蛋白质多聚SUMO化修饰及其功能

泛素（ubiquitin, Ub）是一类高度保守的小蛋白, 可与靶蛋白的赖氨酸残基共价连接, 形成多聚泛素链行使功能. 类似于泛素化修饰过程, 小泛素相关修饰物(small ubiquitin related modifier, SUMO）也可以共价修饰靶蛋白的赖氨酸残基, 从而影响靶蛋白的定位、稳定性以及蛋白间的相互作用, 发挥重要的生理功能. 尽管在多数情况下, 靶蛋白发生的是单SUMO化修饰, 但最近研究发现,SUMO依赖自身的赖氨酸也可以形成多聚链. 与单SUMO化修饰不同的是, 多聚SUMO化修饰的靶蛋白可以进一步被泛素化修饰, 进而诱导靶蛋白的降解. 这是一种新的、特殊的化学修饰形式, 弄清它的生理功能,对于了解细胞的生长、分化以及凋亡等生理过程将具有重要的意义. 本文将就此方面的最新研究进展做一综述.     

泛素化和SUMO化对DEC1的拮抗调控作用

分化的胚软骨表达蛋白1(differentiated embryo-chondrocyte expressed gene 1,DEC1)作为一种时钟蛋白,除了在周期节律的调控中发挥转录抑制作用外,还在能量代谢以及多种肿瘤相关的信号通路的调控中发挥重要作用。此外,蛋白质的翻译后修饰是实现蛋白质功能精细调控的一种重要方式。目前发现,DEC1主要可被两种翻译后修饰,即泛素化和SUMO化修饰。尽管泛素化和SUMO化是两种过程非常类似的蛋白质翻译后修饰方式,但是它们对目的蛋白功能的调控却截然不同。由于泛素化和SUMO化与底物的作用靶点都是赖氨酸(Lys),因此在多数情况下,泛素化和SUMO化以拮抗性的方式调控底物蛋白的功能。鉴于此,该文旨在阐述泛素化和SUMO化修饰对DEC1功能的拮抗调节过程,为了解时钟蛋白DEC1对多种信号通路的调控过程中的分子机制提供新的思路。     

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。     

小泛素相关修饰物SUMO研究进展

蛋白质翻译后修饰对改变蛋白功能、活性或定位都起着非常重要的作用,泛素及其相似蛋白的修饰是其中一种重要形式。与其他诸如磷酸化、乙酰化、糖基化等不同的是,泛素及其相似蛋白的修饰基团本身即是一个小的多肽,通过异肽键与靶蛋白Lys侧链ε-NH2相连,其中小泛素相关修饰物(small ubiquitin—related modifier,SUMO)与蛋白的共价连接是一种新的广泛存在的翻译后修饰形式。SUMO是广泛存在于真核生物中高度保守的蛋白家族,在脊椎动物中有三个SUMO基因,称为SUMO-1,-2,-3,与泛素在二级结构上极其相似,且催化修饰过程的酶体系也具有很高的同源性。然而,与泛素化介导的蛋白酶降解途径不同,SUMO化修饰发挥着更为广泛的功能,如核质转运、细胞周期调控、信号转导、转录活性调控等。     

HIF-1α的可逆性SUMO化修饰

低氧诱导因子1(hypoxia inducible factor-1, HIF-1)是参与调节机体氧平衡的重要转录因子,在细胞低氧应答反应中起核心作用,能调节100多种涉及低氧应激下细胞适应和存活的靶基因．HIF-1由氧敏感的α亚基和在细胞内稳定表达的β亚基组成．其中α亚基可受到多种翻译后化学修饰作用,如在常氧下,HIF-1α通过泛素化蛋白酶修饰并导致其快速降解．最近几年发现的泛素样蛋白家族成员小泛素蛋白样修饰蛋白（SUMO）也能与HIF-1α共价结合．SUMO是一种分子量约为12 kD的小蛋白,从拟南芥到人类普遍存在．SUMO可共价结合许多靶底物蛋白,并对其进行翻译后修饰,该过程称为SUMO化．与泛素化蛋白酶体途径不同的是,SUMO化修饰能在常氧和相对低氧的条件下调节HIF-1α蛋白的稳定性,从而改变其转录活性．SUMO化是一个可逆的动态过程,可被特异性蛋白酶ULP/SENP将其从底物上去除．本文主要就HIF-1α的可逆性SUMO化修饰作一综述．     

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.     

蛋白质SUMO化和泛素化修饰的关系

泛素化和SUMO化是蛋白质翻译后修饰的重要方式,广泛参与调节蛋白质功能和细胞生命活动各个环节。多聚泛素化降解蛋白质,而SUMO化主要调节蛋白质的相互作用和定位等。在不同情况下,SUMO化和泛素化既可协同调节蛋白质功能,也可相互拮抗。最近研究发现,某些底物的SUMO化能够激活体内一类新发现的SUMO依赖的泛素连接酶,启动泛素-蛋白酶体途径降解底物,导致蛋白质SUMO化和汔素化的关系进一步精细化和复杂化。     

SENPs对肿瘤发展的分子调控机理

SUMO化修饰是一种把小泛素相关修饰物(small ubiquition related modifier,SUMO)共价连接到细胞内靶蛋白半胱氨酸残基上的一种蛋白质翻译后修饰。SUMO化修饰参与并调控着多种细胞进程,如转录调控、核转运和信号转导等。SUMO化修饰是一种动态可逆的修饰方式。SUMO特异性蛋白酶(SUMO-specific proteases,SENPs)可以使SUMO化修饰的蛋白质发生去SUMO化,在维持细胞内SUMO化与去SUMO化的平衡中起重要作用。研究表明,SENPs与多种癌症的发生发展密切相关,如SENP1能直接调节多条致癌通路,诱发正常的前列腺上皮细胞状态异常。癌细胞中的SENP3能诱导血管生成。因此,对去SUMO化机制研究可以为开发癌症治疗药物提供新的思路。     

SUMO化修饰对NF-kB信号通路的调控

小泛素相关修饰物(small ubiquitin-related modifier,SUMO)经由一系列酶介导的生化级联反应共价结合于靶蛋白的赖氨酸残基上,稳定靶蛋白免受降解的过程称为SUMO化修饰(SUMOylation).核转录因子kB(nuclear factors kB,NF-kB)是公认的炎症和免疫反应的重要调节因子,并与糖尿病的发生发展密切相关.近年来研究发现,不仅NF-kB抑制蛋白(inhibitor of NF-kB,IkB)的SUMO化修饰参与NF-kB信号通路的调节,而且SUMO酶可以直接调节NF-kB对靶基因的转录.现就SUMO亚型及结构,SUMO化修饰与去SUMO化修饰过程,SUMO、SUMO酶对NF-kB的转录调控及其与糖尿病相关性的最新研究进展作以综述.     

SENP1与前列腺癌

SUMO (small ubiquitin-related modifier)是一种小泛素相关修饰物,能共价结合许多调控基因转录的重要蛋白,包括转录因子、转录辅助因子等．SUMO化修饰对蛋白-蛋白之间的相互作用、亚细胞定位、基因转录的活性以及靶蛋白的稳定性等具有重要的调节作用． SUMO化修饰是一个动态可逆的过程,将SUMO从靶蛋白上去除,称为去SUMO化（desumoylation）,去SUMO化是SUMO特异蛋白酶（SUMO-specific proteases,SENPｓ）的主要功能．由于SUMO化是近几年才发现的一种新的蛋白质翻译后修饰系统,对其生物学功能还不十分清楚．前列腺癌是男性最常见的恶性肿瘤,最近的研究发现,SENP1在前列腺癌细胞中高表达,而且雄激素能诱导SENP1的表达,表明SENP1与前列腺癌的发生、发展密切相关．在本篇综述中,我们将就SENP1作一介绍．     

Identification and molecular properties of SUMO-binding proteins in Arabidopsis

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.     

Functional reconstitution of a tunable E3-dependent sumoylation pathway in Escherichia coli

SUMO (small ubiquitin-related modifier) is a reversible post-translational protein modifier that alters the localization, activity, or stability of proteins to which it is attached. Many enzymes participate in regulated SUMO-conjugation and SUMO-deconjugation pathways. Hundreds of SUMO targets are currently known, with the majority being nuclear proteins. However, the dynamic and reversible nature of this modification and the large number of natively sumoylated proteins in eukaryotic proteomes makes molecular dissection of sumoylation in eukaryotic cells challenging. Here, we have reconstituted a complete mammalian SUMO-conjugation cascade in Escherichia coli cells that involves a functional SUMO E3 ligase, which effectively biases the sumoylation of both native and engineered substrate proteins. Our sumo-engineered E. coli cells have several advantages including efficient protein conjugation and physiologically relevant sumoylation patterns. Overall, this system provides a rapid and controllable platform for studying the enzymology of the entire sumoylation cascade directly in living cells.     

A genome-wide analysis of sumoylation-related biological processes and functions in human nucleus

Protein sumoylation is an important reversible post-translational modification of proteins in the nucleus, and it orchestrates a variety of the cellular processes. Genome-wide analysis of functional abundance and distribution of Small Ubiquitin-related MOdifier (SUMO) substrates may shed a light on how sumoylation is involved in nuclear biological processes and functions. Two interesting questions about sumoylation have emerged: (1) how many SUMO substrates exist in mammalian proteomes, such as human and mouse, (2) and what are their functions and how are they involved in a variety of biological processes? To address these two questions,we present an in silico genome-scale analysis for SUMO substrates in human. Based on the pattern recognition and phylogenetic conservation, we retrieved a list of 2683 potential SUMO substrates conserved in both human and mouse. Then, by functional enrichment analysis, we surveyed the over-represented GO terms and functional domains of them against the whole human proteome. Besides the consistence between our analyses and in vivo or in vitro work, the in silico predicted candidates also point to several potential roles of sumoylation, e.g., perception of sound. These potential SUMO substrates in human are of great value for further in vivo or in vitro experimental analysis.     

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.     

Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation

Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process.     

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.     

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.