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.     

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.     

Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest

Posttranslational modification by ubiquitin and SUMO is recognized as an effective means of controlling the stability, localization or activity of intracellular proteins, thereby contributing to the regulation of many biological processes. Over the past few years, it has become apparent that the two modification systems often communicate and jointly affect the properties of common substrate proteins, in some cases by being targeted to the same site. However, although SUMO and ubiquitin might have very different effects on a given target, their actions can rarely be explained by simple competition. This article gives an overview of target proteins that can serve as substrates for both SUMO and ubiquitin to highlight the diversity of regulatory strategies that result from the crosstalk between the two modification systems.     

The p66 and p12 subunits of DNA polymerase delta are modified by ubiquitin and ubiquitin-like proteins

Modification by ubiquitin-like proteins is now known to be important for the functions of many proteins involved in DNA replication and repair. We have investigated the modification of human DNA polymerase delta by ubiquitin and SUMO proteins. We find that while the p125 and p50 subunits were not modified, the p12 subunit is ubiquitinated and the p66 subunit can be modified by ubiquitin and SUMO3. We show that levels of p12 are regulated by the proteasome, either directly or indirectly, through a mechanism that is not dependent upon p12 ubiquitination. We have mapped two sites of SUMO3-specific modification on the p66 subunit. SUMOylation by SUMO3 but not SUMO2 is unusual: their level of homology is so high that they are normally classified as variants of the same protein. However, our findings show that these two proteins can be distinguished in vivo and may have specific functions.     

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.     

SUMO Chain Formation Is Required for Response to Replication Arrest in S. pombe

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved ββαββαβ fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.     

一种新型SUMO E3连接酶CBX4的化学修饰作用

多梳蛋白家族（polycomb group proteins,PcG）是一类在染色质水平上通过表观遗传修饰抑制靶基因转录的调节因子,它在调节细胞周期、DNA修复、细胞分化、衰老和死亡中起到重要作用。CBX4作为PcG家族中唯一具有SUMO E3 连接酶活性的成员,可以作用于多种底物,包括HIPK2、SIP1、CtBP、CTCF、Dnmt3a和HIF-1α等。底物的SUMO化修饰依赖于特定的结构基础,而且SUMO化的底物功能也会相应发生改变。同时,CBX4还可以被其它分子,如HIPK2, SENP2等进行磷酸化以及去SUMO化等修饰。本篇综述详细阐述了CBX4对底物的SUMO化修饰、自身被修饰及其生物学功能的变化。     

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post‐translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin‐like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO‐modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome‐targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome‐targeting is crucially required for the repair of TRF2‐depleted dysfunctional telomeres by 53BP1‐mediated non‐homologous end joining.     

In Vitro Studies Reveal a Sequential Mode of Chain Processing by the Yeast SUMO (Small Ubiquitin-related Modifier)-specific Protease Ulp2

Sumoylation is a post-translational modification essential in most eukaryotes that regulates stability, localization, activity, or interaction of a multitude of proteins. It is a reversible process wherein counteracting ligases and proteases, respectively, mediate the conjugation and deconjugation of SUMO molecules to/from target proteins. Apart from attachment of single SUMO moieties to targets, formation of poly-SUMO chains occurs by the attachment of additional SUMO molecules to lysine residues in the N-terminal extensions of SUMO. In Saccharomyces cerevisiae there are apparently only two SUMO(Smt3)-specific proteases: Ulp1 and Ulp2. Ulp2 has been shown to be important for the control of poly-SUMO conjugates in cells and to dismantle SUMO chains in vitro, but the mechanism by which it acts remains to be elucidated. Applying an in vitro approach, we found that Ulp2 acts sequentially rather than stochastically, processing substrate-linked poly-SUMO chains from their distal ends down to two linked SUMO moieties. Furthermore, three linked SUMO units turned out to be the minimum length of a substrate-linked chain required for efficient binding to and processing by Ulp2. Our data suggest that Ulp2 disassembles SUMO chains by removing one SUMO moiety at a time from their ends (exo mechanism). Apparently, Ulp2 recognizes surfaces at or near the N terminus of the distal SUMO moiety, as attachments to this end significantly reduce cleavage efficiency. Our studies suggest that Ulp2 controls the dynamic range of SUMO chain lengths by trimming them from the distal ends.     

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.     

Structural and functional characterization of Tomato SUMO1 gene

Small ubiquitin-related modifier (SUMO) genes regulate various functions of target proteins through post-translational modification. The SUMO proteins have a similar 3-dimensional structure as that of ubiquitin proteins and occur through a cascade of enzymatic reactions. In the present study we have cloned a new SUMO gene from Tomato (Solanum lycopersicum L.), cv Saudi-1, named SlS-SUMO1 gene by PCR using specific primers. This gene has SUMO member's features such as C-terminal diglycine (GG) motif as processing site by ULP (ubiquitin-like SUMO protease) and has SUMO consensus ΨKXE/D sequence. Phylogenetic analysis showed that SlS-SUMO1 gene is highly conserved and homologous to Potatoes Ca-SUMO1 and Ca-SUMO2 genes based on sequence similarity. Expression protein of SlS-SUMO1 gene found to be localized in the nucleus, cytoplasm, and nuclear envelop or nuclear pore complex. SUMO conjugating enzyme SCE1a with SlS-SUMO1 protein co-expressed and co-localized in nucleus and formed nuclear subdomains. This study reported that the SlS-SUMO1 gene is a member of SUMO family and its SUMO protein processing using GG motif and activate and transport to nucleus through Sumoylation system in the plant cell.     

SUMO/sentrin: protein modifiers regulating important cellular functions.

Regulation of protein functions can be achieved by posttranslational protein modifications. One of the most studied modifications has been conjugation to ubiquitin, which mainly targets substrate proteins for degradation by the 26 S proteasome. Recently, SUMO/sentrin, a ubiquitin-like protein has been characterized. This evolutionary conserved protein is conjugated to specific proteins in a way similar, but not identical, to ubiquitin and seems also to be involved in the regulation of protein localization or function. An increasing number of SUMO/sentrin substrates are currently described. We focus here on three major substrates of modification by SUMO: RanGAP1, PML, and IkappaBalpha proteins. These different examples illustrate how SUMO conjugation may be involved in the control of the level of critical proteins within the cell or in the modulation of subcellular localization and nucleocytoplasmic trafficking.     

SUMO protein modification

SUMO (small ubiquitin-related modifier) family proteins are not only structurally but also mechanistically related to ubiquitin in that they are posttranslationally attached to other proteins. As ubiquitin, SUMO is covalently linked to its substrates via amide (isopeptide) bonds formed between its C-terminal glycine residue and the epsilon-amino group of internal lysine residues. The enzymes involved in the reversible conjugation of SUMO are similar to those mediating the ubiquitin conjugation. Since its discovery in 1996, SUMO has received a high degree of attention because of its intriguing and essential functions, and because its substrates include a variety of biomedically important proteins such as tumor suppressor p53, c-jun, PML and huntingtin. SUMO modification appears to play important roles in diverse processes such as chromosome segregation and cell division, DNA replication and repair, nuclear protein import, protein targeting to and formation of certain subnuclear structures, and the regulation of a variety of processes including the inflammatory response in mammals and the regulation of flowering time in plants.