UBE1DC1, an ubiquitin-activating enzyme, activates two different ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins are known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway. These modifications lead to the degradation of substrates or change its functional status. The ubiquitin-activating enzyme (E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins. Ubiquitin-activating enzyme E1-domain containing 1 (UBE1DC1) had been proved to activate an ubiquitin-like protein, ubiquitin-fold modifier 1 (Ufm1), by forming a high-energy thioester bond. In this report, UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2, besides Ufm1, both in vitro and in vivo by immunological analysis. It indicated that UBE1DC1 could activate two different ubiquitin-like proteins, SUMO2 and Ufm1, which have no significant similarity with each other. Subcellular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm; whereas UBE1DC1 was mainly distributed in the nucleus when was cotransfected with SUMO2. It presumed that UBE1DC1 greatly activated SUMO2 in the nucleus or transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.     

A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing

Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.     

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.     

Emerging roles of the SUMO pathway in mitosis

SUMO proteins are small ubiquitin-like modifiers found in all eukaryotes that become covalently conjugated to other cellular proteins. The SUMO conjugation pathway is biochemically similar to ubiquitin conjugation, although the enzymes within the pathway act exclusively on SUMO proteins. This post-translational modification controls many processes. Here, I will focus on evidence that SUMOylation plays a critical role(s) in mitosis: Early studies showed a genetic requirement for SUMO pathway components in the process of cell division, while later findings implicated SUMOylation in the control of mitotic chromosome structure, cell cycle progression, kinetochore function and cytokinesis. Recent insights into the targets of SUMOylation are likely to be extremely helpful in understanding each of these aspects. Finally, growing evidence suggests that SUMOylation is a downstream target of regulation through Ran, a small GTPase with important functions in both interphase nuclear trafficking and mitotic spindle assembly.     

Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation

Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.     

The 19S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair.

The 26S proteasome degrades proteins targeted by the ubiquitin pathway, a function thought to explain its role in cellular processes. The proteasome interacts with the ubiquitin-like N terminus of Rad23, a nucleotide excision repair (NER) protein, in Saccharomyces cerevisiae. Deletion of the ubiquitin-like domain causes UV radiation sensitivity. Here, we show that the ubiquitin-like domain of Rad23 is required for optimal activity of an in vitro NER system. Inhibition of proteasomal ATPases diminishes NER activity in vitro and increases UV sensitivity in vivo. Surprisingly, blockage of protein degradation by the proteasome has no effect on the efficiency of NER. This establishes that the regulatory complex of the proteasome has a function independent of protein degradation.     

DeSUMOylating enzymes--SENPs

Modification of proteins by ubiquitin and SUMO (small ubiquitin-like modifiers) is a dynamic and reversible process. Similar to the ubiquitin pathway, where the action of deubiquitinating enzymes removes ubiquitin from ubiquitin-adducts, SUMO is also removed intact from its substrates by proteases belonging to the sentrin-specific proteases (SENPs) family. In addition to their isopeptidase activity, SENPs also execute another essential function as endopeptidases by removing the short C-terminal extension from immature SUMOs. The defining characteristics of SENPs are their predicted conserved molecular scaffold-defined as members of peptidase Clan CE, conserved catalytic mechanism, and their reported activity on SUMO or Nedd8 conjugated proteins (or the respective precursors). We discuss recent progress on the human SENPs and their substrates.     

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.     

Mechanism and function of deubiquitinating enzymes

Attachment of ubiquitin to proteins is a crucial step in many cellular regulatory mechanisms and contributes to numerous biological processes, including embryonic development, the cell cycle, growth control, and prevention of neurodegeneration. In these diverse regulatory settings, the most widespread mechanism of ubiquitin action is probably in the context of protein degradation. Polyubiquitin attachment targets many intracellular proteins for degradation by the proteasome, and (mono)ubiquitination is often required for down-regulating plasma membrane proteins by targeting them to the vacuole (lysosome). Ubiquitin-protein conjugates are highly dynamic structures. While an array of enzymes directs the conjugation of ubiquitin to substrates, there are also dozens of deubiquitinating enzymes (DUBs) that can reverse the process. Several lines of evidence indicate that DUBs are important regulators of the ubiquitin system. These enzymes are responsible for processing inactive ubiquitin precursors, proofreading ubiquitin-protein conjugates, removing ubiquitin from cellular adducts, and keeping the 26S proteasome free of inhibitory ubiquitin chains. The present review focuses on recent discoveries that have led to a better understanding the mechanisms and physiological roles of this diverse and still poorly understood group of enzymes. We also discuss briefly some of the proteases that act on ubiquitin-like protein (UBL) conjugates and compare them to DUBs.     

Assay methods for small ubiquitin-like modifier (SUMO)–SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO–SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein–protein interactions, either in vivo or in vitro, in a high-throughput manner.     

蛋白质的类泛素化修饰

SUMO(small ubiquitin-related modifier)是一类重要的类泛素蛋白,在生物进化过程中高度保守,其三维结构及生化修饰过程与泛素类似,但该两类蛋白质修饰的生物学意义却不尽相同。SUMO化修饰作为一种重要的蛋白质翻译后修饰,广泛参与细胞活动的各个方面,且SUMO化修饰异常与许多人类重大疾病密切相关。     

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.     

蛋白质SUMO化修饰研究进展

SUMO(small ubiquitin-related modifier)是类泛素蛋白家族的重要成员之一,可与多种蛋白结合发挥相应的功能,其分子结构及SUMO化反应途径都与泛素类似,但二者功能完全不同。SUMO化修饰可参与转录调节、核转运、维持基因组完整性及信号转导等多种细胞内活动,是一种重要的多功能的蛋白质翻译后修饰方式。SUMO化修饰功能的失调可能导致某些疾病的发生。     

A SIM-ultaneous role for SUMO and ubiquitin

Ubiquitin and ubiquitin-like proteins (Ubls) share a beta-GRASP fold and have key roles in cellular growth and suppression of genome instability. Despite their common fold, SUMO and ubiquitin are classically portrayed as distinct, and they can have antagonistic roles. Recently, a new family of proteins, the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligases (STUbLs), which directly connect sumoylation and ubiquitylation, has been discovered. Uniquely, STUbLs use SUMO-interaction motifs (SIMs) to recognize their sumoylated targets. STUbLs are global regulators of protein sumoylation levels, and cells lacking STUbLs display genomic instability and hypersensitivity to genotoxic stress. The human STUbL, RNF4, is implicated in several diseases including cancer, highlighting the importance of characterizing the cellular functions of STUbLs.     

The Wnts

Background

The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.

Results

The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis.

Conclusions

The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.     

Viruses and sumoylation: recent highlights

Since its discovery in 1997, SUMO (small ubiquitin-like modifier) has been implicated in a range of activities, indicating that this protein is as important in the cell as ubiquitin is. Although it can function throughout the cell, it appears to be involved more in nuclear functions. The growing list of substrates that are covalently modified by SUMO includes many viral proteins; SUMO appears to facilitate viral infection of cells, making it a possible target for antiviral therapies. It therefore is important to understand how viruses manipulate the cellular sumoylation system and how sumoylation affects viral functions.     

Highly sensitive assays for SUMOylation and small ubiquitin-like modifier-dependent protein-protein interactions

Small ubiquitin-like proteins (SUMO) are recently discovered post-translational modifiers that regulate protein functions and intracellular trafficking. In this study, we are describing two chemoluminescence-based assays, one for SUMOylation and another one for SUMO-mediated protein-protein interactions. These assays can be used to characterize the activity and kinetics of the enzymes that catalyze SUMOylation, and in high-throughput screening for inhibitors of SUMOylation and SUMO-dependent protein-protein interactions. These novel assays represent the most sensitive assays for ubiquitin-like systems published to date. Similar strategies can be used to develop assays for other ubiquitin-like modification systems.