去泛素化酶与肿瘤

泛素化修饰(ubiquitination modification)广泛存在于真核生物,通过26S蛋白酶体降解途径或信号传递等,改变蛋白质稳定性、定位和活性等功能,参与细胞的周期、转录、炎症、肿瘤和免疫等各项功能,是一类复杂的动态调控系统.泛素化调节是一个可逆过程,被泛素连接酶(ubiquitin ligase,E3)...     

去泛素化酶USP11在细胞中的功能作用及其研究进展

Ubiquitin-specific protease 11(USP11)属于半胱氨酸蛋白酶,是去泛素化酶家族(deubiquitinating enzymes,DUBs)的重要成员之一。近年来研究表明USP11能调节细胞内众多蛋白底物的稳定性及功能,包括DNA修复蛋白、病毒RNA复制相关蛋白、TGFβ和NF-κB信号转导通路相关蛋白等,在疾病的发生发展中起着重要的作用。主要综述了USP11的结构、在细胞中的分子功能以及与肿瘤和病毒性疾病的关系,探讨了USP11作为治疗分子靶标的可能性。     

巴西橡胶树去泛素化酶UCHs基因的克隆及功能分析

为探究去泛素化酶中泛素羧基末端水解酶（UCHs）在巴西橡胶树乳管泛素化过程中发挥的潜在功能,从巴西橡胶树中成功分离2个UCHs家族成员（HbUCH-L3和HbUCH-L5）的全长序列,开放阅读框分别长993、558 bp,编码330、185个氨基酸,具有典型的UCHs结构域;qRT-PCR结果表明HbUCH-L3和HbUCH-L5在各组织中均有表达,且在胶乳中表达丰度较低;HbUCHs重组蛋白的体外泛素化底物切割试验表明,HbUCH-L3和HbUCH-L5均具有水解泛素的功能。HbUCHs显著降低C乳清中总蛋白整体泛素化水平,且HbUCH-L3去泛素化酶活性高于HbUCH-L5。由此推测橡胶树UCHs在乳管中参与维持泛素化动态平衡进而发挥其特定的生物学功能,但具体的作用及调控机制尚不清楚。     

针对泛素化与去泛素化酶的化学探针

蛋白质泛素化对真核细胞内几乎所有生理过程都具备调控作用。新的泛素化与去泛素化酶的发现、功能机制研究及相关化学分子干预是该领域的重要科学命题。针对泛素化与去泛素化酶的化学探针开发促进了人们对于蛋白质泛素化的形成、募集、信号传导及脱除过程中所涉及生化事件的理解。本文总结了近年来针对泛素化与去泛素化酶化学探针的发展，归纳了不同类型探针的化学结构及合成方法，并讨论了它们的各类应用，包括筛查泛素依赖的信号传导系统、监控泛素相关酶活性、辅助泛素相关的识别和催化过程的分子机制解析等。     

协同伴侣分子CHIP的E3连接酶活性及其生物学意义

CHIP(carboxy terminus of Hsc70 interacting protein)是一种新发现的具有泛素化连接酶活性的协同分子伴侣,它N端有TPR(the tetratricopeptide repeat)结构,可以和分子伴侣结合,C端有U-box结构,具有E3酶功能.CHIP可以介导一系列重要蛋白质的泛素化降解,从而维持细胞内蛋白质的质量.现就CHIP的结构和功能、CHIP的底物特征、CHIP的生物学作用以及CHIP在疾病发生中的可能作用做一综述.     

细菌中的去泛素化酶及其生物学功能研究进展

在细菌感染过程中,宿主细胞可以利用自身泛素系统对其进行免疫应答。研究发现,在宿主与细菌协同进化过程中,细菌可以编码去泛素化酶靶向宿主泛素系统,降低宿主炎症信号反应,这有利于细菌的生存与繁殖。该文综合介绍了目前在细菌中已发现的去泛素化酶并将其分类总结,此外,该文详细阐述了OTU家族去泛素化酶、CE家族去泛素化酶的切割特异性和生物学功能,还介绍了具有特殊催化活性的去泛素化酶。深入研究去泛素化酶的分子机制将有助于理解其生物学功能,同时可为开发新的治疗药物和抗感染疫苗提供信息。     

组蛋白的泛素化与去泛素化修饰

泛素在真核生物体内广泛存在,泛素化修饰是转录后的修饰方式之一;组蛋白是染色质的主要成分之一,与基因的表达有密切关系。组蛋白的泛素化修饰与经典的蛋白质的泛素调节途径不同,不会导致蛋白质的降解,但是能够招募核小体到染色体、参与X染色体的失活、影响组蛋白的甲基化和基因的转录。组蛋白的去泛素化修饰同样与染色质的结构及基因表达密切相关。组蛋白的泛素化和磷酸化、乙酰化、甲基化修饰之间还存在协同和级联效应。     

分子伴侣结构与功能的研究进展

分子伴侣是细胞内一类能够协助其他多肽进行正常折叠、组装、转运、降解的蛋白,并在 DNA的复制、转录,细胞骨架功能,细胞内的信号转导等广泛的领域,都发挥着重要的生理作用,其结构与功能异常会导致多种相关的疾病。简要综述了分子伴侣结构与功能方面的研究进展。     

泛素连接酶和去泛素化酶在帕金森病中的作用

中脑黑质多巴胺能神经元特异性损伤和α突触核蛋白聚集的分子机制是帕金森病（Parkinson’s disease,PD）研究领域亟待解决的问题。蛋白质异常聚集很大程度上是由于泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）功能障碍引起的。蛋白质泛素化由一系列泛素化酶级联反应促进，并受去泛素化酶（deubiquitylases,DUBs）的反向调节。泛素化和去泛素化过程异常导致蛋白质异常聚集和包涵体形成，进而损伤神经元。近来研究报道，蛋白质的泛素化和去泛素化修饰在PD的发病机制中发挥重要作用。E3泛素连接酶促进蛋白质的泛素化，有利于α突触核蛋白的清除、促进多巴胺能神经元的存活、维持线粒体的功能等。DUBs可以去掉底物蛋白质的泛素化修饰，抑制α突触核蛋白的降解，调控线粒体的功能和神经元内铁的稳态。本文以E3泛素连接酶和DUBs为切入点，综述了蛋白质泛素化和去泛素化修饰参与多巴胺能神经元损伤机制的最新研究进展。     

分子内分子伴侣机制的研究进展

在原核生物、真核生物及病毒中,一些蛋白质的折叠不符合Anfinsen原则,即依靠自身的氨基酸序列是不够的,还需一段被称为分子内分子伴侣(IMC)的肽段来协助折叠．根据机制不同,IMC可分为两类：第一类IMC引导成熟肽折叠为具有空间结构的蛋白质;第二类IMC协助成熟肽的多聚化而使其获得生物学功能．IMC能提供比分子伴侣更契合的结构,更有效地引导成熟肽折叠,是一种更优的折叠策略．研究IMC分子机制,不仅能够确定IMC上哪些残基的协同作用引导成熟肽折叠,而且可通过改变或修饰其侧链来改造成熟肽,拓展传统的蛋白质工程．     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

Chaperonin-Affected Folding of Globular Proteins

We studied the effect of GroEL on the kinetic refolding of-lactalbumin by stopped-flow fluorescence techniques. We usedwild-type GroEL and its ATPase-defficient mutant D398A, and studied thebinding constants between GroEL and the molten globule foldingintermediate at various concentrations of ADP and ATP. The results arecompared with titration of GroEL with the nucleotides, ADP, ATP-analogs(ATP-S and AMP-PNP) and ATP, which have shown that bothADP and the ATP analogs are bound to GroEL in a non-cooperativemanner but that ATP shows a cooperative effect. Similarly, the bindingconstant between GroEL and the folding intermediate decreased in acooperative manner with an increase in ATP concentration although itshowed non-cooperative decrease with respect to ADP concentration. Itis shown that the allosteric control of GroEL by the nucleotides isresponsible for the above behavior of GroEL and that the observeddifference between the ATP- and ADP-induced transitions of GroEL isbrought about by a small difference in an allosteric parameter (the ratio ofthe nucleotide affinities of GroEL in the high-affinity and the low-affinitystates), i.e., 4.1 for ATP and 2.6 for ADP.     

去泛素化酶在肿瘤上皮间质转化中的作用

肿瘤的侵袭和转移是加剧肿瘤恶化的主要原因,也是导致患者预后不良的根本原因。近年来大量研究发现,大部分肿瘤的转移都依赖于上皮间质转化(epithelial-mesenchymal transition, EMT)的发生,此外EMT也与肿瘤干性和肿瘤耐药等诸多肿瘤恶性行为密切相关,因此有效的抑制EMT的发生将可能极大的有利于肿瘤的治疗。去泛素化酶(deubiquitinating enzymes, DUBs)的主要功能之一就是通过移除底物蛋白质上泛素链,避免其通过泛素蛋白酶体途径降解,来维持细胞内蛋白质水平的动态平衡。去泛素化酶作为调节蛋白质泛素化修饰的一类重要酶类,其异常表达或酶活性的改变通常都会导致疾病的发生。众多研究发现,部分去泛素化酶在肿瘤侵袭和转移过程中表达失衡,在肿瘤转移的过程中扮演着重要的角色。EMT是指由上皮型细胞转变为间质型细胞的动态细胞生物学过程,在该过程中涉及到例如Snial1、Slug、ZEB1等EMT相关转录因子和细胞表面的例如E-钙黏着蛋白、N-钙黏着蛋白等分子标志物表达水平的变化。这些蛋白质通常具有不稳定性,易被降解等特征。EMT过程的发生,涉及到许多蛋白质稳定性的调节,而去泛素化酶作为一类维持蛋白质稳定的重要酶类,在调节这些蛋白质的稳定性方面发挥着重要的作用。EMT的发生也与TGF-β通路、Wnt通路等细胞内众多信号通路的异常活化密不可分,去泛素化酶通过介导这些信号通路的活化,从而间接的调节EMT发生发展。去泛素化酶通过调节EMT相关分子或EMT相关信号通路等多种方式直接或间接影响EMT进展,因此,通过靶向于去泛素化酶抑制肿瘤的侵袭和转移,将为肿瘤治疗提供新的治疗手段和方案,从而有效的推动肿瘤的治疗。本文主要就去泛素化酶在调节EMT相关分子以及信号通路等方面,阐述去泛素化酶在EMT过程中所发挥的重要作用及其作为肿瘤治疗靶点的可能性。     

叶绿体J蛋白研究进展

J蛋白是广泛存在于细胞内的一种分子伴侣。它作为Hsp70的辅伴侣分子有着广泛而复杂的生物学功能。本文概述了J蛋白的相关概念、结构、种类、分布及其作用机制,并重点讨论了其在叶绿体内的功能。最后对有关J蛋白研究中需要解决的问题做了展望。     

热休克蛋白对细胞凋亡的调控作用

热休克蛋白属于细胞内分子伴侣蛋白,除涉及细胞内一些蛋白质分子构象和稳定性的调节之外,热休克蛋白对细胞应激、代谢、增殖以及凋亡等生理过程均具有重要的调控作用。研究表明热休克蛋白对细胞凋亡的调控机制是复杂的,可直接作用于与凋亡相关的蛋白质,也可以通过影响细胞信号传递而间接影响凋亡的发生。由于热休克蛋白对细胞凋亡的调控机制大多依赖于其分子伴侣功能,阻断热休克蛋白的伴侣功能已经成为研究药物诱导肿瘤细胞凋亡的重要靶点。     

The Effect of Clp Proteins on DnaK-Dependent Refolding of Bacterial Luciferases

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK–DnaJ–GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.     

The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network

The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation. sHsps share the conserved α-crystallin domain (ACD) and gain functional specificity through variable and largely disordered N- and C-terminal extensions (NTE, CTE). They form large, polydisperse oligomers through multiple, weak interactions between NTE/CTEs and ACD dimers. Sequence variations of sHsps and the large variability of sHsp oligomers enable sHsps to fulfill diverse tasks in the PQC network. sHsp oligomers represent inactive yet dynamic resting states that are rapidly deoligomerized and activated upon stress conditions, releasing substrate binding sites in NTEs and ACDs Bound substrates are usually isolated in large sHsp/substrate complexes. This sequestration activity of sHsps represents a third strategy of the proteostasis network. Substrate sequestration reduces the burden for other PQC components during immediate and persistent stress conditions. Sequestered substrates can be released and directed towards refolding pathways by ATP-dependent Hsp70/Hsp100 chaperones or sorted for degradation by autophagic pathways. sHsps can also maintain the dynamic state of phase-separated stress granules (SGs), which store mRNA and translation factors, by reducing the accumulation of misfolded proteins inside SGs and preventing unfolding of SG components. This ensures SG disassembly and regain of translational capacity during recovery periods.     

去泛素化酶在肾细胞癌中的作用

肾细胞癌（renal cell carcinoma,RCC）是成人肾脏的原发性恶性肿瘤。泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）对控制蛋白质水平和调节生理病理过程至关重要。去泛素化酶（deubiquitinases,DUBs）是UPS的关键成分,特别是从靶蛋白中去除泛素链,通过严格调节正常生理学中泛素化和去泛素化之间的平衡,对蛋白质稳态和质量控制显示出至关重要的作用。越来越多的研究表明,功能异常的DUBs与RCC的进展和转移有关。根据底物的不同,一些DUB可能会抑制RCC,而另一些则促进。本文综述了RCC相关DUB的最新研究进展,描述了其分类、功能作用,总结了DUB在RCC中的作用和作用机制,并讨论了靶向DUBs用于癌症治疗。     

植物中的核糖体失活蛋白及其抗病毒机制

植物中的核糖体失活蛋白是一类分布于植物体内的毒蛋白,其作用于真核细胞大亚基28S导致核糖体失活,抑制蛋白质的生物合成,从而对细胞产生毒害作用.文章简述了植物核糖体失活蛋白的酶活性和抗病毒的可能分子机制.     

The Abi Proteins and Their Involvement in Bacteriocin Self-Immunity

The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other''s bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.Bacteriocins are ribosomally synthesized antimicrobial peptides and proteins produced by a wide variety of bacterial genera. The majority of bacteriocins from Gram-positive bacteria are classified into two groups: the class I lantibiotics, containing posttranslationally modified peptides with ring-forming lanthionine or methyllanthionine residues, and the nonmodified class II peptide bacteriocins (8, 33, 34). Class II bacteriocins are further subdivided into pediocin-like bacteriocins (class IIa), two-peptide bacteriocins (class IIb), and nonpediocin one-peptide bacteriocins (class IIc) (33). Bacteriocin-producing bacteria normally possess a mechanism of immunity to protect themselves from their own bacteriocins, and such self-immunity is often mediated by a dedicated protein (32). For a few bacteriocin systems, the mechanisms by which these proteins confer immunity have been elucidated. For instance, immunity to the lantibiotic nisin (class I) involves a combined action which includes (i) sequestering of bacteriocins on the bacterial cell membrane by a protein called NisI and (ii) removal of the bacteriocins from cells by a dedicated ABC transporter (NisFEG) (39, 44). On the other hand, proteins conferring immunity to pediocin-like bacteriocins (class IIa) as well as lactococcins A and B (class IIc) have been shown to bind directly to the bacteriocin receptor and thereby inhibit pore formation (13). Hitherto, no immunity mechanism is known for any class IIb two-peptide bacteriocins.Recently, we reported that several bacteriocin loci encode proteins belonging to the Pfam Abi protein family (Pfam accession no. PF02517) (14). These loci include the plantaricin (pln) locus of Lactobacillus plantarum, encoding two two-peptide bacteriocins (12), the multibacteriocin pnc locus of Streptococcus pneumoniae (25), and the streptolysin S (sag) locus found in group A streptococci (35) (Fig. ​(Fig.1A).1A). Some of the Abi proteins encoded in these loci (PlnI in L. plantarum, PncO in S. pneumoniae, and SagE in Streptococcus pyogenes) are probable bacteriocin self-immunity proteins on the basis of gene knockout studies (10, 25) and genetic organization (i.e., being closely associated with bacteriocin structural genes), while others (e.g., PlnP and PlnTUVW in L. plantarum and PncP in S. pneumoniae) have completely unknown functions.Open in a separate windowFIG. 1.Abi-associated bacteriocin and bacteriocin-like loci. (A) Three known bacteriocin loci containing Abi genes, including the pln locus of L. plantarum (14), the pnc locus of S. pneumoniae (25), and the sag locus of S. pyogenes (10). (B) Seven potential new bacteriocin loci identified by genome mining for bacteriocin-associated Abi genes. Abi genes are shown as black arrows, bacteriocin structural genes are shown in red, ABC transporter genes are shown in blue, regulatory genes are shown in green, and other genes are shown in white. Gene names or locus tags are shown below the arrows. The genes are drawn approximately to scale. The boxed arrows in the L. sakei 23K genome represent a disrupted histidine protein kinase gene.The Abi family, also known as the CAAX amino-terminal protease family, consists of putative membrane-bound metalloproteases from both eukaryotes and prokaryotes (38), with the majority being bacterial proteins (90%). The Abi family is recognized by three highly conserved motifs (38): motif 1, consisting of two neighboring and invariant glutamate residues and a conserved arginine separated by three residues (EEXXXR, where X denotes any amino acid); motif 2, consisting of a conserved phenylalanine and a conserved histidine separated by three residues (FXXXH); and motif 3, with an invariant histidine. The three conserved motifs are thought to constitute the active site of the Abi protease, and their importance in proteolytic activity has been demonstrated by mutational analysis of the yeast Abi protease RCE1 (15). In eukaryotic cells, Abi family proteins are involved in membrane targeting of proteins harboring the C-terminal sequence CAAX (C, cysteine; A, aliphatic amino acid; and X, any amino acid) via a process known as prenylation, which consists of the following three sequential reactions (23): (i) a geranylgeranyltransferase/farnesyltransferase attaches a prenyl group (lipid anchor) to the cysteine in the fourth-to-last position, (ii) a CAAX protease of the Abi family cleaves off the three C-terminal amino acids (-AAX), and (iii) an isoprenylcysteine carboxyl methyltransferase then attaches a methyl group to the C-terminal cysteine.Despite being widespread in prokaryotic genomes, the function of Abi proteins has not been investigated much in bacteria, with the exception of gene knockout experiments on the Abi genes sagE and pncO, which implies their involvement in bacteriocin self-immunity in streptococci (10, 25). Another Abi-like gene, prsW in Bacillus subtilis, has been studied to some extent (16). PrsW does not belong to any Pfam family but contains the same three motifs as the Abi proteins, with the exception of the conserved histidine in motif 2, which has been replaced by a glutamate in this protein. PrsW is a protease involved in response to antimicrobial peptides via a process known as regulated intracellular proteolysis (16). In this process, PrsW together with another protease (YluC) cleaves an anti-σ factor (RsiW) to release σ^W, which subsequently regulates gene expression in a manner that protects the cells from antimicrobial peptides. However, the PrsW target protein RsiW in B. subtilis does not harbor the C-terminal consensus sequence CAAX found for Abi target proteins in eukaryotes.Pfam (http://pfam.sanger.ac.uk/) is a comprehensive collection of protein families and domains. For each protein family in Pfam, a profile hidden Markov model (profile HMM) is built from an alignment of sequences from nonredundant representatives of the family (seed sequences), and this profile HMM is then used in an iterative fashion to find new members of the protein family (18, 43). The Abi family in Pfam contains a large number of sequences (1,966 by September 2009). However, several proteins containing Abi or Abi-like motifs are somehow not detected by the current search tool. Examples of this include PrsW from B. subtilis and PlnL from L. plantarum, which apparently contain all three motifs but somehow do not fit into Pfam''s Abi family. These observations suggest that the profile HMM for Abi may be based upon a slightly skewed sample of seed sequences, resulting in a low sensitivity. We provide here an updated profile HMM to detect Abi-like proteins in prokaryotes that are omitted in the present protein family. Furthermore, searches for novel Abi-associated bacteriocin loci were also performed in silico. Several potentially novel bacteriocin loci were identified, and one of them was assessed further by experimentation. The role of bacteriocin-associated Abi genes in self-immunity was also addressed by heterologous expression and site-directed mutagenesis.