泛素连接酶MDM2促进外源NOLC1的泛素化和降解

目的研究泛素连接酶MDM2对其调节因子NOLC1的泛素化和降解。方法克隆原核表达了NOLC1全长蛋白和其核定位信号区域,在体外泛素化体系中利用有泛素连接酶活性的重组MDM2研究其对NOLC1的泛素化。在哺乳动物细胞中,研究MDM2对外源转染的NOLC1的降解。结果在体外泛素化体系中MDM2泛素化NOLC1全长和其核定位信号区域;在哺乳动物细胞中,MDM2促进外源NOLC1降解超过70%,且NOLC1的降解通过蛋白酶体途经实现。结论 MDM2促进外源NOLC1的泛素化和降解,为研究MDM2-TP53-NOLC1之间的相互调节提供了新的线索。     

泛素-蛋白酶体途径及意义

泛素-蛋白酶体途径介导的蛋白降解是机体调节细胞内蛋白水平与功能的一个重要机制。负责执行这个调控过程的组成成分包括泛素及其启动酶系统和蛋白酶体系统。泛素启动酶系统负责活化泛素,并将其结合到待降解的蛋白上,形成靶蛋白多聚泛素链,即泛素化。蛋白酶体系统可以识别已泛素化的蛋白并将其降解。此外,细胞内还有另一类解离泛素链分子的去泛素化蛋白酶形成反向调节。泛素-蛋白酶体途径涉及许多细胞的生理过程,其调节异常与多种疾病的发生有关。     

泛素结合结构域在泛素化底物鉴定中的应用

泛素化(Ubiquitylation)是目前真核细胞内已知的最复杂的翻译后修饰。泛素化底物的识别需要一类特异性受体蛋白介导,这些受体蛋白往往包含一个或多个泛素结合结构域(UBDs)。UBD-泛素间的特异性结合决定了泛素化底物功能的特异性。目前已发现20多种UBDs超家族可识别泛素化底物上的特异性功能团进而传递信号。因此深入了解UBD的识别机制对新UBD的发现及泛素化底物的鉴定具有重要意义。     

P53泛素化的非Mdm2依赖途径

泛素化、磷酸化、甲基化和乙酰化均是P53蛋白最重要的修饰形式。其中泛素化对P53蛋白功能的调控起中心作用。Mdm2具有E3连接酶活性，能使P53蛋白发生泛素化而降解。但近年研究发现其他几种E3泛素连接酶也能使P53蛋白发生泛素化而降解，提示存在P53蛋白泛素化的非Mdm2依赖途径。     

泛素羧基端水解酶L1在人类疾病发生发展中的调控作用

泛素-蛋白酶系统(ubiquitin proteasome system,UPS)在细胞分裂、细胞信号转导以及细胞程序死亡的过程中起到非常重要的作用.此系统主要是靠泛素化酶和去泛素化酶来实施对于目的蛋白的快速精确调控.其中,泛素羧基端水解酶L1 (ubiquitin carboxy-terminal hydrolase L1,UCH-L1)是去泛素化酶家族中的一个重要的分子,是由223个氨基酸组成的一类半胱氨酸水解酶,通过识另和裂解目标蛋白上羧基末端第76位甘氨酸,可把泛素分子从目标蛋白的多聚泛素化链上切割下来,阻止目标蛋白被UPS系统降解,从而对目标蛋白的降解代谢负向调控.由此而产生游离的泛素单体,则进一步循环参与下一个目标蛋白的泛素化代谢.UCH-L1是一个多功能的分子,除了去泛素化酶功能,还有稳定泛素单体,泛素连接酶及参与细胞骨架蛋白调控,细胞微管形成等多功能作用.     

E3泛素连接酶在核转录共抑制因子SnoN蛋白降解中的作用

目前已知泛素-蛋白酶体途径是真核细胞内蛋白降解的主要途径,能选择性降解细胞内多种蛋白（如细胞周期蛋白、蛋白激酶、信号分子等）.其中,E3泛素连接酶因决定了靶蛋白底物的特异性,而成为该途径中起决定作用的关键酶[1].近年研究发现,核转录共抑制因子Ski相关活性蛋白N（SnoN）蛋白的泛素化降解在调控SnoN蛋白表达中起着重要作用[2].表现为多种水平多个环节上的调控,而且这种调控与肿瘤、发育障碍、中枢神经系统疾病、纤维化疾病等密切相关.本文拟对E3泛素连接酶介导SnoN蛋白泛素化的研究进展作一综述.     

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化和泛素化的关系进一步精细化和复杂化.     

泛素-蛋白酶体途径与EBV潜伏蛋白

泛素-蛋白酶体途径是真核细胞内重要的蛋白质调控系统,参与调节细胞周期进程、细胞增生与分化,以及信号传导等多种细胞生理过程,因此,细胞内蛋白泛素化降解是蛋白质重要的转录后修饰方式。EBV编码多种病毒蛋白,通过泛素．蛋白酶体途径调节病毒的潜伏,使病毒能在免疫活性较高的宿主体中存活。LMP1和LMP2A可能作为泛素．蛋白酶体途径的底物而受其调控,EBNA1则充当蛋白酶体降解过程中的阻滞剂。对它们在该途径中不同作用的深入了解将促使我们发展治疗EBV相关癌症的新策略。     

SCF E3泛素化连接酶的研究进展

Skp1-Cullins-F-box蛋白复合物(SCF复合物)E3泛素化连接酶是最大的泛素化连接酶家族成员,该蛋白复合体可以促进多种蛋白通过泛素化降解,对细胞周期、DNA复制等多种生物过程具有重要的调节作用。SCF E3泛素化连接酶在人类肿瘤中的异常激活可以诱导细胞无限制的增殖和基因组的不稳定,因此,E3泛素化连接酶复合物成员可以作为抗肿瘤治疗的靶点。本文主要介绍SCF E3泛素化连接酶作为抗癌药物的靶点和新型肿瘤抑制剂MLN4924的抗肿瘤作用。     

蛋白质泛素化降解途径

泛素系统(UPS)广泛存在于真核生物中，是精细的特异性的蛋白质降解系统。它由泛素、26S蛋白酶体、多种酶（如E1、E2、E3、去泛素酶）构成。在泛素系统中，泛素（Ubiquitin Ub）是一种序列保守的小分子蛋白，蛋白质与泛素结合后，被蛋白酶体以ATP依赖的方式降解。E1、E2酶分别称为泛素活化酶和泛素载体酶，使泛素通过Ub-腺苷酸中间产物形成E2-Ub巯基酯。泛素连接酶E3负责连接泛素和特异性的底物，这样泛素化的底物可以被26S蛋白酶体降解为若干肽段。泛素系统通过特异性的降解蛋白质，调节细胞分化、免疫反应，参与转录、离子通道、分泌的调控及神经元网络、细胞器的形成等等，泛素系统还与人类某些疾病有关。本文对泛素系统的成员、作用机制和功能的研究进展作了介绍。     

Ubiquitin protein ligase activity of the anti-apoptotic baculovirus protein Op-IAP3

The baculovirus inhibitor of apoptosis protein (IAP) Op-IAP3 is required to prevent apoptosis during infection of insect cells by Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) and inhibits apoptosis when overexpressed in insect and mammalian cells. Although previous reports have demonstrated that the RING domain is important for the anti-apoptotic function of Op-IAP3, the function of this domain in Op-IAP3 has not been studied. Here, the ability of Op-IAP3 to function as an E3 ubiquitin protein ligase was examined. Op-IAP3 expressed in the insect cell line Spodoptera frugiperda (Sf21) was ubiquitinated, but only if the RING domain was intact. In addition, co-expression of Op-IAP3 and the pro-apoptotic Drosophila protein HID resulted in the ubiquitination of HID. Recombinant Op-IAP3 protein also promoted the ubiquitination of both itself and recombinant HID protein in vitro, and the ubiquitination of HID required both the RING and BIR2 of Op-IAP3. Thus, we conclude that Op-IAP3 is a functional E3 ubiquitin ligase, and the ability to ubiquitinate pro-apoptotic cellular proteins such as HID may play an important role in the anti-apoptotic function of Op-IAP3.     

Ubiquitination of Lyn-kinase in rat basophilic leukemia RBL-2H3 cells

Receptor-mediated signal transduction pathways of cells involved in allergy and inflammations are extremely significant. Lyn is a member of the Src family of non-receptor protein tyrosine kinases and is associated with a number of cell surface receptors, including the B-cell antigen receptor and immunoglobulin E receptor (FcepsilonRI). Lyn is necessary for FcepsilonRI-mediated mast cell activation. To investigate how the level of Lyn is maintained in mast cell activation, it was studied whether Lyn binds to ubiquitin and is ubiquitinated for proteasomal degradation in cells. In the yeast two hybrid system, Lyn specifically interacted with ubiquitin in vivo. Furthermore, Lyn bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. Pulse-chase experiments indicated intracellular degradation of Lyn was associated with the generation of a high molecular weight complex in the presence of proteasome-specific inhibitor, lactacystin. This high molecular weight complex cross-reacted with anti-Lyn and anti-ubiquitin demonstrating the ubiquitination Lyn. Overexpression of Lyn and ubiquitin in COS 7.2 cells also resulted in the ubiquitination of Lyn in the presence of lactacystin, supporting the ubiquitination of Lyn by a proteasome specific pathway.     

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.     

Mutation and copy number analysis of LNX1 and Numbl in nervous system tumors

Alterations at chromosome locus 4q12 are frequently found in gliomas; this locus contains the receptor tyrosine kinase--encoding genes KIT, PDGFRA, and KDR (alias VEGFR2). Notable among the genes at this locus is LNX1, the ligand of Numb protein X. LNX1 encodes a PDZ domain containing protein, which interacts with the cell fate determinant Numbl, a Numb homolog-like gene involved in the maintenance of neural progenitor cells during embryonic neurogenesis. We performed a mutation analysis for LNX1 and Numbl genes. In addition, gene copy numbers of LNX1, Numbl, and KIT in human nervous system tumors were analyzed by chromogenic in situ hybridization. Tissue samples from 90 patients were screened for LNX1 and Numbl mutations, and tissue sections from 56 samples were analyzed for gene amplification status. Our analysis revealed missense mutations in LNX1 exons 3 and 5 and a single-nucleotide polymorphism in Numbl exon 6. In addition, polyglutamine repeat polymorphism was found in Numbl exon 10. Chromogenic in situ hybridization showed gene amplification of LNX1 in 10%, Numbl in 5%, and KIT in 6% of nervous system tumors. Both gene sequence alterations and amplifications of LNX1 and Numbl are present in a subset of human gliomas, and the role of these genes in neurogenesis suggests that they may contribute to development of glial tumors.     

Evading the proteasome: absence of lysine residues contributes to pertussis toxin activity by evasion of proteasome degradation

Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.     

应用组织芯片研究PBK/TOPK在人体正常及肿瘤组织中的表达

目的观察PBK/TOPK在人体正常组织和常见恶性肿瘤组织的分布和表达,探讨作为肿瘤标志物的可能性。方法应用免疫组织化学EnV ision法,在含有人体19种正常组织的组织芯片和含有19种恶性肿瘤组织的组织芯片上进行PBK/TOPK染色,检测PBK/TOPK蛋白的表达。结果在19种正常组织中,只有头皮的汗腺、睾丸的精原细胞、肝脏的胆管上皮、食管黏膜下腺体、胰腺外分泌部的导管上皮、前列腺的基底细胞和肾脏的远曲小管8种组织有不同数量细胞明确阳性表达。在19种恶性肿瘤组织中,乳腺癌、子宫颈癌、甲状腺癌等多种肿瘤组织中存在PBK/TOPK的大量、强阳性表达。结论PBK/TOPK在少数正常组织特定细胞中的明确表达及其在多种恶性肿瘤细胞的强阳性表达,为其成为研究肿瘤起源与发生的标志物提供了依据。     

Xic1 degradation in Xenopus egg extracts is coupled to initiation of DNA replication

CDK2 activity is regulated by phosphorylation/dephosphorylation, subcellular localization, cyclin levels, and cyclin dependent kinase inhibitors (CKIs). Using Xenopus egg extracts, we find that degradation of Xic1, a Xenopus p21(cip1)/p27(kip1) family member, is coupled to initiation of DNA replication. Xic1 turnover requires the formation of a prereplication complex (pre-RC). Additionally, downstream initiation factors including CDK2, Cdc7, and Cdc45, but not RPA or DNA polymerase alpha, are necessary for activating the degradation system. Xic1 degradation is attenuated following completion of DNA replication. Unlike degradation of p27(kip1) in mammalian cells, CDK2 activity is not directly involved in Xic1 degradation and interactions between Xic1 and CDK2/cyclin E are dispensable for Xic1 turnover. Interestingly, a C-terminal region (162-192) of Xic1 is essential and apparently sufficient for triggering Xic1 ubiquitination prior to degradation. These observations demonstrate that a direct link exists between DNA replication and CKI degradation.     

An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.     

Bacterial plasminogen receptors: in vitro evidence for a role in degradation of the mammalian extracellular matrix.

The potential of bacterium-bound plasmin to degrade mammalian extracellular matrix and to enhance bacterial penetration through basement membrane was assessed with the adherent strain SH401-1 of Salmonella enterica serovar Typhimurium. Typhimurium SH401-1 was able to bind plasminogen and to enhance the tissue-type plasminogen activator-mediated activation of the single-chain plasminogen to the two-chain plasmin. The end product, the enzymatically active, bacterium-bound plasmin activity, was also formed in a normal human plasma milieu in the presence of exogenous tissue-type plasminogen activator, indicating that plasmin was protected from the plasminogen activator inhibitors and plasmin inhibitors of plasma. Plasmin bound on Typhimurium cells degraded 125I-labeled laminin as well as 3H-labeled extracellular matrix prepared from the human endothelial cell line EA.hy926. The degradations were not seen with Typhimurium cells without plasminogen and were inhibited by the low-molecular-weight plasmin inhibitor aprotinin. Plasmin bound on Typhimurium cells also potentiated penetration of bacterial cells through the basement membrane preparation Matrigel reconstituted on membrane filters. The results give in vitro evidence for degradation of the mammalian extracellular matrix by bacterium-bound plasmin and for a pathogenetic role for bacterial plasminogen receptors.