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.

Chemical synthesis of on demand-activated SUMO-based probe by a photocaged glycine-assisted strategy.

The transiently-activated SUMO probes are conducive to understand the dynamic control of SENPs activity. Here, we developed a photocaged glycine-assisted strategy for the construction of on demand-activated SUMO-ABPs. The light-sensitive groups installed at G92 and G64 backbone of SUMO-2 can temporarily block probes activity and hamper aspartimide formation, respectively, which enabled the efficient synthesis of inert SUMO-2 propargylamide (PA). The probe could be activated to capture SENPs upon photo-irradiation not only in vitro but also in intact cells, providing opportunities to further perform intracellular time-resolved proteome-wide profiling of SUMO-related enzymes.

The SMC5/6 complex subunit MMS21 regulates stem cell proliferation in rice.

KEY MESSAGE: SMC5/6 complex subunit OsMMS21 is involved in cell cycle and hormone signaling and required for stem cell proliferation during shoot and root development in rice. The structural maintenance of chromosome (SMC)5/6 complex is required for nucleolar integrity and DNA metabolism. Moreover, METHYL METHANESULFONATE SENSITIVITY GENE 21 (MMS21), a SUMO E3 ligase that is part of the SMC5/6 complex, is essential for the root stem cell niche and cell cycle transition in Arabidopsis. However, its specific role in rice remains unclear. Here, OsSMC5 and OsSMC6 single heterozygous mutants were generated using CRISPR/Cas9 technology to elucidate the function of SMC5/6 subunits, including OsSMC5, OsSMC6, and OsMMS21, in cell proliferation in rice. ossmc5/ + and ossmc6/ + heterozygous single mutants did not yield homozygous mutants in their progeny, indicating that OsSMC5 and OsSMC6 both play necessary roles during embryo formation. Loss of OsMMS21 caused severe defects in both the shoot and roots in rice. Transcriptome analysis showed a significant decrease in the expression of genes involved in auxin signaling in the roots of osmms21 mutants. Moreover, the expression levels of the cycB2-1 and MCM genes, which are involved the cell cycle, were significantly lower in the shoots of the mutants, indicating that OsMMS21 was involved in both hormone signaling pathways and the cell cycle. Overall, these findings indicate that the SUMO E3 ligase OsMMS21 is required for both shoot and root stem cell niches, improving the understanding of the function of the SMC5/6 complex in rice.

An "up" oriented methionine-aromatic structural motif in SUMO is critical for its stability and activity.

Protein structural bioinformatic analyses suggest preferential associations between methionine and aromatic amino acid residues in proteins. Ab initio energy calculations highlight a conformation-dependent stabilizing interaction between the interacting sulfur-aromatic molecular pair. However, the relevance of buried methionine-aromatic motifs to protein folding and function is relatively unexplored. The Small Ubiquitin-Like Modifier (SUMO) is a ß-grasp fold protein and a common posttranslational modifier that affects diverse cellular processes, including transcriptional regulation, chromatin remodeling, metabolic regulation, mitosis, and meiosis. SUMO is a member of the Ubiquitin-Like (UBL) protein family. Herein, we report that a highly conserved and buried methionine-phenylalanine motif is a unique signature of SUMO proteins but absent in other homologous UBL proteins. We also detect that a specific "up" conformation between the methionine-phenylalanine pair of interacting residues in SUMO is critical to its ß-grasp fold. The noncovalent interactions of SUMO with its ligands are dependent on the methionine-phenylalanine pair. MD simulations, NMR, and biophysical and biochemical studies suggest that perturbation of the methionine-aromatic motif disrupts native contacts, modulates noncovalent interactions, and attenuates SUMOylation activity. Our results highlight the importance of conserved orientations of Met-aromatic structural motifs inside a protein core for its structure and function.

DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex.

Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6-Nse2 molecules engaged on DNA.

High level expression and purification of recombinant 3ABC non-structural protein of foot-and-mouth disease virus using SUMO fusion system.

The detection of antibody to non-structural protein (NSP) of Foot-and-mouth disease virus (FMDV) is the reliable diagnostic method for differentiating infected from vaccinated animals (DIVA). For this purpose, the detection of antibodies to non-structural 3ABC protein is suitable for identification of virus activity in the animals exposed to FMDV infection. However, large-scale production of recombinant 3ABC protein is challenging due to the formation of inclusion bodies in Escherichia coli and low yield due to protein aggregation during in vitro refolding. In this study, 3ABC gene was fused with SUMO (small ubiquitin-like modifiers) fusion system which significantly enhanced expression of recombinant 3ABC protein in E. coli. The solubility of the recombinant 6xHis-SUMO 3ABC fusion protein was improved by mild detergent treatment and purified through Ni-NTA chromatography under non-denaturing conditions which yielded 9 mg protein obtained from 1-L bacterial fermentation culture. The diagnostic potential of recombinant 3ABC protein was also tested by ELISA that provided reliable diagnostic performance (DSn = 92%, DSp = 94%) upon comparison with commercially available kit. The thermal stability of fusion protein was also tested which presented reliable performance at different temperatures. In conclusion, we presented SUMO fusion for the enhanced expression in E. coli and purification of active recombinant 3ABC protein using non-denaturing conditions without refolding steps. This protein can be used as a suitable diagnostic antigen to detect antibodies following FMDV infection.

The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition.

Proteins of the small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair. Recently, numerous insights into regulatory mechanisms of the SUMO modification pathway have emerged. Although SUMO-conjugating enzymes can discriminate between SUMO targets, many substrates possess characteristics that facilitate their modification. Other post-translational modifications also regulate SUMO conjugation, suggesting that SUMO signalling is integrated with other signal transduction pathways. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analysing the function of SUMO and substrate conjugation in distinct cellular pathways.

A conserved and buried edge-to-face aromatic interaction in small ubiquitin-like modifier (SUMO) has a role in SUMO stability and function.

Aromatic amino acids buried at a protein's core are often involved in mutual paired interactions. Ab initio energy calculations have highlighted that the conformational orientations and the effects of substitutions are important for stable aromatic interactions among aromatic rings, but studies in the context of a protein's fold and function are elusive. Small ubiquitin-like modifier (SUMO) is a common post-translational modifier that affects diverse cellular processes. Here, we report that a highly conserved aromatic triad of three amino acids, Phe36-Tyr51-Phe64, is a unique SUMO signature that is absent in other ubiquitin-like homologous folds. We found that a specific edge-to-face conformation between the Tyr51-Phe64 pair of interacting aromatics is vital to the fold and stability of SUMO. Moreover, the noncovalent binding of SUMO-interacting motif (SIM) at the SUMO surface was critically dependent on the paired aromatic interactions buried at the core. NMR structural studies revealed that perturbation of the Tyr51-Phe64 conformation disrupts several long-range tertiary contacts in SUMO, leading to a heterogeneous and dynamic protein with attenuated SUMOylation both in vitro and in cells. A subtle perturbation of the edge-to-face conformation by a Tyr to Phe substitution significantly decreased stability, SUMO/SIM affinity, and the rate of SUMOylation. Our results highlight that absolute co-conservation of specific aromatic pairs inside the SUMO protein core has a role in its stability and function.

Casein kinase-2-mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2.

Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.

Experimental comparison of energy landscape features of ubiquitin family proteins.

Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. Here, we used chemical denaturation in comparing the folding-unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W-pseudo wild-type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant-dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two-state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow-down their folding rates. The denaturant-dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent-accessible surface area, as quantified by the Tanford ß u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding-unfolding rates within a structurally homologous protein family.

Amide temperature coefficients in characterizing the allosteric effects of ligand binding on local stability in proteins.

Proteins can stabilize upon binding a ligand. Due to allosteric effects, the changes in stability can occur at regions far from the protein:ligand interface. Efficient methods to measure the changes in local stability upon ligand binding will be useful to understand allostery and may be helpful in protein engineering. In this work, we suggest the measurement of backbone amide temperature coefficients to probe the effect of ligand binding on the local stability of ß-sheet rich proteins. The method was applied for two protein:ligand complexes with different binding affinities. The protein includes a beta-sheet network connected by hydrogen bonds. The measured temperature coefficient data captured the stabilizing effect of ligand binding, which propagated across the beta-sheet network of the protein. Intriguingly, the impact on the local and global stability of the protein was proportional to the strength of protein:ligand interaction.

Regulation of the Ebola Virus VP24 Protein by SUMO.

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation.

Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.

The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase.

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.

Role of the CBP catalytic core in intramolecular SUMOylation and control of histone H3 acetylation.

The histone acetyl transferases CREB-binding protein (CBP) and its paralog p300 play a critical role in numerous cellular processes. Dysregulation of their catalytic activity is associated with several human diseases. Previous work has elucidated the regulatory mechanisms of p300 acetyltransferase activity, but it is not known whether CBP activity is controlled similarly. Here, we present the crystal structure of the CBP catalytic core encompassing the bromodomain (BRD), CH2 (comprising PHD and RING), HAT, and ZZ domains at 2.4-Å resolution. The BRD, PHD, and HAT domains form an integral structural unit to which the RING and ZZ domains are flexibly attached. The structure of the apo-CBP HAT domain is similar to that of acyl-CoA-bound p300 HAT complexes and shows that the acetyl-CoA binding site is stably formed in the absence of cofactor. The BRD, PHD, and ZZ domains interact with small ubiquitin-like modifier 1 (SUMO-1) and Ubc9, and function as an intramolecular E3 ligase for SUMOylation of the cell cycle regulatory domain 1 (CRD1) of CBP, which is located adjacent to the BRD. In vitro HAT assays suggest that the RING domain, the autoregulatory loop (AL) within the HAT domain, and the ZZ domain do not directly influence catalytic activity, whereas the BRD is essential for histone H3 acetylation in nucleosomal substrates. Several lysine residues in the intrinsically disordered AL are autoacetylated by the HAT domain. Upon autoacetylation, acetyl-K1596 (Ac-K1596) binds intramolecularly to the BRD, competing with histones for binding to the BRD and acting as a negative regulator that inhibits histone H3 acetylation.

Site-Selective Lysine Reactions Guided by Protein-Peptide Interaction.

Site-selective lysine post-translational modifications such as acetylation, methylation, hydroxylation, and isopeptide formation mediate the precise control of important signaling events in cells with unmistakable accuracy. This unparalleled site selectivity (modification of a single lysine in a particular protein in the proteome) is still a challenge for non-enzymatic protein reactions; the difficulty lies in the differentiation of the lysine ε-amino group from other reactive groups and in the precise pinpointing of one particular lysine ε-amino group out of many other lysine ε-amino groups and the N-terminal amine of the protein that have similar chemical reactivity. Here, we have explored proximal lysine conjugation reactions through peptide-guided fluorodinitrobenzene, isothiocyanate, and phenyl ester reactions and have validated the site-specific targeting of the ε-amino group of one single lysine in natural proteins that contain multiple lysine residues. This precise site selectivity is a result of the proximity-induced reactivity guided by a specific protein-peptide interaction: the binding interaction preorganizes an amine-reactive group in the peptide and one of the lysine side chain ε-amino groups of the protein into close proximity, thereby confining the reactivity to a selected area of the target protein. The binding-guide lysine reactions were first examined on an SH3 domain and then tested on several ubiquitin-like proteins such as SUMO, Atg8 protein family, plant ATG8, and mammalian LC3 proteins that contain at least seven lysine residues on the surface. Exquisite site selectivity was confirmed in all of the proteins tested. A set of amine reactions were tested for their feasibility in the site-selective lysine reaction. Selected amine-reactive groups were optimized, and the reaction sites on the LC3 protein were confirmed by mass spectrometry.

NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3).

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the ß2 -loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of 15 N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx729 DPEEIIVLSDSD740 ), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by ß2 -loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of ß2 -loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.

Chemical Modification of the N Termini of Unprotected Peptides for Semisynthesis of Modified Proteins by Utilizing a Hydrophilic Protecting Group.

A simple and efficient strategy for the selective modification of the peptide N terminus with an unnatural amino acid is described. A peptide having a SUMO-HisTag-TEV sequence (SUMO: small ubiquitin-related modifier, TEV: tobacco etch virus) preceding the N terminus of the target peptide was designed. Recombinant expression in E. coli and subsequent SUMO protease cleavage yielded the HisTag-TEV-target peptide. Partial protection of the lysine side chains of this peptide with d-glucopyranosyloxycarbonyl and removal of the HisTag-TEV sequence by TEV protease yielded the partially protected peptide with a free N-terminal amine. Coupling of selenocysteine selectively at the N terminus and subsequent acidic deprotection of the carbohydrate protecting groups yielded a modified peptide that can be used for native chemical ligation (NCL). As a proof of concept, the modification of a longer recombinant peptide with selenocysteinylserine (GalNAc) at the N terminus was demonstrated.

Cloning and high-level SUMO-mediated fusion expression of a serine protease inhibitor from Hyphantria cunea Drury that exhibits activity against papain.

Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli, resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2-12), temperature (20-50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure.

Proteome-wide Mapping of Endogenous SUMOylation Sites in Mouse Testis.

SUMOylation is a reversible post-translational modification involved in various critical biological processes. To date, there is limited approach for endogenous wild-type SUMO-modified peptides enrichment and SUMOylation sites identification. In this study, we generated a high-affinity SUMO1 antibody to facilitate the enrichment of endogenous SUMO1-modified peptides from Trypsin/Lys-C protease digestion. Following secondary Glu-C protease digestion, we identified 53 high-confidence SUMO1-modified sites from mouse testis by using high-resolution mass spectrometry. Bioinformatics analyses showed that SUMO1-modified proteins were enriched in transcription regulation and DNA repair. Nab1 was validated to be an authentic SUMOylated protein and Lys479 was identified to be the major SUMOylation site. The SUMOylation of Nab1 enhanced its interaction with HDAC2 and maintained its inhibitory effect on EGR1 transcriptional activity. Therefore, we provided a novel approach to investigating endogenous SUMOylation sites in tissue samples.