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.

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.

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.

The SUMO components in rheumatoid arthritis.

Small ubiquitin-like modifier (SUMO) proteins can reversibly attach covalently or non-covalently to lysine residues of various substrates. The processes are named SUMOylation and de-SUMOylation, which maintain a dynamic balance in the physiological state, and are regulated by SUMO components. However, the dysregulation of components disturbs the balance and alters the functions of target proteins, which causes the occurrence of diseases. To date, certain SUMO components, including SUMO-1, SUMO-2/3, SAE1/Uba2, Ubc9, PIASs (protein inhibitors of activated signal transducer and activator of transcription) and SENPs (SUMO-specific proteases), have been found to participate in the pathogenesis of RA and their potential value as therapeutic targets also have been highlighted. In addition, single nucleotide polymorphisms (SNPs) in the SUMO components have been reported to be associated with disease susceptibility. Until now, only the SNP site of SUMO-4 has been reported in RA. Here we provided a systematic overview of the general characteristics of SUMO components and highlighted a summary of their impact on RA.

SUMOylation regulates the number and size of promyelocytic leukemia-nuclear bodies (PML-NBs) and arsenic perturbs SUMO dynamics on PML by insolubilizing PML in THP-1 cells.

The functional roles of protein modification by small ubiquitin-like modifier (SUMO) proteins are not well understood compared to ubiquitination. Promyelocytic leukemia (PML) proteins are good substrates for SUMOylation, and PML-nuclear bodies (PML-NBs) may function as a platform for the PML SUMOylation. PML proteins are rapidly modified both with SUMO2/3 and SUMO1 after exposure to arsenite (As3+) and SUMOylated PML are further ubiquitinated and degraded by proteasomes. However, effects of As3+ on SUMO dynamics on PML-NBs are not well investigated. In the present study, we report that (1) the number and size of PML-NBs were regulated by SUMO E1-activating enzyme, (2) SUMO2/3 co-localized with PML irrespective of As3+ exposure and was restricted to PML-nuclear bodies (PML-NBs) via covalent binding in response to As3+, and (3) As3+-induced biochemical changes in PML were not modulated by ubiquitin-proteasome system (UPS) in THP-1 cells. Undifferentiated and differentiated THP-1 cells responded to As3+ similarly and PML proteins were changed from the detergent soluble to the insoluble form and further SUMOylated with SUMO2/3 and SUMO1. ML792, a SUMO E1 inhibitor, decreased the number of PML-NBs and reciprocally increased the size irrespective of exposure to As3+, which itself slightly decrease both the number and size of PML-NBs. TAK243, a ubiquitin E1 inhibitor, did not change the PML-NBs, while SUMOylated proteins accumulated in the TAK243-exposed cells. Proteasome inhibitors did not change the As3+-induced SUMOylation levels of PML. Co-localization and further restriction of SUMO2/3 to PML-NBs were confirmed by PML-transfected CHO-K1 cells. Collectively, SUMOylation regulates PML-NBs and As3+ restricts SUMO dynamics on PML by changing its solubility.

Carrot mottle virus ORF4 movement protein targets plasmodesmata by interacting with the host cell SUMOylation system.

Plant virus movement proteins (MPs) facilitate virus spread in their plant hosts, and some of them are known to target plasmodesmata (PD). However, how the MPs target PD is still largely unknown. Carrot mottle virus (CMoV) encodes the ORF3 and ORF4 proteins, which are involved in CMoV movement. In this study, we used CMoV as a model to study the PD targeting of a plant virus MP. We showed that the CMoV ORF4 protein, but not the ORF3 protein, modified PD and led to the virus movement. We found that the CMoV ORF4 protein interacts with the host cell small ubiquitin-like modifier (SUMO) 1, 2 and the SUMO-conjugating enzyme SCE1, resulting in the ORF4 protein SUMOylation. Downregulation of mRNAs for NbSCE1 and NbSUMO impaired CMoV infection. The SUMO-interacting motifs (SIMs) LVIVF, VIWV, and a lysine residue at position 78 (K78) are required for the ORF4 protein SUMOylation. The mutation of these motifs prevented the protein to efficiently target PD, and further slowed or completely abolished CMoV systemic movement. Finally, we found that some of these motifs are highly conserved among umbraviruses. Our data suggest that the CMoV ORF4 protein targets PD by interacting with the host cell SUMOylation system.

Enhanced Live-Cell Delivery of Synthetic Proteins Assisted by Cell-Penetrating Peptides Fused to DABCYL.

Live-cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell-penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca-arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and a Ub variant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.

Replication protein A (RPA) sumoylation positively influences the DNA damage checkpoint response in yeast.

The DNA damage response relies on protein modifications to elicit physiological changes required for coping with genotoxic conditions. Besides canonical DNA damage checkpoint-mediated phosphorylation, DNA damage-induced sumoylation has recently been shown to promote genotoxin survival. Cross-talk between these two pathways exists in both yeast and human cells. In particular, sumoylation is required for optimal checkpoint function, but the underlying mechanisms are not well-understood. To address this question, we examined the sumoylation of the first responder to DNA lesions, the ssDNA-binding protein complex replication protein A (RPA) in budding yeast (Saccharomyces cerevisiae). We delineated the sumoylation sites of the RPA large subunit, Rfa1 on the basis of previous and new mapping data. Findings using a sumoylation-defective Rfa1 mutant suggested that Rfa1 sumoylation acts in parallel with the 9-1-1 checkpoint complex to enhance the DNA damage checkpoint response. Mechanistically, sumoylated Rfa1 fostered an interaction with a checkpoint adaptor protein, Sgs1, and contributed to checkpoint kinase activation. Our results suggest that SUMO-based modulation of a DNA damage sensor positively influences the checkpoint response.

Inhibiting ubiquitination causes an accumulation of SUMOylated newly synthesized nuclear proteins at PML bodies.

Protein ubiquitination and SUMOylation are required for the maintenance of cellular protein homeostasis, and both increase in proteotoxic conditions (e.g. heat shock or proteasome inhibition). However, we found that when ubiquitination was blocked in several human cell lines by inhibiting the ubiquitin-activating enzyme with TAK243, there was an unexpected, large accumulation of proteins modified by SUMO2/3 chains or SUMO1, but not by several other ubiquitin-like proteins. This buildup of SUMOylated proteins was evident within 3-4 h. It required the small ubiquitin-like modifier (SUMO)-conjugating enzyme, UBC9, and the promyelocytic leukemia protein (PML) and thus was not due to nonspecific SUMO conjugation by ubiquitination enzymes. The SUMOylated proteins accumulated predominantly bound to chromatin and were localized to PML nuclear bodies. Because blocking protein synthesis with cycloheximide prevented the buildup of SUMOylated proteins, they appeared to be newly-synthesized proteins. The proteins SUMOylated after inhibition of ubiquitination were purified and analyzed by MS. In HeLa and U2OS cells, there was a cycloheximide-sensitive increase in a similar set of SUMOylated proteins (including transcription factors and proteins involved in DNA damage repair). Surprisingly, the inhibition of ubiquitination also caused a cycloheximide-sensitive decrease in a distinct set of SUMOylated proteins (including proteins for chromosome modification and mRNA splicing). More than 80% of the SUMOylated proteins whose levels rose or fell upon inhibiting ubiquitination inhibition underwent similar cycloheximide-sensitive increases or decreases upon proteasome inhibition. Thus, when nuclear substrates of the ubiquitin-proteasome pathway are not efficiently degraded, many become SUMO-modified and accumulate in PML bodies.

Desumoylation of RNA polymerase III lies at the core of the Sumo stress response in yeast.

Post-translational modification by small ubiquitin-like modifier (Sumo) regulates many cellular processes, including the adaptive response to various types of stress, referred to as the Sumo stress response (SSR). However, it remains unclear whether the SSR involves a common set of core proteins regardless of the type of stress or whether each particular type of stress induces a stress-specific SSR that targets a unique, largely nonoverlapping set of Sumo substrates. In this study, we used MS and a Gene Ontology approach to identify differentially sumoylated proteins during heat stress, hyperosmotic stress, oxidative stress, nitrogen starvation, and DNA alkylation in Saccharomyces cerevisiae cells. Our results indicate that each stress triggers a specific SSR signature centered on proteins involved in transcription, translation, and chromatin regulation. Strikingly, whereas the various stress-specific SSRs were largely nonoverlapping, all types of stress tested here resulted in desumoylation of subunits of RNA polymerase III, which correlated with a decrease in tRNA synthesis. We conclude that desumoylation and subsequent inhibition of RNA polymerase III constitutes the core of all stress-specific SSRs in yeast.

A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies.

The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403-621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416-621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying l-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M l-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

Failed mitochondrial import and impaired proteostasis trigger SUMOylation of mitochondrial proteins.

Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins.

Discovery and engineering of enhanced SUMO protease enzymes.

Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.

The SUMO protease SENP1 and the chromatin remodeler CHD3 interact and jointly affect chromatin accessibility and gene expression.

The small ubiquitin-like modifier (SUMO) post-translationally modifies lysine residues of transcription factors and co-regulators and thereby contributes to an important layer of control of the activities of these transcriptional regulators. Likewise, deSUMOylation of these factors by the sentrin-specific proteases (SENPs) also plays a role in gene regulation, but whether SENPs functionally interact with other regulatory factors that control gene expression is unclear. In the present work, we focused on SENP1, specifically, on its role in activation of gene expression investigated through analysis of the SENP1 interactome, which revealed that SENP1 physically interacts with the chromatin remodeler chromodomain helicase DNA-binding protein 3 (CHD3). Using several additional methods, including GST pulldown and co-immunoprecipitation assays, we validated and mapped this interaction, and using CRISPR-Cas9-generated CHD3- and SENP1-KO cells (in the haploid HAP1 cell line), we investigated whether these two proteins are functionally linked in regulating chromatin remodeling and gene expression. Genome-wide ATAC-Seq analysis of the CHD3- and SENP1-KO cells revealed a large degree of overlap in differential chromatin openness between these two mutant cell lines. Moreover, motif analysis and comparison with ChIP-Seq profiles in K562 cells pointed to an association of CHD3 and SENP1 with CCCTC-binding factor (CTCF) and SUMOylated chromatin-associated factors. Lastly, genome-wide RNA-Seq also indicated that these two proteins co-regulate the expression of several genes. We propose that the functional link between chromatin remodeling by CHD3 and deSUMOylation by SENP1 uncovered here provides another level of control of gene expression.

Nucleolar and spindle-associated protein 1 (NUSAP1) interacts with a SUMO E3 ligase complex during chromosome segregation.

The mitotic spindle is composed of dynamic microtubules and associated proteins that together direct chromosome movement during mitosis. The spindle plays a vital role in accurate chromosome segregation fidelity and is a therapeutic target in cancer. Nevertheless, the molecular mechanisms by which many spindle-associated proteins function remains unknown. The nucleolar and spindle-associated protein NUSAP1 is a microtubule-binding protein implicated in spindle stability and chromosome segregation. We show here that NUSAP1 localizes to dynamic spindle microtubules in a unique chromosome-centric pattern, in the vicinity of overlapping microtubules, during metaphase and anaphase of mitosis. Mass spectrometry-based analysis of endogenous NUSAP1 interacting proteins uncovered a cell cycle-regulated interaction between the RanBP2-RanGAP1-UBC9 SUMO E3 ligase complex and NUSAP1. Like NUSAP1 depletion, RanBP2 depletion impaired the response of cells to the microtubule poison Taxol. NUSAP1 contains a conserved SAP domain (SAF-A/B, Acinus, and PIAS). SAP domains are common among many other SUMO E3s, and are implicated in substrate recognition and ligase activity. We speculate that NUSAP1 contributes to accurate chromosome segregation by acting as a co-factor for RanBP2-RanGAP1-UBC9 during cell division.

Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation.

Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.

Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy.

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase.

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.