A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.     

SUMO assay with peptide arrays on solid support: insights into SUMO target sites

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.     

SUMO and SUMOylation in plants

The traditional focus on the central dogma of molecular biology, from gene through RNA to protein, has now been replaced by the recognition of an additional mechanism. The new regulatory mechanism, post-translational modifications to proteins, can actively alter protein function or activity introducing additional levels of functional complexity by altering cellular and sub-cellular location, protein interactions and the outcome of biochemical reaction chains. Modifications by ubiquitin (Ub) and ubiquitin-like modifiers systems are conserved in all eukaryotic organisms. One of them, small ubiquitin-like modifier (SUMO) is present in plants. The SUMO mechanism includes several isoforms of proteins that are involved in reactions of sumoylation and de-sumoylation. Sumoylation affects several important processes in plants. Outstanding among those are responses to environmental stresses. These may be abiotic stresses, such as phosphate deficiency, heat, low temperature, and drought, or biotic stressses, as well including defense reactions to pathogen infection. Also, the regulations of flowering time, cell growth and development, and nitrogen assimilation have recently been added to this list. Identification of SUMO targets is material to characterize the function of sumoylation or desumoylation. Affinity purification and mass spectrometric identification have been done lately in plants. Further SUMO noncovalent binding appears to have function in other model organisms and SUMO interacting proteins in plants will be of interest to plant biologists who dissect the dynamic function of SUMO. This review will discuss results of recent insights into the role of sumoylation in plants.     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

SUMO on the road to neurodegeneration

Sumoylation is a post-translational modification by which small ubiquitin-like modifiers (SUMO) are covalently conjugated to target proteins. This reversible pathway provides a rapid and efficient way to modulate the subcellular localization, activity and stability of a wide variety of substrates. Similar to its well-known cousin ubiquitin, SUMO co-localize with the neuronal inclusions associated with several neurodegenerative diseases, including multiple system atrophy, Huntington's disease and other related polyglutamine disorders. The identification of huntingtin, ataxin-1, tau and alpha-synuclein as SUMO substrates further supports the involvement of sumoylation in the pathogenesis of this family of neurological diseases. In addition to direct targeting of these constituent proteins, sumoylation also impacts other disease pathways such as oxidative stress, protein aggregation and proteasome-mediated degradation. This review highlights the recent advances in understanding the contributions of SUMO to neurodegeneration and the underlying pathogenic mechanisms of these diseases.     

Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are approximately 96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralog-selective modification.     

SUMO modification of septin-interacting proteins in Candida albicans

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.     

SUMO: a history of modification

The small ubiquitin-like modifier (SUMO) is covalently linked to a variety of proteins and is deconjugated by SUMO-specific proteases. A characteristic of SUMO modification is that the biological consequences of conjugation do not appear proportionate to the small fraction of substrate that is modified. SUMO conjugation appears to alter the long-term fate of the modified protein even though the SUMO may be rapidly deconjugated. Thus an unmodified protein with a history of SUMO modification may have different properties from a protein that never has been modified. Here, the diverse effects of SUMO modification are discussed and models proposed to explain SUMO actions.     

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.     

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.     

Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2

SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.     

Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.     

Analysis of Human Cytomegalovirus-Encoded SUMO Targets and Temporal Regulation of SUMOylation of the Immediate-Early Proteins IE1 and IE2 during Infection

Post-translational modification of proteins by members of the small ubiquitin-like modifier (SUMO) is involved in diverse cellular functions. Many viral proteins are SUMO targets and also interact with the cellular SUMOylation system. During human cytomegalovirus (HCMV) infection, the immediate-early (IE) proteins IE1 and IE2 are covalently modified by SUMO. IE2 SUMOylation promotes its transactivation activity, whereas the role of IE1 SUMOylation is not clear. We performed in silico, genome-wide analysis to identify possible SUMOylation sites in HCMV-encoded proteins and evaluated their modification using the E. coli SUMOylation system and in vitro assays. We found that only IE1 and IE2 are substantially modified by SUMO in E. coli, although US34A was also identified as a possible SUMO target in vitro. We also found that SUMOylation of IE1 and IE2 is temporally regulated during viral infection. Levels of SUMO-modified form of IE1 were increased during the early phase of infection, but decreased in the late phase when IE2 and its SUMO-modified forms were expressed at high levels. IE2 expression inhibited IE1 SUMOylation in cotransfection assays. As in IE2 SUMOylation, PIAS1, a SUMO E3 ligase, interacted with IE1 and enhanced IE1 SUMOylation. In in vitro assays, an IE2 fragment that lacked covalent and non-covalent SUMO attachment sites, but was sufficient for PIAS1 binding, effectively inhibited PIAS1-mediated SUMOylation of IE1, indicating that IE2 expression negatively regulates IE1 SUMOylation. We also found that the IE2-mediated downregulation of IE1 SUMOylation correlates with the IE1 activity to repress the promoter containing the interferon stimulated response elements. Taken together, our data demonstrate that IE1 and IE2 are the main viral SUMO targets in HCMV infection and that temporal regulation of their SUMOylation may be important in the progression of this infection.     

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.     

Misregulation of 2 microm circle copy number in a SUMO pathway mutant

Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1Delta siz2Delta double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing cells as well as sectors of enlarged cells that are arrested in G(2)/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2 microm circle. siz1Delta siz2Delta mutants have up to 40-fold-higher levels of 2 microm than do wild-type strains. Furthermore, 2 microm is responsible for the siz1Delta siz2Delta mutant's obvious growth defects, as siz1Delta siz2Delta [cir(0)] strains, which lack 2 microm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2 microm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2 microm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2 microm copy number in the siz1Delta siz2Delta strain.     

Lamin A tail modification by SUMO1 is disrupted by familial partial lipodystrophy–causing mutations

Lamin filaments are major components of the nucleoskeleton that bind LINC complexes and many nuclear membrane proteins. The tail domain of lamin A directly binds 21 known partners, including actin, emerin, and SREBP1, but how these interactions are regulated is unknown. We report small ubiquitin-like modifier 1 (SUMO1) as a major new posttranslational modification of the lamin A tail. Two SUMO1 modification sites were identified based on in vitro SUMOylation assays and studies of Cos-7 cells. One site (K420) matches the SUMO1 target consensus; the other (K486) does not. On the basis of the position of K486 on the lamin A Ig-fold, we hypothesize the SUMO1 E2 enzyme recognizes a folded structure–dependent motif that includes residues genetically linked to familial partial lipodystrophy (FPLD). Supporting this model, SUMO1-modification of the lamin A tail is reduced by two FPLD-causing mutations, G465D and K486N, and by single mutations in acidic residues E460 and D461. These results suggest a novel mode of functional control over lamin A in cells.     

The CTCF Insulator Protein Is Posttranslationally Modified by SUMO

The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.