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。     

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化修饰与肿瘤调控

SUMO化修饰是一种重要的蛋白质翻译后修饰方式,在细胞周期调控、细胞代谢、基因转录、DNA损伤和修复等众多细胞生物学过程中,对底物蛋白质的表达、定位和活性进行调控。蛋白质SUMO化修饰是动态可逆的过程,去SUMO化修饰由SUMO特异性蛋白酶(SENPs)家族成员所催化。由于受到SUMO化修饰的底物蛋白种类众多、功能多样,SUMO化修饰能够在整体和特定蛋白质修饰层面,参与调控肿瘤的发生发展,并且这种调控机制非常复杂,比如调控细胞周期的进程、DNA损伤和基因组不稳定性、肿瘤代谢与生长、抗肿瘤免疫等。SENPs家族成员是底物蛋白质SUMO化修饰程度的决定者,该研究团队对SENPs家族成员在肿瘤中的作用开展了系列研究,因此该文也将以SENP1和SENP3为例,对SENPs在肿瘤进程中的作用及其作用机制展开介绍。     

SUMO蛋白酶活性片段的表达、纯化及活性测定

利用PCR技术人工合成编码酿酒酵母泛素样特异性蛋白酶1 (Ubiquitin-like specific protease 1,Ulp1)第403到621个氨基酸残基之间的DNA片段Ulp1p,并连接到大肠杆菌表达载体pET-3c中,构建出重组表达质粒pET-Ulp1p。将重组质粒转化至大肠杆菌BL21(DE3)中,氨苄青霉素抗性筛选转化子。经IPTG诱导4h后, SDS-PAGE结果显示,Ulp1p为可溶性表达,表达量占菌体总蛋白的50.8%。通过Ni-NTA凝胶亲和层析和G-25凝胶层析联用可以获得纯度大于95%的Ulp1p。Western-blotting分析表明,Ulp1p能与6xHis抗体产生免疫反应。以重组蛋白SUMO-hEGF(人表皮生长因子)和GST-SUMO-MT(金属硫蛋白)为底物进行酶切分析,结果显示,Ulp1p能特异性水解这两种SUMO融合蛋白,其比活为1.386 x104U/mg。     

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。     

HIF-1α的泛素化和SUMO化修饰

低氧诱导因子-1（hypoxia-inducible factor-1,HIF-1）是异二聚体的转录因子,由氧敏感的α亚基和在细胞内稳定表达的β亚基组成,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.泛素是一种由76个氨基酸残基组成的保守性多肽,广泛存在真核生物中.SUMO是泛素样蛋白家族成员,分子量约为12 kD的小蛋白,从拟南芥到人类普遍存在.泛素和SUMO可共价结合许多靶底物蛋白,对其进行翻译后修饰,该过程分别称为泛素化与SUMO化.近来研究显示,HIF-1α的翻译后修饰如泛素化、SUMO化可调节其的稳定性,从而改变HIF 1α的转录激活活性.本文主要就HIF-1α泛素化及SUMO化修饰等问题作一综述.     

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.     

串联双SUMO融合促进重组CRM197蛋白在大肠杆菌中高效可溶表达及其纯化与表征

白喉毒素突变体蛋白(cross-reacting material 197, CRM197)是一种常用的多糖结合疫苗载体,其在大肠杆菌体系中往往以包涵体形式表达。为了改善通过包涵体复性来制备CRM197而导致的收率低、操作烦琐等不足,研究设计了一种基于SUMO标签串联的策略,改善重组CRM197的可溶性,同时使用Origami B (DE3)大肠杆菌宿主,成功使重组CRM197蛋白上清液可溶比例提高至95%以上。同时简化了CRM197蛋白的获取途径,经两步Ni亲和层析和一步酶切除去融合标签等手段建立了从破菌上清液中纯化CRM197的方法,获得纯度高于98%的CRM197且收率大于80%。通过圆二色光谱及荧光发射光谱分析等证实纯化所得CRM197空间结构折叠正确。抗原性ELISA实验结果表明所制得的CRM197蛋白与CRM197标准品蛋白抗原性相当。研究结果表明串联双SUMO标签对重组CRM197在大肠杆菌中高效、可溶表达具有促进作用,并建立了快速纯化体系,获得了具有较好免疫原性的CRM197蛋白,为改善重组蛋白在原核表达体系的可溶表达设计提供了新思路。     

植物中的小类泛素修饰因子(SUMO)修饰系统

小类泛素修饰因子(SUMO)是一种可以通过异肽键修饰其他蛋白质的小分子多肽,是在动物中发现的类似于泛素的蛋白质修饰方式。它参与调节了植物对逆境反应、病源防御、脱落酸信号传递以及成花诱导等许多过程,是植物正常生长发育中必不可少的蛋白质修饰方式。文章就其研究进展作一简要介绍。     

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.     

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR.     

The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation

The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling.     

SUMO-TAT-Klf4融合蛋白的原核表达及纯化

Klf4是体细胞诱导成多能干细胞的重要转录因子之一,参与细胞增殖分化。本研究通过将小鼠Klf4基因、穿膜肽TAT以及小分子泛素样修饰蛋白SUMO融合并转入大肠杆菌Rosseta(DE3)中进行表达,经PCR鉴定和酶切验证后,用IPTG诱导后表达出约78 k D的融合蛋白,利用Ni-NTA亲和层析胶纯化获得了高质量的SUMO-TAT-Klf4蛋白,使用Western blotting检测显示特异性良好。本研究成功构建了pET3c-SUMO-TAT-Klf4原核表达载体,有利于获得大量的Klf4蛋白,并以期提高其穿透细胞膜的能力,为后续利用重编程因子融合蛋白诱导体细胞成为iPS细胞奠定基础。     

Memo蛋白的融合表达和纯化

目的:一些临床预后较差的恶性肿瘤一般具有很高的转移性和侵袭性,近来的研究表明,Memo蛋白与恶性肿瘤的这些特性有十分密切的关系。本研究拟克隆、融合表达和纯化Memo蛋白。方法:用PCR方法从乳腺文库中扩增Memo蛋白编码序列,并将其以正确相位与pGST载体中的GST编码序列融合,构建成重组质粒pGST-Memo。将此重组质粒转化大肠杆菌DH5α后,用谷胱甘肽-Sepharose4B纯化融合蛋白,并用Western印迹检测融合蛋白的表达。结果:构建了Memo蛋白的融合表达载体,并在大肠杆菌DH5α中得到表达,经谷胱甘肽-Sepharose4B亲和层析获得了纯化的GST-Memo融合蛋白。Western印迹检测表明,此蛋白可与GST抗体反应。结论:Memo蛋白的克隆、融合表达和纯化,为进一步研究恶性肿瘤的转移性、侵袭性及其相关机理提供了有用的材料。     

Activation of the Double-stranded RNA-dependent Protein Kinase PKR by Small Ubiquitin-like Modifier (SUMO)

The dsRNA-dependent kinase PKR is an interferon-inducible protein with ability to phosphorylate the α subunit of the eukaryotic initiation factor (eIF)-2 complex, resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. Here we analyzed the modification of PKR by the small ubiquitin-like modifiers SUMO1 and SUMO2 and evaluated the consequences of PKR SUMOylation. Our results indicate that PKR is modified by both SUMO1 and SUMO2, in vitro and in vivo. We identified lysine residues Lys-60, Lys-150, and Lys-440 as SUMOylation sites in PKR. We show that SUMO is required for efficient PKR-dsRNA binding, PKR dimerization, and eIF2α phosphorylation. Furthermore, we demonstrate that SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA, whereas a PKR SUMOylation mutant is impaired in its ability to inhibit protein synthesis and shows reduced capability to control vesicular stomatitis virus replication and to induce apoptosis in response to vesicular stomatitis virus infection. In summary, our data demonstrate the important role of SUMO in processes mediated by the activation of PKR.     

Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.     

凋亡素融合蛋白的可溶性表达及活性分析

目的:构建PET-28a-SPA原核表达载体,在大肠杆菌BL21(DE3)中实现其高效可溶性表达,测定对肿瘤细胞的凋亡效果。方法:本实验在获得凋亡蛋白融合基因的基础上,成功地构建了重组表达质粒PET-28a-SPA,将阳性重组质粒转化表达受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,表达产物经聚丙烯酰胺凝胶电泳检测和Western blot检测,并采用MTT法检测其对肿瘤细胞的增殖抑制。结果:表达产物经聚丙烯酰胺凝胶电泳检测,凋亡蛋白融合基因获得高效表达,软件分析表明表达蛋白占菌体蛋白20%左右。上清表达量约为10%。上清蛋白经纯化后,Western blot结果显示,利用凋亡蛋白单克隆抗体可以很好地和所表达的蛋白带特异性结合,并且对A549肺癌细胞及Hela细胞具有一定的凋亡作用。结论:所获凋亡蛋白以高效胞质可溶形式表达,为其研制有效的肿瘤免疫治疗靶向药物提供一定的基础。