In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.     

Chip-based analysis of SUMO (small ubiquitin-like modifier) conjugation to a target protein

A chip-based analysis of protein interactions and modifications in cell signaling pathways has been of great potential in drug discovery, diagnostics, and cell biology, because it enables rapid and high-throughput biological assays with a small amount of samples. We report a chip-based analysis of sumoylation, the post-translational modification (PTM) process that involves covalent attachment of the small ubiquitin-like modifier (SUMO) protein to a target protein through multiple enzyme reactions in eukaryotic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for sumoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip sumoylation of Ran GTPase-activating protein 1 (RanGAP1) domain resulted in highly specific fluorescence intensity compared to that of its mutant (K524A) irrelevant to SUMO conjugation. The on-chip sumoylation was also verified and quantified by using the surface plasmon resonance(SPR) spectroscopy. As the exemplary study for a parallel analysis of sumoylation, fluorescent detection of sumoylation was conducted in a microarray format on a glass slide. The chip-based analysis developed here is expected to be applicable to assay for screening of target proteins from existing protein pools and proteome arrays in a high throughput manner.     

The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress

Small ubiquitin-like modifier (SUMO) is a member of the superfamily of ubiquitin-like polypeptides that become covalently attached to various intracellular target proteins as a way to alter their function, location, and/or half-life. Here we show that the SUMO conjugation system operates in plants through a characterization of the Arabidopsis SUMO pathway. An eight-gene family encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation, and release. A diverse array of conjugates could be detected, some of which appear to be SUMO isoform-specific. The levels of SUMO1 and -2 conjugates but not SUMO3 conjugates increased substantially following exposure of seedlings to stress conditions, including heat shock, H(2)O(2), ethanol, and the amino acid analog canavanine. The heat-induced accumulation could be detected within 2 min from the start of a temperature upshift, suggesting that SUMO1/2 conjugation is one of the early plant responses to heat stress. Overexpression of SUMO2 enhanced both the steady state levels of SUMO2 conjugates under normal growth conditions and the subsequent heat shock-induced accumulation. This accumulation was dampened in an Arabidopsis line engineered for increased thermotolerance by overexpressing the cytosolic isoform of the HSP70 chaperonin. Taken together, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in plants with initial data indicating that one important function may be in stress protection and/or repair.     

The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii

SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.     

The small ubiquitin-like modifier (SUMO) and SUMO-conjugating system of Chlamydomonas reinhardtii

The availability of the complete DNA sequence of the Chlamydomonas reinhardtii genome and advanced computational biology tools has allowed elucidation and study of the small ubiquitin-like modifier (SUMO) system in this unicellular photosynthetic alga and model eukaryotic cell system. SUMO is a member of a ubiquitin-like protein superfamily that is covalently attached to target proteins as a post-translational modification to alter the localization, stability, and/or function of the target protein in response to changes in the cellular environment. Three SUMO homologs (CrSUMO96, CrSUMO97, and CrSUMO148) and three novel SUMO-related proteins (CrSUMO-like89A, CrSUMO-like89B, and CrSUMO-like90) were found by diverse gene predictions, hidden Markov models, and database search tools inferring from Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana SUMOs. Among them, CrSUMO96, which can be recognized by the A. thaliana anti-SUMO1 antibody, was studied in detail. Free CrSUMO96 was purified by immunoprecipitation and identified by mass spectrometry analysis. A SUMO-conjugating enzyme (SCE) (E2, Ubc9) in C. reinhardtii was shown to be functional in an Escherichia coli-based in vivo chimeric SUMOylation system. Antibodies to CrSUMO96 recognized free and conjugated forms of CrSUMO96 in Western blot analysis of whole-cell extracts and nuclear localized SUMOylated proteins with in situ immunofluorescence. Western blot analysis showed a marked increase in SUMO conjugated proteins when the cells were subjected to environmental stresses, such as heat shock and osmotic stress. Related analyses revealed multiple potential ubiquitin genes along with two Rub1 genes and one Ufm1 gene in the C. reinhardtii genome.     

Small ubiquitin-like modifier (SUMO) modification of natively unfolded proteins tau and alpha-synuclein

Sumoylation is an important post-translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization, and stability of target proteins. We have examined the covalent attachment of the small ubiquitin-like modifier (SUMO) proteins to tau and alpha-synuclein, two natively unfolded proteins that define several neurodegenerative diseases. Both brain proteins were preferentially modified by SUMO1, as compared with SUMO2 or SUMO3. Tau contains two SUMO consensus sequences, and mutational analyses identified Lys(340) as the major sumoylation site. Although both tau and alpha-synuclein are targets for proteasomal degradation, only tau sumoylation was affected by inhibitors of the proteasome pathway. Tau is a microtubule-associated protein, whose ability to bind and stabilize microtubules is negatively regulated by phosphorylation. Treatment with the phosphatase inhibitor, okadaic acid, or the microtubule depolymerizing drug, colchicine, up-regulated tau sumoylation. This suggests that SUMO modification may preferentially target a free soluble pool of the substrate. These findings revealed a new, possibly regulatory, modification of tau and alpha-synuclein that may also have implications for their pathogenic roles in neurodegenerative diseases.     

Mutational analysis of the central centromere targeting domain of human centromere protein C, (CENP-C)

Human centromere protein C (CENP-C) is an essential component of the inner kinetochore plate. A central region of CENP-C can bind DNA in vitro and is sufficient for targeting the protein to centromeres in vivo, raising the possibility that this domain mediates centromere localization via direct DNA binding. We performed a detailed molecular dissection of this domain to understand the mechanism by which CENP-C assembles at centromeres. By a combination of PCR mutagenesis and transient expression of GFP-tagged proteins in HeLa cells, we identified mutations that disrupt centromere localization of CENP-C in vivo. These cluster in a 12 amino acid region adjacent to the core domain required for in vitro DNA binding. This region is conserved between human and mouse, but is divergent or absent in invertebrate and plant CENP-C homologues. We suggest that these 12 amino acids are essential to confer specificity to DNA binding by CENP-C in vivo, or to mediate interaction with another as yet unidentified centromere component. A differential yeast two-hybrid screen failed to identify interactions specific to this sequence, but nonetheless identified 14 candidate proteins that interact with the central region of CENP-C. This collection of mutations and interacting proteins comprise a useful resource for further elucidating centromere assembly.     

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.     

Swapping small ubiquitin-like modifier (SUMO) isoform specificity of SUMO proteases SENP6 and SENP7

SUMO proteases can regulate the amounts of SUMO-conjugated proteins in the cell by cleaving off the isopeptidic bond between SUMO and the target protein. Of the six members that constitute the human SENP/ULP protease family, SENP6 and SENP7 are the most divergent members in their conserved catalytic domain. The SENP6 and SENP7 subclass displays a clear proteolytic cleavage preference for SUMO2/3 isoforms. To investigate the structural determinants for such isoform specificity, we have identified a unique sequence insertion in the SENP6 and SENP7 subclass that is essential for their proteolytic activity and that forms a more extensive interface with SUMO during the proteolytic reaction. Furthermore, we have identified a region in the SUMO surface determinant for the SUMO2/3 isoform specificity of SENP6 and SENP7. Double point amino acid mutagenesis on the SUMO surface allows us to swap the specificity of SENP6 and SENP7 between the two SUMO isoforms. Structure-based comparisons combined with biochemical and mutagenesis analysis have revealed Loop 1 insertion in SENP6 and SENP7 as a platform to discriminate between SUMO1 and SUMO2/3 isoforms in this subclass of the SUMO protease family.     

Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins

Reversible covalent modification of proteins with a small ubiquitin-related modifier (SUMO) is emerging as an important system contributing to dynamic regulation of protein function. To enhance our understanding of the cell regulatory systems impacted by sumoylation, we used affinity chromatography-coupled high pressure liquid chromatography/tandem mass spectrometry for unbiased identification of candidate cellular SUMO substrate proteins. Here we describe the identification of 21 candidate sumoylated proteins from whole-cell lysates of HEK-293 cells. The nature of the proteins identified is consistent with a role for sumoylation in diverse cell regulatory systems but highlights regulation of chromatin organization and gene expression as major systems targeted by the sumoylation machinery.     

Rap80 protein recruitment to DNA double-strand breaks requires binding to both small ubiquitin-like modifier (SUMO) and ubiquitin conjugates

Ubiquitin (Ub) modifications at sites of DNA double-strand breaks (DSBs) play critical roles in the assembly of signaling and repair proteins. The Ub-interacting motif (UIM) domain of Rap80, which is a component of the BRCA1-A complex, interacts with Ub Lys-63 linkage conjugates and mediates the recruitment of BRCA1 to DSBs. Small ubiquitin-like modifier (SUMO) conjugation also occurs at DSBs and promotes Ub-dependent recruitment of BRCA1, but its molecular basis is not clear. In this study, we identified that Rap80 possesses a SUMO-interacting motif (SIM), capable of binding specifically to SUMO2/3 conjugates, and forms a tandem SIM-UIM-UIM motif at its N terminus. The SIM-UIM-UIM motif binds to both Ub Lys-63 linkage and SUMO2 conjugates. Both the SIM and UIM domains are required for efficient recruitment of Rap80 to DSBs immediately after damage and confer cellular resistance to ionizing radiation. These findings propose a model in which SUMO and Ub modification is coordinated to recruit Rap80 and BRCA1 to DNA damage sites.     

Insights into high affinity small ubiquitin-like modifier (SUMO) recognition by SUMO-interacting motifs (SIMs) revealed by a combination of NMR and peptide array analysis

The small ubiquitin-like modifiers (SUMOs) regulate many essential cellular functions. Only one type of SUMO-interacting motif (SIM) has been identified that can extend the β-sheet of SUMO as either a parallel or an antiparallel strand. The molecular determinants of the bound orientation and paralogue specificity of a SIM are unclear. To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. In addition, the SIM sequence requirements have been investigated by peptide arrays in comparison with another high affinity SIM that binds in the opposing orientation. We found that antiparallel binding SIMs tolerate more diverse sequences, whereas the parallel binding SIMs prefer the more strict sequences consisting of (I/V)DLT that have a preference in high affinity SUMO2 and -3 binding. Comparison of two high affinity SUMO1-binding SIMs that bind in opposing orientations has revealed common SUMO1-specific interactions needed for high affinity binding. This study has significantly advanced our understanding of the molecular determinants underlining SUMO-SIM recognition.     

In vitro galactation of human serum albumin: analysis of the protein's galactation sites by mass spectrometry

The posttranslational modification of proteins by sugars has been demonstrated in diabetes and classical galactosemia. In diabetes, the glycation process occurs as a result of d-glucose nonenzymatically reacting with proteins such as albumin and hemoglobin, used today as important tools to monitor the efficiency of dietary control and therapy during treatment of diabetes. In classical galactosemia, d-galactose contributes to the formation of glycated proteins as well, suggesting that, akin to diabetes with glucated proteins, the monitoring of galactated proteins may facilitate management of patients with galactosemia. The objectives of this study were (i) to galactate human serum albumin (HSA) in vitro; (ii) to determine, by a sodium borohydride-dependent mass peptide mapping method, the galactation sites in HSA; and (iii) to compare HSA’s galactation sites with the protein’s reported glucation sites. Treatment of galactated HSA with sodium borohydride stabilized the condensed sugars on the protein and yielded discrete fragmentation patterns by tandem mass spectrometry, allowing reliable identification of HSA’s galactation sites. Liquid chromatography/electrospray ionization/mass spectrometry, in combination with tandem mass spectrometry, revealed that the principal sites of galactation in HSA were the ε-amino groups of lysine residues 12, 233, 281/276, 414, and 525. Lysyl residues 12, 233, 276, and 525 were previously reported as privileged sites for the nonenzymatic binding of d-glucose with HSA.     

Small ubiquitin-like modifier (SUMO) modification of E1 Cys domain inhibits E1 Cys domain enzymatic activity

Although it is well established that ubiquitin-like modifications are tightly regulated, it has been unclear how their E1 activities are controlled. In this study, we found that the SAE2 subunit of the small ubiquitin-like modifier (SUMO) E1 is autoSUMOylated at residue Lys-236, and SUMOylation was catalyzed by Ubc9 at several additional Lys residues surrounding the catalytic Cys-173 of SAE2. AutoSUMOylation of SAE2 did not affect SUMO adenylation or formation of E1·SUMO thioester, but did significantly inhibit the transfer of SUMO from E1 to E2 and overall SUMO conjugations to target proteins due to the altered interaction between E1 and E2. Upon heat shock, SUMOylation of SAE2 was reduced, which corresponded with an increase in global SUMOylation, suggesting that SUMOylation of the Cys domain of SAE2 is a mechanism for "storing" a pool of E1 that can be quickly activated in response to environmental changes. This study is the first to show how E1 activity is controlled by post-translational modifications, and similar regulation likely exists across the homologous E1s of ubiquitin-like modifications.