SUMOylation Research Articles

Solution structure of human SUMO2

Solution structure of human SUMO2

Target and resistance-related proteins of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand on myeloma cell lines.

Recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) has become a potential therapeutic drug for multiple myeloma (MM). However, the exact targets and resistance mechanisms of rmhTRAIL on MM cells remain to be elucidated. The present study aimed to investigate the target and resistance-related proteins of rmhTRAIL on myeloma cell lines. A TRAIL-sensitive myeloma cell line, RPMI 8226, and a TRAIL-resistance one, U266, were chosen and the differentially expressed proteins between the two cell lines were analyzed prior and subsequent to rmhTRAIL administration by a liquid chromatography-tandem mass spectrometry method. The results showed that following TRAIL treatment, 6 apoptosis-related proteins, calpain small subunit 1 (CPNS1), peflin (PEF1), B-cell receptor-associated protein 31 (BAP31), apoptosis-associated speck-like protein containing CARD (ASC), BAG family molecular chaperone regulator 2 (BAG2) and chromobox protein homolog 3 (CBX3), were upregulated in RPMI 8226 cells while no change was identified in the U266 cells. Furthermore, small ubiquitin-related modifier 1 and several other ubiquitin proteasome pathway (UPP)-related proteins expressed higher levels in TRAIL-resistant cells U266 compared to the RPMI-8226 cells prior and subsequent to rmhTRAIL treatment. These results suggested that CPNS1, PEF1, BAP31, ASC, BAG2 and CBX3 were possibly target proteins of rmhTRAIL on RPMI 8226 cells, while UPP may have a vital role in mediating TRAIL-resistance in U266 cells.

Expression and purification of human epidermal growth factor (hEGF) fused with GB1

ABSTRACTFusion expression is a promising strategy for the production of bioactive peptides in Escherichia coli. In this study, we constructed a new recombinant expression plasmid containing the coding sequence of 56-residue B1 domain of streptococcal protein G (GB1). For easy purification and cleavage of the recombinant proteins, except GB1, an engineered hexahistidine and tobacco etch virus (TEV) protease recognition sites were included in the fusion sequence. Next, we cloned the coding sequence of human epidermal growth factor (hEGF) into this new plasmid and produced the recombinant hEGF in E. coli. The bioactive hEGF is a 53-amino acid peptide and is stabilized by three intramolecular disulphide bonds. Compared with glutathione S-transferase, thioredoxin and small ubiquitin-related modifier, GB1 greatly improved the expression and solubility of hEGF. Moreover, the recombinant hEGF bound to the nickel nitrilotriacetic acid resin column, was easily cleaved by TEV protease and the free hEGF was released. The results showed that this new plasmid was appropriate for recombinant production of small bioactive peptides, such as hEGF, which contains a high proportion of hydrophobic residues and intramolecular disulphide linkages.

IL-6 induced proliferation and cytotoxic activity of CD8(+) T cells is elevated by SUMO2 overexpression.

T cells play an important role in adaptive immune responses that destroy pathogens or infected cells. Therefore, regulation of T cell activity is important in various diseases, such as autoimmune diseases, hypersensitivity, and cancer. The conjugation of small ubiquitin-related modifier (SUMO) is a post-translational protein modification that regulates activity, stability, and subcellular translocation of target proteins. In this study, CD8(+) T cells overexpressing SUMO2 showed greater proliferation and cytotoxic activity against tumor cells in the presence of IL-6 than wild-type CD8(+) T cells in vitro. These CD8(+) T cell functions were suppressed during treatment with MEK1 or PI3K-specific inhibitors. Therefore, our findings suggest that IL-6-derived signaling pathways, including the MEK1 and PI3K pathways, are upregulated by SUMO2 overexpression. However, transgenic expression of SUMO2 in T cells did not modulate Th1/2 balance. Collectively, our results showed that SUMO2-Tg promotes cytotoxic activity against tumor cells by increasing the proliferation and cytotoxicity of CD8(+) T cells.

SUMO4 163 G>A variation is associated with kidney disease in Indian subjects with type 2 diabetes.

Genetic susceptibility probably plays a role in the development and/or progression of diabetic kidney disease. Small ubiquitin-related modifier 4 (SUMO4) mRNA is expressed in human kidney. Substitution of methionine with valine at codon 55 (M55V) of SUMO4 gene induces higher nuclear factor-kB activity, which is known to mediate the development of kidney disease in individuals with diabetes. We investigated the association between the SUMO4 M55V (rs237025, c.163 G>A) and kidney disease in north Indian subjects with diabetes. A case-control analysis was performed using genomic DNA samples from 216 diabetic patients without nephropathy (DM) and 201 diabetic with nephropathy (DN). The SUMO4 c.163 G>A polymorphism was genotyped using polymerase chain reaction amplification followed by restriction digestion. The duration of diabetes was significantly greater in DN. The genotypic and allelic frequencies were different in DM and DN groups: GG genotype was significantly more frequent in DN as compared to DM (p = 0.018, OR 1.72, 95% CI 1.1-2.7). Similarly the G allele was more frequent in DN compared to DM (p = 0.017, OR 1.4, 95% CI 1.1-1.8). This study suggests that SUMO4 c.163 G>A polymorphism is associated with the susceptibility to diabetic nephropathy in north Indian subjects with type 2 diabetes.

Development and evaluation of a highly reliable assay for SUMO-specific protease inhibitors

SUMOylation, as a post-translational modification of proteins, plays essential regulatory roles in a variety of pathological conditions. In the dynamic process of SUMOylation and deSUMOylation, SENPs (SUMO-specific proteases), in charge of deconjugation of SUMO (small ubiquitin-related modifier) from substrate proteins, have recently been found to be potential therapeutic targets for cancer treatment. A reliable and practical assay is much needed to accelerate the discovery of SENPs inhibitors. We established a quantitative assay based on readily available SDS–PAGE-Coomassie system using RanGAP-SUMO as the substrate, thus avoiding the use of expensive fluorescent dyes or the error-prone fluorescent reporter. Its reproducibility and reliability were also evaluated in this report.

Polymorphisms of small ubiquitin-related modifier genes are associated with risk of Alzheimer's disease in Korean: A case-control study

Sumoylation regulates transcription factor transactivation, protein-protein interactions, and appropriate subcellular localization of certain proteins. Previous studies have shown that sumoylation of amyloid precursor protein (APP) is associated with decreased levels of amyloid beta (Aβ) proteins, suggesting that sumoylation may play a role in the pathogenesis of Alzheimer's disease (AD). We investigated the association between polymorphisms of the SUMO genes and the risk of AD. Our study subjects consisted of 144 AD patients and 335 healthy controls without dementia. We focused on tagged single nucleotide polymorphisms (tagSNPs) of the SUMO1 and SUMO2 genes. The tagSNPs were amplified by PCR and sequenced. We used binary logistic regression to calculate odds ratios (ORs) with 95% confidence intervals (CIs) for the associations between SUMO gene polymorphisms and the risk of AD. We found that rs12472035 polymorphism of SUMO1 was significantly associated with an increased risk of AD in male group (the CT genotype of rs12472035: adjusted OR=8.737, 95% CI=2.041–37.41, p-value=0.003). In addition, two polymorphisms of SUMO2 were significantly associated with an increased risk of AD in female group (the GA genotype of rs35271045: adjusted OR=2.879, 95% CI=1.399–5.924, p-value=0.004; and the TC genotype of rs9913676: adjusted OR=2.460, 95% CI=1.197–5.057, p-value=0.014). Furthermore, three combinations were associated with an increased risk of AD. Our data suggest that three individual polymorphisms and three combinations may be potential risk factors for AD in Korean population.

Functional analysis of small ubiquitin-modifier (SUMO) in orange-spotted grouper (Epinephelus coioides)

Small ubiquitin-like modifiers (SUMOs), structurally similar to ubiquitin, ligated to lysine residues within sumoylation target proteins. Sumoylation and ubiquitination exhibit similar biological processes for post-translational modification regulation, apoptosis and protein stability which was participated in a number of cellular processes such as nuclear transport, transcriptional regulation, apoptosis and protein stability. In the present study, the cDNA of orange-spotted grouper SUMO, terms osgSUMO, was cloned by the combination of homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approaches. The SUMO1 and SUMO2-yellow fluorescent protein (YFP) were distributed in the cytoplasm. Our results show that SUMO1 was capable of decreasing coat protein (CP) of nucleus localization ratio, not SUMO2. Thus, we demonstrate here that SUMO1 was able to interact with CP by using analysis of fluorescence resonance energy transfer (FRET). Taken together, grouper SUMO can partially influence on localization of CP.

Small ubiquitin-related modifier 1 is involved in hepatocellular carcinoma progression via mediating p65 nuclear translocation.

Small ubiquitin-related modifier (SUMO) proteins participate in a post-translational modification called SUMOylation and regulate a variety of intracellular processes, such as targeting proteins for nuclear import. The nuclear transport of p65 results in the activation of NF-κB, and p65 contains several SUMO interacting motifs (SIMs). However, the relationship between p65 and SUMO1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we demonstrated the potential roles of SUMO1 in HCC via the regulation of p65 subcellular localization. We found that either SUMO1- or p65-positive immunoreactivity was remarkably increased in the nuclei of tumor tissues in HCC patients compared with non-tumor tissues, and further analysis suggested a correlation between SUMO1- and nuclear p65-positive immunoreactivities (R = 0.851, P = 0.002). We also verified the interaction between p65 and SUMO1 in HCC by co-immunoprecipitation. TNF-α and hypoxia increased SUMO1 protein levels and enhanced SUMO1-modified p65 SUMOylation. Moreover, the knockdown of SUMO1 decreased p65 nuclear translocation and inhibited NF-κB transcriptional activity. Further the results of this study revealed that the knockdown of SUMO1 suppressed the proliferation and migration of hepatoma cells. These results suggest that SUMO1 contributes to HCC progression by promoting p65 nuclear translocation and regulating NF-κB activity.

A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein.

Ubiquitin-like protease 1 (Ulp1) of Saccharomyces cerevisiae emerges as a fundamental tool to obtain the natural N-terminal target protein by cleavage of the small ubiquitin-related modifier (SUMO) fusion protein. However, the costly commercial Ulp1 and its complicated procedures limit its application in the preparation of the target protein with natural N-terminal sequence. Here, we describe the preparation of bioactive codon-optimized recombinant truncated Ulp1 (Leu403-Lys621) (rtUlp1) of S. cerevisiae in Escherichia coli using only one-step with Ni–NTA affinity chromatograph, and the application of rtUlp1 to cleave the SUMO fusion protein by simply mixing the purified rtUlp1, SUMO fusion protein and DL-Dithiothreitol in Tris–HCl buffer. The optimal expression level of non-fusion protein rtUlp1 accounts for approximately 50 % of the total cellular protein and 36 % of the soluble form by addition of isopropyl β-D-l-thiogalactopyranoside at a final concentration of 0.4 mM at 18 °C for 20 h. The purification of target protein rtUlp1 was conducted by Ni–NTA affinity chromatography. The final yield of rtUlp1 was 45 mg/l in flask fermentation with a purity up to 95 %. Furthermore, the high purity of rtUlp1 could effectively cleave the SUMO-tTβRII fusion protein (SUMO gene fused to truncated transforming growth factor-beta receptor type II gene) with the above simplified approach, and the specific activity of the rtUlp1 reached up to 2.8 × 104 U/mg, which is comparable to the commercial Ulp1. The preparation and application strategy of the rtUlp1 with commonly available laboratory resources in this study will be convenient to the cleavage of the SUMO fusion protein to obtain the natural N-terminal target protein, which can be implemented in difficult-to-express protein functional analysis.

Inhibition of protein SUMOylation by natural quinones

Conjugation of small ubiquitin-related modifier (SUMO) to target proteins (SUMOylation) is a posttranslational modification that regulates a wide range of biological processes, including transcription, intracellular transport, DNA repair, DNA replication and cell signaling.1 SUMOylation is catalyzed by a multi-step enzymatic cascade that resembles ubiquitination: the process involves an E1-activating enzyme (a heterodimer consisting of Aos1 and Uba2), an E2-conjugating enzyme (Ubc9) and various E3 ligases.1 E3s catalyze SUMO conjugation to target proteins and may contribute to substrate specificity. SUMO is subsequently cleaved from target proteins by SUMO-specific isopeptidases. Because SUMO modifications have been implicated in a variety of disorders such as cancers2 and neurodegenerative diseases,3 the enzymes involved in SUMOylation are promising targets for therapeutic agents.

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.

USP7 is a SUMO deubiquitinase essential for DNA replication.

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents.

Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif

Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

A rapid solubility-optimized screening procedure for recombinant subtilisins in E. coli

Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic expression in E. coli, and a casein-based functional screen. The procedure is plate-format compatible and can be completed in only four days, starting from the gene of interest in a suitable cloning vector. The expression vector suite includes six vectors with combinations of maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) for increased solubility, and polyhistidine tags for downstream purification. We used enhanced green fluorescent protein and four Bacilli subtilisins to validate the screening procedure and our results show that proteins were expressed, soluble and active. Interestingly, the highest activities were consistently achieved with either MBP or SUMO fusions, thus demonstrating the merit of including solubility tags. In conclusion, the results demonstrate that our approach can be used to efficiently screen for new subtilisins, and suggest that the approach may also be used to screen for proteins with other activities.

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.

Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems

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

In vitro assay to determine SUMOylation sites on protein substrates.

Protein SUMOylation regulates the activity of a wide range of cellular substrates, and the identification of small ubiquitin-related modifier (SUMO)-modified sites is often required to understand how this modification affects protein function. However, the site-specific identification of modified lysine residues by mass spectrometry (MS) remains challenging because of the dynamic nature of this modification, its low stoichiometry and the relatively large SUMO remnant left on peptide backbones after tryptic digestion. Here we report a versatile method to identify sites and to profile the extent of modification on recombinant proteins from in vitro SUMOylation assays. We define the steps required for sample preparation, and we describe how to perform proper controls and conduct the liquid chromatography-MS (LC-MS) and bioinformatics analyses. Native protein substrates can be used for the assay, although we recommend the use of His-tagged proteins to facilitate removal of contaminants. The procedure was developed for human SUMO paralogs, and it requires <2 d for completion.

SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus in Saccharomyces cerevisiae.

In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2Δ and ulp2Δ slx5Δ cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2Δ,slx5Δ, and ulp2Δ slx5Δ cells. The increase in sumoylation of Net1 and Tof2 in ulp2Δ, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2 Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2Δ cells and both factors largely regain this association in ulp2Δ slx5Δ A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2Δ cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2Δ cells, which is also rescued by elimination of Slx5 Additionally, overexpression of Sir2, another RFB-associated factor, suppresses the growth defect of ulp2Δ cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2Δ cellular defects.

High-level expression of prolyl endopeptidase in Pichia pastoris using PLA2 as a fusion partner

In our previous studies, a prolyl endopeptidase (PEP) gene from Aspergillus oryzae (MOH) was cloned and expressed in Pichia pastoris; however, the recombinant protein expression level of MOH was very low. In the present study, the PEP expression level was successfully improved by constructing fusion expression proteins with four fusion partners, namely, Streptomyces violaceoruber Phospholipase A2 (PLA2), cellulose-binding domain (CBD), small ubiquitin-related modifier (SUMO) and maltose binding protein (MBP). The enzyme activities of the recombinant fusion proteins CLMH, SLMH, MLMH and PLMH were increased to 3.8-, 2.7-, 4.9- and 7.4-fold compared with that of the parent MOH. Moreover, the extracellular protein content of CLMH, SLMH, MLMH, PLMH were 1.42-, 1.25-, 1.67- and 1.83-fold higher compared with that of MOH. Both PLMH and MOH showed the highest activity at pH 5.5, the highest stability at pH 6.0 and maximal activity at 40°C.