Protein SUMOylation Research Articles

An application of topological data analysis in predicting sumoylation sites.

Sumoylation is a reversible post-translational modification that regulates certain significant biochemical functions in proteins. The protein alterations caused by sumoylation are associated with the incidence of some human diseases. Therefore, identifying the sites of sumoylation in proteins may provide a direction for mechanistic research and drug development. Here, we propose a new computational approach for identifying sumoylation sites using an encoding method based on topological data analysis. The features of our model captured the key physical and biological properties of proteins at multiple scales. In a 10-fold cross validation, the outcomes of our model showed 96.45% of sensitivity (Sn), 94.65% of accuracy (Acc), 0.8946 of Matthew's correlation coefficient (MCC), and 0.99 of area under curve (AUC). The proposed predictor with only topological features achieves the best MCC and AUC in comparison to the other released methods. Our results suggest that topological information is an additional parameter that can assist in the prediction of sumoylation sites and provide a novel perspective for further research in protein sumoylation.

The secreted tyrosine phosphatase PtpA promotes Staphylococcus aureus survival in RAW 264.7 macrophages through decrease of the SUMOylation host response.

Staphylococcus aureus uses numerous strategies to survive and persist in the intracellular environment of professional phagocytes, including modulation of the SUMOylation process. This study aims to understand how S. aureus alters host SUMOylation to enhance its intracellular survival in professional phagocytes. Our results indicate that S. aureus strain Newman utilizes PtpA-driven phosphorylation to decrease the amount of SUMOylated proteins in murine macrophages to facilitate its survival in this immune cell type.

Mycobacteria modulate SUMOylation to suppresses protective responses in dendritic cells.

Post translational modifications (PTMs) are exploited by various pathogens in order to escape host immune responses. SUMOylation is one of the PTMs which is involved in regulation of a variety of cellular responses. However, the effects of host SUMOylation on pathogenic bacteria largely remain elusive. We, therefore, investigated the role of SUMOylation in regulating defense responses in dendritic cells (DCs) during mycobacterial infection. Dendritic Cells of female BALB/c mice and THP-1 macrophages were used. Western blotting was performed to measure the expression of level of SUMO1, pSTAT1, pp38, pERK, Beclin-1, LC3, Bax and Cytochrome C. For bacterial burden confocal microscopy and CFU (Colony Forming Unit) were used. Flow cytometry was used for ROS and co-stimulatory molecules measurement. Cytokine level were measured using ELISA. We show that stimulation of Bone Marrow Derived Dendritic Cells (BMDCs) with mycobacterial antigen Rv3416 or live infection with Mycobacterium bovis BCG increases the SUMOylation of host proteins. Inhibition of SUMOylation significantly decreased intracellular bacterial loads in DCs. Additionally, inhibiting SUMOylation, induces protective immune responses by increasing oxidative burst, pro-inflammatory cytokine expression and surface expression of T cell co-stimulatory molecules, and activation of pSTAT1 and Mitogen Activated Protein Kinases (MAPK) proteins- pp38 and pERK. SUMOylation inhibition also increased apoptosis and autophagy in BMDCs. Intriguingly, mycobacteria increased SUMOylation of many of the above molecules. Furthermore, inhibiting SUMOylation in DCs primed T cells that in turn attenuated bacterial burden in infected macrophages. These findings demonstrate that SUMOylation pathway is exploited by mycobacteria to thwart protective host immune responses.

SENP6 regulates localization and nuclear condensation of DNA damage response proteins by group deSUMOylation

The SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage response proteins, including BRCA1-BARD1, 53BP1, BLM and ERCC1-XPF. SENP6 maintains these proteins in a hypo-SUMOylated state under unstressed conditions and counteracts their polySUMOylation after hydroxyurea-induced stress. Co-depletion of RNF4 leads to a further increase in SUMOylation of BRCA1, BARD1 and BLM, suggesting that SENP6 antagonizes targeting of these proteins by RNF4. Functionally, depletion of SENP6 results in uncoordinated recruitment and persistence of SUMO2/3 at UVA laser and ionizing radiation induced DNA damage sites. Additionally, SUMO2/3 and DNA damage response proteins accumulate in nuclear bodies, in a PML-independent manner driven by multivalent SUMO-SIM interactions. These data illustrate coordinated regulation of SUMOylated DNA damage response proteins by SENP6, governing their timely localization at DNA damage sites and nuclear condensation state.

Aspergillus SUMOylation mutants exhibit chromosome segregation defects including chromatin bridges.

Functions of protein SUMOylation remain incompletely understood in different cell types. Via forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO activation enzyme UbaB in the filamentous fungus Aspergillus nidulans. The ubaBQ247*, ΔubaB, and ΔsumO mutants all produce abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. The bridges are enclosed by nuclear membrane containing peripheral nuclear pore complex proteins that normally get dispersed during mitosis, and the bridges are also surrounded by cytoplasmic microtubules typical of interphase cells. Time-lapse sequences further indicate that most bridges persist through interphase prior to the next mitosis, and anaphase chromosome segregation can produce new bridges that persist into the next interphase. When the first mitosis happens at a higher temperature of 42°C, SUMOylation deficiency produces not only chromatin bridges but also many abnormally shaped single nuclei that fail to divide. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO targets being nuclear proteins. Finally, although the budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, loss of SUMOylation does not cause any obvious defect in dynein-mediated transport of nuclei and early endosomes, indicating that SUMOylation is unnecessary for dynein activation in A. nidulans.

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.

Mechanisms of Modulation of Mitochondrial Architecture.

Mitochondrial network architecture plays a critical role in cellular physiology. Indeed, alterations in the shape of mitochondria upon exposure to cellular stress can cause the dysfunction of these organelles. In this scenario, mitochondrial dynamics proteins and the phospholipid composition of the mitochondrial membrane are key for fine-tuning the modulation of mitochondrial architecture. In addition, several factors including post-translational modifications such as the phosphorylation, acetylation, SUMOylation, and o-GlcNAcylation of mitochondrial dynamics proteins contribute to shaping the plasticity of this architecture. In this regard, several studies have evidenced that, upon metabolic stress, mitochondrial dynamics proteins are post-translationally modified, leading to the alteration of mitochondrial architecture. Interestingly, several proteins that sustain the mitochondrial lipid composition also modulate mitochondrial morphology and organelle communication. In this context, pharmacological studies have revealed that the modulation of mitochondrial shape and function emerges as a potential therapeutic strategy for metabolic diseases. Here, we review the factors that modulate mitochondrial architecture.

SUMOylation of AnxA6 facilitates EGFR-PKCα complex formation to suppress epithelial cancer growth

BackgroundThe Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells.MethodsCells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC–MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6K299R plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFRT790M/L858R was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model.ResultsAnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6K299R showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6K299R conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6K299R mutation-expressing cells were much greater than that of AnxA6-expressing cells.ConclusionsBesides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect.4V8MbH9D2-V-dxkGbe_YjXVideo

Deficiency of protein inhibitor of activated STAT3 exacerbates atherosclerosis by modulating VSMC phenotypic switching

Background and aimsPhenotypic switching of vascular smooth muscle cells (VSMCs) plays an essential role in the development of atherosclerosis. Protein inhibitor of activated STAT (Pias) regulates VSMCs phenotype via acting as sumo E3 ligase to promote protein sumoylation. Our previous study indicated that Pias3 expression decreased in atherosclerotic lesions. Therefore, this study aimed to explore the role of Pias3 on VSMCs phenotype switching during atherosclerosis. MethodsApoE−/− and ApoE−/−Pias3−/− double-deficient mice were fed with high-fat/high-cholesterol diet to induce atherosclerosis. Aorta tissues and primary VSMCs were collected to assess plaque formation and VSMCs phenotype. In vitro, Pias3 was overexpressed in A7r5, a VSMCs cell line, by transfection with Pias3 plasmid. Real-time quantitative PCR, immunoblotting, immunoprecipitation, were used to analyze the effect of Pias3 on VSMCs phenotypic switching. ResultsPias3 deficiency significantly exacerbated atherosclerotic plaque formation and promoted VSMCs phenotypic switching to a synthetic state within lesion. In vitro, overexpressing Pias3 in VSMCs increased the expression of contractile markers (myosin heavy chain 11, calponin 1), while it decreased the level of synthetic marker (vimentin). Additionally, Pias3 overexpression blocked PDGF-BB-induced VSMCs proliferation and migration. Immunoprecipitation and mass spectrometry results showed that Pias3 enhanced sumoylation and ubiquitination of vimentin, and shortened its half-life. Moreover, the ubiquitination level of vimentin was impaired by 2-D08, a sumoylation inhibitor. This suggests that Pias3 might accelerate the ubiquitination-degradation of vimentin by promoting its sumoylation. ConclusionsThese results indicate that Pias3 might ameliorate atherosclerosis progression by suppressing VSMCs phenotypic switching and reducing vimentin protein stability.

The essential functions of protein SUMOylation in cell proliferation.

The essential functions of protein SUMOylation in cell proliferation.

Monoclonal antibody-based systematic identification of SUMO1-modification sites reveals TFII-I SUMOylation is involved in tumor growth.

SUMOylation plays an essential role in diverse physiological and pathological processes. Identification of wild-type SUMO1-modification sites by mass spectrometry is still challenging. In this study, we produced a monoclonal SUMO1C-K antibody recognizing SUMOylated peptides and proposed an efficient streamline for identification of SUMOylation sites. We identified 471 SUMOylation sites in 325 proteins from five raw data. These identified sites exhibit a high positive rate when evaluated by mutation-verified SUMOylation sites. We identified many SUMOylated proteins involved in mitochondrial metabolism and non-membrane-bounded organelles formation. We proposed a SUMOylation motif, ΨKXD/EP, where proline is required for efficient SUMOylation. We further revealed SUMOylation of TFII-I was stimulated by growth signals and was required for nucleus-localization of p-ERK1/2. Mutation of SUMOylation sites of TFII-I suppressed tumor cell growth in vitro and in vivo. Taken together, we provided a strategy for personalized identification of wild-type SUMO1-modification sites and revealed the physiological significance of TFII-I SUMOylation in this study.

SUMOylation at the inner nuclear membrane facilitates nuclear envelope biogenesis during mitosis.

As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.

ERα promotes SUMO1 transcription by binding with the ERE and enhances SUMO1-mediated protein SUMOylation in breast cancer.

Estrogen plays a crucial role in the tumorigenesis of breast cancer (BC), and epigenetic modification by SUMOylation is essential for cancer development. However, the mechanism underlying estrogen's actions on protein SUMOylation and its effect on BC development are still incompletely understood. SUMO1 in BC cell lines was verified via real-time quantitative PCR (RT-qPCR) and western blot. Cell proliferation and colony formation assays was also performed to evaluate SUMOylation as mediated by SUMO1. Luciferase activity to examine whether E2 promoted the transcription of SUMO1, and chromatin immunoprecipitation (ChIP) assay to determine the binding of estrogen receptor alpha (ERα) to SUMO1 were conduction, and an animal model was used to evaluate the effects of E2-ERα-enhanced SUMO1 transcription. E2 promoted SUMO1 mRNA and protein expression levels in a dose- and time-dependent manner in ER-positive BC cells; it exerted no influence on SUMO2/3 expression; in E2-induced SUMO1 transcription, ERα, but not ERβ, was essential to the process. In addition, E2-ERα upregulated the transcription of SUMO1 by binding with an estrogen-response element half-site (1/2ERE, in the -134 to -123 bp region) of the SUMO1 promoter, and E2-ERα induced SUMO1 transcription-enhanced cellular viability in ER-positive BC cells. To further determine SUMOylation as mediated by SUMO1 in ER-positive BC, we evaluated novel SUMO1 target proteins such as Ras and demonstrated that E2 increased Ras SUMOylation and cellular proliferation by affecting downstream signaling-pathway transduction. Finally, our data revealed that E2-ERα enhanced SUMO1 transcription to promote tumor growth in a BC orthotopic tumor model. Collectively, our results showed that E2 promoted the transcription and protein expression of SUMO1 via ERα binding to a 1/2ERE in the SUMO1 promoter, and that E2-ERα also augmented SUMO1-mediated Ras SUMOylation and mediated cellular responses in ER-positive BC. We therefore achieved significant insights into the mechanism involved in ER-positive BC development and provided a novel target for its treatment.

The Cajal body marker protein coilin is SUMOylated and possesses SUMO E3 ligase-like activity

Cajal bodies (CBs) are subnuclear domains that contribute to the biogenesis of several different classes of ribonucleoproteins (RNPs), including small nuclear RNPs. Only some cell types contain abundant CBs, such as neuronal cells and skeletal muscle, but CBs are invariant features of transformed cells. In contrast, coilin, the CB marker protein, is a ubiquitously expressed nuclear protein, but the function of coilin in cell types that lack CBs is not well understood. We have previously shown that coilin promotes microRNA biogenesis by promoting phosphorylation of DGCR8, a component of the microprocessor. Here, we identify seven additional residues of DGCR8 with decreased phosphorylation upon coilin knockdown. In addition to phosphorylation, the addition of a small ubiquitin-like modifier (SUMO) to DGCR8 also increases its stability. Because of coilin’s role in the promotion of DGCR8 phosphorylation, we investigated whether coilin is involved in DGCR8 SUMOylation. We show that coilin knockdown results in global decrease of protein SUMOylation, including decreased DGCR8 and Sp100 (a PML body client protein) SUMOylation and decreased SMN expression. Alternatively, we found that coilin expression rescued Sp100 SUMOylation and increased DGCR8 and SMN levels in a coilin knockout cell line. Furthermore, we found that coilin facilitates RanGAP1 SUMOylation, interacts directly with components of the SUMOylation machinery (Ubc9 and SUMO2), and, itself, is SUMOylated in vitro and in vivo. In summary, we have identified coilin as a regulator of DGCR8 phosphorylation and a promotor of protein SUMOylation with SUMO E3 ligase-like activity.

Protein SUMOylation: Current updates and insights to elucidate potential roles of SUMO in plants

Protein SUMOylation: Current updates and insights to elucidate potential roles of SUMO in plants

Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic

Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesized through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9∼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9∼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9∼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2∼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterize the resulting mimetic of human UBC9∼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9-SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerization via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9∼SUMO in some cellular contexts.

Deciphering the role of cytoplasmic domain of Channelrhodopsin in modulation of the interactome and SUMOylome of Chlamydomonas reinhardtii

Translocation of channelrhodopsins (ChRs) is mediated by the intraflagellar transport (IFT) machinery. However, the functional role of the network involving photoreceptors, IFT and other proteins in controlling algal ciliary motility is still not fully delineated. In the current study, we have identified two important motifs at the C-terminus of ChR1, VXPX and LKNE. VXPX is a known ciliary targeting sequence in animals, and LKNE is a well-known SUMOylation motif. To the best of our knowledge, this study gives prima facie insight into the role of SUMOylation in Chlamydomonas. We prove that VMPS of ChR1 is important for interaction with GTPase CrARL11. We show that SUMO motifs are present in the C-terminus of putative ChR1s from green algae. Performing experiments with n-Ethylmaleimide (NEM) and Ubiquitin-like protease 1 (ULP-1), we show that SUMOylation may modulate ChR1 protein in Chlamydomonas. Experiments with 2D08, a known sumoylation blocker, increased the concentration of ChR1 protein. Finally, we show the endogenous SUMOylated proteins (SUMOylome) of C. reinhardtii, identified by using immunoprecipitation followed by nano-LC-MS/MS detection. This report establishes a link between evolutionarily conserved SUMOylation and ciliary machinery for the maintenance and functioning of cilia across the eukaryotes. Our enriched SUMOylome of C. reinhardtii comprehends the proteins related to ciliary development and photo-signaling, along with the orthologue(s) associated to human ciliopathies as SUMO targets.

Pervasive SUMOylation of heterochromatin and piRNA pathway proteins

Genome regulation involves complex protein interactions that are often mediated through post-translational modifications (PTMs). SUMOylation-modification by the small ubiquitin-like modifier (SUMO)-has been implicated in numerous essential processes in eukaryotes. In Drosophila, SUMO is required for viability and fertility, with its depletion from ovaries leading to heterochromatin loss and ectopic transposon andgene activation. Here, we developed a proteomics-based strategy to uncover the Drosophila ovarian "SUMOylome," which revealed that SUMOylation is widespread among proteins involved in heterochromatin regulation and different aspects of the Piwi-interacting small RNA (piRNA) pathway that represses transposons. Furthermore, we show that SUMOylation of several piRNA pathway proteins occurs in a Piwi-dependent manner. Together, these data highlight broad implications of protein SUMOylation in epigenetic regulation and indicate novel roles of this modification in the cellular defense against genomic parasites. Finally, this work provides a resource for the study of SUMOylation in other biological contexts in the Drosophila model.

SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA.

Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA.

Paradoxes of Cellular SUMOylation Regulation: A Role of Biomolecular Condensates?

Protein SUMOylation is a major post-translational modification essential for maintaining cellular homeostasis. SUMOylation has long been associated with stress responses as a diverse array of cellular stress signals are known to trigger rapid alternations in global protein SUMOylation. In addition, while there are large families of ubiquitination enzymes, all small ubiquitin-like modifiers (SUMOs) are conjugated by a set of enzymatic machinery comprising one heterodimeric SUMO-activating enzyme, a single SUMO-conjugating enzyme, and a small number of SUMO protein ligases and SUMO-specific proteases. How a few SUMOylation enzymes specifically modify thousands of functional targets in response to diverse cellular stresses remains an enigma. Here we review recent progress toward understanding the mechanisms of SUMO regulation, particularly the potential roles of liquid-liquid phase separation/biomolecular condensates in regulating cellular SUMOylation during cellular stresses. In addition, we discuss the role of protein SUMOylation in pathogenesis and the development of novel therapeutics targeting SUMOylation. SIGNIFICANCE STATEMENT: Protein SUMOylation is one of the most prevalent post-translational modifications and plays a vital role in maintaining cellular homeostasis in response to stresses. Protein SUMOylation has been implicated in human pathogenesis, such as cancer, cardiovascular diseases, neurodegeneration, and infection. After more than a quarter century of extensive research, intriguing enigmas remain regarding the mechanism of cellular SUMOylation regulation and the therapeutic potential of targeting SUMOylation.