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。     

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.     

Cytoplasmic sumoylation by PIAS-type Siz1-SUMO ligase

SUMO (small ubiquitin-related modifier), a 12 kDa protein with distant similarity to ubiquitin, covalently binds to many proteins in eukaryotic cells. In contrast to ubiquitination, which mainly regulates proteasome-dependent degradation and protein sorting, sumoylation is known to regulate assembly and disassembly of protein complexes, protein localization and stability, and so on. SUMO is primarily localized to the nucleus, and many SUMO substrates are nuclear proteins involved in DNA transaction. However, certain roles of SUMO conjugates have been shown outside the nucleus. Particularly in budding yeast, SUMO is also localized to the bud-neck in a cell cycle-dependent manner. The first and prominent SUMO substrates are septins, evolutionally conserved proteins required for cytokinesis in yeast. Recent analysis of human septin structure would greatly facilitate the study of the functions of these SUMO conjugates. SUMO modification of septins is regulated by cell cycle-dependent nuclear transport of PIAS-type Siz1 (SUMO E3) and Ulp1 desumoylation enzyme in yeast. Domains outside the SUMO-ligase core (SP-RING) of Siz1 ensure its regulations. Furthermore, newly discovered ubiquitin ligases that specifically recognize poly-SUMO conjugates could lead to degradation of SUMO conjugates. Thus, protein modifications seem to be regulated in an unexpectedly complex manner. In this review, we focus on various regulations in yeast septin sumoylation and discuss its possible functions.     

SUMO, ubiquitin's mysterious cousin

Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO regulates various cellular processes, such as nuclear transport, signal transduction, stress response and cell-cycle progression. But, in contrast to ubiquitylation, sumoylation does not tag proteins for degradation, but seems to enhance their stability or modulate their subcellular compartmentalization.     

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 modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

Ubiquitin-dependent proteolytic control of SUMO conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.     

Evolution of a signalling system that incorporates both redundancy and diversity: Arabidopsis SUMOylation

The reversible post-translational modifier, SUMO (small ubiquitin-related modifier), modulates the activity of a diverse set of target proteins, resulting in important consequences to the cellular machinery. Conjugation machinery charges the processed SUMO so that it can be linked via an isopeptide bond to a target protein. The removal of SUMO moieties from conjugated proteins by isopeptidases regenerates pools of processed SUMOs and unmodified target proteins. The evolutionarily conserved SUMO-conjugating proteins, E1 and E2, recognize a diverse set of Arabidopsis SUMO proteins using them to modify protein substrates. In contrast, the deSUMOylating enzymes differentially recognize the Arabidopsis SUMO proteins, resulting in specificity of the deconjugating machinery. The specificity of the Arabidopsis deSUMOylating enzymes is further diversified by the addition of regulatory domains. Therefore the SUMO proteins, in this signalling system, have evolved to contain information that allows not only redundancy with the conjugation system but also diversity with the deconjugating enzymes.     

SUMO与乳腺癌

小泛素修饰物（Small ubiquitin-like modifier,SUMO）是结构上与泛素类似的一种修饰蛋白,能与一些特定的靶蛋白共价连接。与泛素介导蛋白质的降解不同,SUMO化修饰调控主要对靶蛋白的功能,如在蛋白质的稳定性、细胞定位、信号转导、基因转录调控等方面均发挥着重要的作用。最近的研究表明：SUMO与乳腺癌的发生发展密切相关,它是通过SUMO化修饰参与并影响雌激素受体信号通路来实现的,本文将就此做一综述。     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

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.     

Characterization of a novel posttranslational modification in polypyrimidine tract-binding proteins by SUMO1

Polypyrimidine tract-binding protein 1 (PTBP1) and its brainspecific homologue, PTBP2, are associated with pre-mRNAs and influence pre-mRNA processing, as well as mRNA metabolism and transport. They play important roles in neural differentiation and glioma development. In our study, we detected the expression of the two proteins in glioma cells and predicted that they may be sumoylated using SUMOplot analyses. We confirmed that PTBP1 and PTBP2 can be modified by SUMO1 with co-immunoprecipitation experiments using 293ET cells transiently co-expressing SUMO1 and either PTBP1 or PTBP2. We also found that SUMO1 modification of PTBP2 was enhanced by Ubc9 (E2). The mutation of the sumoylation site (Lys137) of PTBP2 markedly inhibited its modification by SUMO1. Interestingly, in T98G glioma cells, the level of sumoylated PTBP2 was reduced compared to that of normal brain cells. Overall, this study shows that PTBP2 is posttranslationally modified by SUMO1. [BMB Reports 2014; 47(4): 233-238]     

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.     

Structural basis for regulation of poly‐SUMO chain by a SUMO‐like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A viral ubiquitin ligase has substrate preferential SUMO targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.     

A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast

Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins. Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport. Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates. However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far. By the use of conditional yeast mutants, we examined nuclear protein import in vivo. We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction. In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected. Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import. In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking.