sumoylation modification of LKB1 and fatty acid metabolism is critical in renal fibrosis.

Liver kinase B1 (LKB1) is a serine/threonine kinase controlling cell homeostasis. Among post-translational modification, Sumoylation is vital for LKB1 activating adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), the key regulator in energy metabolism. Of note, AMPK-regulated fatty acid metabolism is highly involved in maintaining normal renal function. However, the regulative mechanisms of LKB1 Sumoylation remain elusive. In this study, we demonstrated that ß-catenin, a notorious signal in renal fibrosis, inhibited the Sumoylation of LKB1, thereby disrupting fatty acid oxidation in renal tubular cells and triggering renal fibrosis. Mechanically, we found that Sumo3 was the key mediator for LKB1 Sumoylation in renal tubular cells, which was transcriptionally inhibited by ß-catenin/Transcription factor 4 (TCF4) signaling. Overexpression of Sumo3, not Sumo1 or Sumo2, restored ß-catenin-disrupted fatty acid metabolism, and retarded lipid accumulation and fibrogenesis in the kidney. In vivo, conditional knockout of ß-catenin in tubular cells effectively preserved fatty acid oxidation and blocked lipid accumulation by maintaining LKB1 Sumoylation and AMPK activation. Furthermore, ectopic expression of Sumo3 strongly inhibited Wnt1-aggravated lipid accumulation and fibrogenesis in unilateral ischemia-reperfusion mice. In patients with chronic kidney disease, we found a loss of Sumo3 expression, and it was highly related to LKB1 repression. This contributes to fatty acid metabolism disruption and lipid accumulation, resulting in renal fibrosis. Overall, our study revealed a new mechanism in fatty acid metabolism dysfunction and provided a new therapeutic target pathway for regulating Sumo modification in renal fibrosis.

SUMOylation of Warts kinase promotes neural stem cell reactivation.

A delicate balance between neural stem cell (NSC) quiescence and proliferation is important for adult neurogenesis and homeostasis. Small ubiquitin-related modifier (SUMO)-dependent post-translational modifications cause rapid and reversible changes in protein functions. However, the role of the SUMO pathway during NSC reactivation and brain development is not established. Here, we show that the key components of the SUMO pathway play an important role in NSC reactivation and brain development in Drosophila. Depletion of SUMO/Smt3 or SUMO conjugating enzyme Ubc9 results in notable defects in NSC reactivation and brain development, while their overexpression leads to premature NSC reactivation. Smt3 protein levels increase with NSC reactivation, which is promoted by the Ser/Thr kinase Akt. Warts/Lats, the core protein kinase of the Hippo pathway, can undergo SUMO- and Ubc9-dependent SUMOylation at Lys766. This modification attenuates Wts phosphorylation by Hippo, leading to the inhibition of the Hippo pathway, and consequently, initiation of NSC reactivation. Moreover, inhibiting Hippo pathway effectively restores the NSC reactivation defects induced by SUMO pathway inhibition. Overall, our study uncovered an important role for the SUMO-Hippo pathway during Drosophila NSC reactivation and brain development.

Circular RNA-encoded oncogenic PIAS1 variant blocks immunogenic ferroptosis by modulating the balance between SUMOylation and phosphorylation of STAT1.

BACKGROUND: The clinical response rate to immune checkpoint blockade (ICB) therapy in melanoma remains low, despite its widespread use. Circular non-coding RNAs (circRNAs) are known to play a crucial role in cancer progression and may be a key factor limiting the effectiveness of ICB treatment. METHODS: The circRNAs that were downregulated after coadministration compared with single administration of PD-1 inhibitor administration were identified through RNA-seq and Ribo-seq, and thus the circPIAS1 (mmu_circ_0015773 in mouse, has_circ_0008378 in human) with high protein coding potential was revealed. Fluorescence in situ hybridization (FISH) assays were conducted to determine the localization of circPIAS1 in human and mouse melanoma cells, as well as its presence in tumor and adjacent tissues of patients. Validation through dual-luciferase reporter assay and LC-MS/MS confirmed the ability of circPIAS1 to encode a novel 108 amino acid polypeptide (circPIAS1-108aa). Specific antisense oligonucleotides (ASOs) targeting the junction site of circPIAS1 were developed to reduce its intracellular levels. Proliferation changes in melanoma cells were assessed using CCK8, EdU, and colony formation assays. The impact of circPIAS1-108aa on the ferroptosis process of melanoma cells was studied through GSH, MDA, and C11-BODIPY staining assays. Western Blot, Immunoprecipitation (IP), and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques were employed to investigate the impact of circPIAS1-108aa on the P-STAT1/SLC7A11/GPX4 signaling pathway, as well as its influence on the balance between STAT1 SUMOylation and phosphorylation. Additionally, a melanoma subcutaneous transplanted tumor mouse model was utilized to examine the combined effect of reducing circPIAS1 levels alongside PD-1 inhibitor. RESULTS: Compared with the group treated with PD-1 inhibitor alone, circPIAS1 was significantly down-regulated in the coadministration group and demonstrated higher protein coding potential. CircPIAS1, primarily localized in the nucleus, was notably upregulated in tumor tissues compared to adjacent tissues, where it plays a crucial role in promoting cancer cell proliferation. This circRNA can encode a unique polypeptide consisting of 108 amino acids, through which it exerts its cancer-promoting function and impedes the effectiveness of ICB therapy. Mechanistically, circPIAS1-108aa hinders STAT1 phosphorylation by recruiting SUMO E3 ligase Ranbp2 to enhance STAT1 SUMOylation, thereby reactivating the transduction of the SLC7A11/GPX4 signaling pathway and restricting the immunogenic ferroptosis induced by IFNγ. Furthermore, the combination of ASO-circPIAS1 with PD-1 inhibitor effectively inhibits melanoma growth and significantly enhances the efficacy of immune drugs in vivo. CONCLUSIONS: Our study uncovers a novel mechanism regarding immune evasion in melanoma driven by a unique 108aa peptide encoded by circPIAS1 in melanoma that dramatically hinders immunogenic ferroptosis triggered by ICB therapy via modulating the balance between SUMOylation and phosphorylation of STAT1. This work reveals circPIAS1-108aa as a critical factor limiting the immunotherapeutic effects in melanoma and propose a promising strategy for improving ICB treatment outcomes.

Multifaceted roles of SUMO in DNA metabolism.

Sumoylation, a process in which SUMO (small ubiquitin like modifier) is conjugated to target proteins, emerges as a post-translational modification that mediates protein-protein interactions, protein complex assembly, and localization of target proteins. The coordinated actions of SUMO ligases, proteases, and SUMO-targeted ubiquitin ligases determine the net result of sumoylation. It is well established that sumoylation can somewhat promiscuously target proteins in groups as well as selectively target individual proteins. Through changing protein dynamics, sumoylation orchestrates multi-step processes in chromatin biology. Sumoylation influences various steps of mitosis, DNA replication, DNA damage repair, and pathways protecting chromosome integrity. This review highlights examples of SUMO-regulated nuclear processes to provide mechanistic views of sumoylation in DNA metabolism.

Highly sensitive site-specific SUMOylation proteomics in Arabidopsis.

SUMOylation-the attachment of a small ubiquitin-like modifier (SUMO) to target proteins-plays roles in controlling plant growth, nutrient signalling and stress responses. SUMOylation studies in plants are scarce because identifying SUMOylated proteins and their sites is challenging. To date, only around 80 SUMOylation sites have been identified. Here we introduce lysine-null SUMO1 into the Arabidopsis sumo1 sumo2 mutant and establish a two-step lysine-null SUMO enrichment method. We identified a site-specific SUMOylome comprising over 2,200 SUMOylation sites from 1,300 putative acceptors that function in numerous nuclear processes. SUMOylation marks occur on several motifs, differing from the canonical ψKxE motif in distant eukaryotes. Quantitative comparisons demonstrate that SUMOylation predominantly enhances the stability of SUMO1 acceptors. Our study delivers a highly sensitive and efficient method for site-specific SUMOylome studies and provides a comprehensive catalogue of Arabidopsis SUMOylation, serving as a valuable resource with which to further explore how SUMOylation regulates protein function.

Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of the androgen receptor in prostate cancer cells.

The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.

Dysregulated gene expression of SUMO machinery components induces the resistance to anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes.

Background: The majority of patients with lung cancer exhibit drug resistance after anti-PD-1 immunotherapy, leading to shortened patient survival time. Previous studies have suggested an association between epigenetic abnormalities such as methylation and clinical response to anti-PD-1 immunotherapy, while the role of SUMOylation in resistance to anti-PD-1 antibody immunotherapy is still unclear. Methods: Here, the mRNA expression of 15 SUMO machinery components in PBMC from lung cancer patients receiving anti-PD-1 immunotherapy were analyzed using real-time PCR. Base on the percentage change in mRNA levels, the relationship between the expression of SUMO machinery components and outcomes of anti-PD-1 immunotherapy, and the influencing factors of SUMOylation were evaluated. PBMC was treated with different concentrations of 2-D08 (a specific inhibitor of SUMOylation) in vitro, and analyzed the activation and the death rates of lymphocyte subsets by flow cytometry analysis. Results: A predictive method, base on the gene expression of three SUMO machinery components (SUMO1, SUMO3 and UBE2I), were developed to distinguish non-responders to PD-1 inhibitors. Furthermore, the number of lymphocytes in peripheral blood significantly reduced in the dysregulated SUMOylation groups (the percentage change >100 or -50 ~ -100 groups). In vitro studies confirmed that lightly low SUMOylation level improved the activation status of T and NK lymphocytes, but extremely low SUMOylation level lead to the increased death rates of lymphocytes. Conclusion: Our findings implied that dysregulated gene expression of SUMO machinery components could induce the resistance of anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes. These data might provide effective circulating biomarkers for predicting the efficacy of anti-PD-1 immunotherapy, and uncovered a novel regulatory mechanism of resistance to anti-PD-1 immunotherapy.

SARS-CoV-2 Nucleocapsid Protein Induces Tau Pathological Changes That Can Be Counteracted by SUMO2.

Neurologic manifestations are an immediate consequence of SARS-CoV-2 infection, the etiologic agent of COVID-19, which, however, may also trigger long-term neurological effects. Notably, COVID-19 patients with neurological symptoms show elevated levels of biomarkers associated with brain injury, including Tau proteins linked to Alzheimer's pathology. Studies in brain organoids revealed that SARS-CoV-2 alters the phosphorylation and distribution of Tau in infected neurons, but the mechanisms are currently unknown. We hypothesize that these pathological changes are due to the recruitment of Tau into stress granules (SGs) operated by the nucleocapsid protein (NCAP) of SARS-CoV-2. To test this hypothesis, we investigated whether NCAP interacts with Tau and localizes to SGs in hippocampal neurons in vitro and in vivo. Mechanistically, we tested whether SUMOylation, a posttranslational modification of NCAP and Tau, modulates their distribution in SGs and their pathological interaction. We found that NCAP and Tau colocalize and physically interact. We also found that NCAP induces hyperphosphorylation of Tau and causes cognitive impairment in mice infected with NCAP in their hippocampus. Finally, we found that SUMOylation modulates NCAP SG formation in vitro and cognitive performance in infected mice. Our data demonstrate that NCAP induces Tau pathological changes both in vitro and in vivo. Moreover, we demonstrate that SUMO2 ameliorates NCAP-induced Tau pathology, highlighting the importance of the SUMOylation pathway as a target of intervention against neurotoxic insults, such as Tau oligomers and viral infection.

SUMO promotes DNA repair protein collaboration to support alternative telomere lengthening in the absence of PML.

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, it is still unknown whether-and if so, how-SUMO supports ALT beyond APB formation. Here, we show that SUMO condensates that contain DNA repair proteins enable telomere maintenance in the absence of APBs. In PML knockout ALT cell lines that lack APBs, we found that SUMOylation is required for manifesting ALT features independent of PML and APBs. Chemically induced telomere targeting of SUMO produces condensate formation and ALT features in PML-null cells. This effect requires both SUMOylation and interactions between SUMO and SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are associated with the accumulation of DNA repair proteins, including Rad52, Rad51AP1, RPA, and BLM, at telomeres. Furthermore, Rad52 can undergo phase separation, enrich SUMO at telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that SUMO condensate formation promotes collaboration among DNA repair factors to support ALT telomere maintenance without PML. Given the promising effects of SUMOylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

DHX9 SUMOylation is required for the suppression of R-loop-associated genome instability.

RNA helicase DHX9 is essential for genome stability by resolving aberrant R-loops. However, its regulatory mechanisms remain unclear. Here we show that SUMOylation at lysine 120 (K120) is crucial for DHX9 function. Preventing SUMOylation at K120 leads to R-loop dysregulation, increased DNA damage, and cell death. Cells expressing DHX9 K120R mutant which cannot be SUMOylated are more sensitive to genotoxic agents and this sensitivity is mitigated by RNase H overexpression. Unlike the mutant, wild-type DHX9 interacts with R-loop-associated proteins such as PARP1 and DDX21 via SUMO-interacting motifs. Fusion of SUMO2 to the DHX9 K120R mutant enhances its association with these proteins, reduces R-loop accumulation, and alleviates survival defects of DHX9 K120R. Our findings highlight the critical role of DHX9 SUMOylation in maintaining genome stability by regulating protein interactions necessary for R-loop balance.

Site-specific identification and quantitation of endogenous SUMOylation based on SUMO-specific protease and strong anion exchange chromatography.

Small ubiquitin-like modifier (SUMO) modification regulates various eukaryotic cellular processes and plays a pivotal role in interferon (IFN)-mediated antiviral defense. While immunoprecipitation enrichment method is widely used for proteome-wide analysis of endogenous SUMOylation, the inability to target all SUMO forms and high cost of antibodies limited its further application. Herein, we proposed an antibody-free enrichment method based on SUMO-specific protease and strong anion exchange chromatography (SPAX) to globally profile the endogenous SUMOylation. The SUMO1/2/3-modified peptides could be simultaneously enriched by SAX chromatography by utilizing its electrostatic interaction with SUMO1/2/3 remnants, which contained multiple aspartic acids (D) and glutamic acids (E). To remove the co-enriched D/E-containing peptides which might interfere with the detection of low-abundance SUMOylated peptides, SUMO-specific protease was used to cleave the SUMO1/2/3 remnants from enriched SUMOylated peptides. As the deSUMOylated peptides lost SUMO remnants, their interaction with SAX materials became weaker, and the D/E-containing peptides could thus be depleted through the second SAX separation. The SPAX method identified over twice the SUMOylated sites than using SAX method only, greatly improving the identification coverage of endogenous SUMOylated sites. Our strategy was then applied to the site-specific identification and quantification of endogenous SUMOylation in A549 cells stimulated by IFN-γ for the first time. A total of 226 SUMOylated sites on 146 proteins were confidently identified, among which multiple up-regulated sites were involved in IFN-mediated antiviral defense, demonstrating the great promise of SPAX to globally profile and discover endogenous SUMOylation with significant biological functions.

The role and mechanism of SUMO modification in liver disease.

Liver disease affects millions of people in the world, and China has the highest prevalence of liver disease in the world. Small ubiquitin-related modifier (SUMO) modification is a highly conserved post-translational modification of proteins. They are widely expressed in a variety of tissues, including the heart, liver, kidney and lung. SUMOylation of protein plays a key role in the occurrence and development of liver disease. Therefore, this study reviewed the effects of SUMO protein on non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), viral hepatitis, hepatic fibrosis (HF), hepatocellular carcinoma (HCC), and other liver diseases to provide novel strategies for targeted treatment of liver disease.

Expression of SUMO and NF-κB genes in hepatitis B virus-associated hepatocellular carcinoma patients: An observational study.

Alterations in signaling pathways and modulation of cell metabolism are associated with the pathogenesis of cancers, including hepatocellular carcinoma (HCC). Small ubiquitin-like modifier (SUMO) proteins and NF-κB family play major roles in various cellular processes. The current study aims to determine the expression profile of SUMO and NF-κB genes in HCC tumors and investigate their association with the clinical outcome of HCC. The expression of 5 genes - SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 - was quantified in tumor and adjacent non-tumor tissues of 58 HBV-related HCC patients by real-time quantitative PCR and was analyzed for the possible association with clinical parameters of HCC. The expression of SUMO2 was significantly higher in HCC tumor tissues compared to the adjacent non-tumor tissues (P = .01), while no significant difference in SUMO1, SUMO3, NF-κB p65, and NF-κB p50 expression was observed between HCC tumor and non-tumor tissues (P > .05). In HCC tissues, a strong correlation was observed between the expression of SUMO2 and NF-κB p50, between SUMO3 and NF-κB p50, between SUMO3 and NF-κB p65 (Spearman rho = 0.83; 0.82; 0.772 respectively; P < .001). The expression of SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 was decreased in grade 3 compared to grades 1 and 2 in HCC tumors according to the World Health Organization grades system. Our results highlighted that the SUMO2 gene is upregulated in tumor tissues of patients with HCC, and is related to the development of HCC, thus it may be associated with the pathogenesis of HCC.

Charting the evolutionary path of the SUMO modification system in plants reveals molecular hardwiring of development to stress adaptation.

SUMO modification is part of the spectrum of Ubiquitin-like (UBL) systems that give rise to proteoform complexity through post-translational modifications (PTMs). Proteoforms are essential modifiers of cell signaling for plant adaptation to changing environments. Exploration of the evolutionary emergence of Ubiquitin-like (UBL) systems unveils their origin from prokaryotes, where it is linked to the mechanisms that enable sulfur uptake into biomolecules. We explore the emergence of the SUMO machinery across the plant lineage from single-cell to land plants. We reveal the evolutionary point at which plants acquired the ability to form SUMO chains through the emergence of SUMO E4 ligases, hinting at its role in facilitating multicellularity. Additionally, we explore the possible mechanism for the neofunctionalization of SUMO proteases through the fusion of conserved catalytic domains with divergent sequences. We highlight the pivotal role of SUMO proteases in plant development and adaptation, offering new insights into target specificity mechanisms of SUMO modification during plant evolution. Correlating the emergence of adaptive traits in the plant lineage with established experimental evidence for SUMO in developmental processes, we propose that SUMO modification has evolved to link developmental processes to adaptive functions in land plants.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

The physical and evolutionary energy landscapes of devolved protein sequences corresponding to pseudogenes.

Protein evolution is guided by structural, functional, and dynamical constraints ensuring organismal viability. Pseudogenes are genomic sequences identified in many eukaryotes that lack translational activity due to sequence degradation and thus over time have undergone "devolution." Previously pseudogenized genes sometimes regain their protein-coding function, suggesting they may still encode robust folding energy landscapes despite multiple mutations. We study both the physical folding landscapes of protein sequences corresponding to human pseudogenes using the Associative Memory, Water Mediated, Structure and Energy Model, and the evolutionary energy landscapes obtained using direct coupling analysis (DCA) on their parent protein families. We found that generally mutations that have occurred in pseudogene sequences have disrupted their native global network of stabilizing residue interactions, making it harder for them to fold if they were translated. In some cases, however, energetic frustration has apparently decreased when the functional constraints were removed. We analyzed this unexpected situation for Cyclophilin A, Profilin-1, and Small Ubiquitin-like Modifier 2 Protein. Our analysis reveals that when such mutations in the pseudogene ultimately stabilize folding, at the same time, they likely alter the pseudogenes' former biological activity, as estimated by DCA. We localize most of these stabilizing mutations generally to normally frustrated regions required for binding to other partners.

A UL26-PIAS1 complex antagonizes anti-viral gene expression during Human Cytomegalovirus infection.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

GPS-SUMO 2.0: an updated online service for the prediction of SUMOylation sites and SUMO-interacting motifs.

Small ubiquitin-like modifiers (SUMOs) are tiny but important protein regulators involved in orchestrating a broad spectrum of biological processes, either by covalently modifying protein substrates or by noncovalently interacting with other proteins. Here, we report an updated server, GPS-SUMO 2.0, for the prediction of SUMOylation sites and SUMO-interacting motifs (SIMs). For predictor training, we adopted three machine learning algorithms, penalized logistic regression (PLR), a deep neural network (DNN), and a transformer, and used 52 404 nonredundant SUMOylation sites in 8262 proteins and 163 SIMs in 102 proteins. To further increase the accuracy of predicting SUMOylation sites, a pretraining model was first constructed using 145 545 protein lysine modification sites, followed by transfer learning to fine-tune the model. GPS-SUMO 2.0 exhibited greater accuracy in predicting SUMOylation sites than did other existing tools. For users, one or multiple protein sequences or identifiers can be input, and the prediction results are shown in a tabular list. In addition to the basic statistics, we integrated knowledge from 35 public resources to annotate SUMOylation sites or SIMs. The GPS-SUMO 2.0 server is freely available at https://sumo.biocuckoo.cn/. We believe that GPS-SUMO 2.0 can serve as a useful tool for further analysis of SUMOylation and SUMO interactions.

Broad-spectrum ubiquitin/ubiquitin-like deconjugation activity of the rhizobial effector NopD from Bradyrhizobium (sp. XS1150).

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection.