TSG101基因研究进展

TSG101基因是近年来发现的一个新的抑癌基因候选者，定位于11p15.1-p15.2，其产物TSG101 蛋白具有多种重要功能，例如调控蛋白及囊泡运输，与细胞存活增殖有关等,其N端与泛素结合酶（UBC）有一定的同源性。近年来的研究表明，TSG101基因与多种肿瘤密切相关，其转录本在某些肿瘤细胞中发生了异常剪接；TSG101也有可能作为一种显性负调节子参与泛素系统对细胞周期的调节。它还参与艾滋病毒的感染过程。本文综述了该基因的最新研究进展。     

TSG101基因研究进展

人 TSG10 1基因是近年来发现的一个新的抑瘤基因候选者 ,定位于 11p15 .1- 15 .2 ,其编码产物TSG10 1蛋白 N端区与泛素连接酶催化区同源。近年来的研究表明 ,TSG10 1基因与多种肿瘤密切相关 ,其产物TSG10 1蛋白具有多种重要功能 ,由于其参与 HIV病毒的感染过程 ,使得研制和开发针对 HIV病毒的基因药物来治疗艾滋病成为可能     

TSG101基因在肿瘤发生中的角色

TSG101编码一个多结构域的蛋白，其N端区与泛素结合酶同源。该蛋白是细胞内涵体分选复合物1的组成部分，同时也涉及到一系列细胞功能。近年来的研究表明：TSG101在乳腺癌等多种肿瘤中过度表达，是肿瘤进展过程中的正性调节器。     

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。     

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。     

TSG101小干扰RNA对SH-SY5Y细胞生长和药物敏感性的作用

目的：探讨TSG101基因对人神经母细胞瘤细胞生长和药物敏感性的调节作用。方法:构建TSG101基因的小干扰RNA载体并将其转导入SH-SY5Y细胞，G418筛选后获得稳定转染的阳性克隆后，应用RT-PCR和Western blotting进行鉴定；MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化；DNA ladder法和流式细胞仪检测细胞转染前后对顺铂诱导的凋亡的变化；Western blotting检测细胞转染前后凋亡相关蛋白Bcl-2、Bax，耐药相关蛋白P-gp、MRP的表达变化。结果:成功构建了TSG101的小干扰RNA载体并将其转染SH-SY5Y细胞；筛选到稳定的TSG101低表达的神经母细胞瘤细胞模型；MTT和流式细胞仪检测结果显示，转染TSG101小干扰RNA后的细胞的生长速度显著减慢于对照组（P<0.05），且G1期的细胞比率显著高于对照组（P<0.05）；DNA ladder法和流式细胞仪检测结果显示，用10 mg/L CDDP处理36 h后，转染TSG101小干扰RNA后的细胞的凋亡数显著多于对照组（P<0.05），形成DNA条带；Western blotting显示，转染TSG101小干扰RNA后的细胞中Bcl-2和P-gp的表达明显低于对照组。结论：下调TSG101基因能抑制神经母细胞瘤细胞生长，增加细胞对化疗药物的敏感性，提示TSG101在基因治疗中具有较好的临床应用前景。     

类泛素蛋白ISG15调控感染与肿瘤的新进展

IFN刺激基因15(interferon-stimulated gene 15,ISG15)编码的蛋白ISG15是最早被发现的一种类泛素蛋白,在生物体内以单体和复合体形式存在。和泛素一样,ISG15的单体和其共价修饰的蛋白参与并调节了复杂的生物学过程。在病毒、细菌感染以及肿瘤发生过程中,ISG15单体和底物蛋白的共价修饰水平均出现不同程度改变,这表明ISG15在固有免疫的调控中发挥重要作用。越来越多的研究表明ISG15和其共价修饰系统已成为疾病预防和治疗的重要靶点。文章介绍了ISG15的发现、结构特点以及其共价修饰系统,并阐述了近年来ISG15在抗病毒、抗菌和肿瘤发生过程中的重要作用。     

肿瘤易感基因101在肝细胞肝癌组织中的表达及意义

目的:检测TSG101(TSG101)在肝细胞肝癌(HCC)组织中表达的临床意义。方法:应用免疫组织化学及Westernblot方法检测TSG101蛋白在肝癌及其对应非癌肝组织组织的表达情况,并分析其在肿瘤中表达水平与患者年龄、性别、TNM分期及转移等临床病理资料之间的关系。结果:免疫组化及Western blot均显示TSG101在肝癌组织表达水平显著高于其对应非癌肝组织(P<0.05)。TSG101高表达与患者TNM分期及侵袭转移显著相关(P<0.05),而与患者性别、年龄及血清HBsAg水平无明显相关性(P>0.05)。多因素回归分析同样提示TSG101阳性表达率与患者TNM分期及转移相关(P<0.05)。结论:TSG101在肝细胞癌表达水平显著高于其对应非癌肝组织,其在肝癌表达水平与患者TNM分期及转移密切相关。     

TSG101在胰腺癌组织的表达及临床意义

目的:检测肿瘤易感基因101(TSG101)在胰腺癌组织中表达水平及其与癌患者临床病理特征的关系。方法:应用免疫组织化学方法检测TSG101蛋白在胰腺癌及其对应正常胰腺组织的表达情况,并分析其在肿瘤中表达水平与患者性别、分化、临床分期及转移等临床病理资料之间的相关性。结果:TSG101在胰腺肿瘤组织中表达率为47/58(81.0%)明显高于正常胰腺组织表达率7/58(12.1%)(P<0.05),统计学分析提示TSG101在胰腺癌组织表达率明显高于正常胰腺组织(P<0.05),在高分化胰腺癌组织中表达明显强于中、低分化肿瘤组织,在转移性胰腺癌中表达率高于非转移性肿瘤组织(P<0.05),而与患者性别及临床分期间差异无统计学意义(P>0.05)。结论:TSG101在胰腺癌组织中表达与其分化程度及转移呈正相关。     

Parkin蛋白及其底物与帕金森病的研究进展

帕金森病(PD)是一种发病率较高的神经退行性疾病,遗传、环境等多种因素均参与其病理生理过程.在PD致病相关蛋白中,PARK2基因编码的Parkin蛋白作为E3泛素连接酶,可与多种底物分子发生相互作用.本文旨在综述 Parkin 蛋白及其底物 septin、Pael-R、Cyclin E、p38、Synphilin-1、...     

A role of tumor susceptibility gene 101 (TSG101) in innate immune response of crayfish Procambarus clarkii

Tumor susceptibility gene 101 (TSG101) is a multi-functional gene involved in cell growth and proliferation in vertebrates. However, its role in the innate immune response of crustaceans remains unclear. Here, a TSG101 gene was identified in crayfish Procambarus clarkii with an open reading frame of 1320 bp that encoded a predicted 48.3-kDa protein highly homologous to those in other invertebrates. TSG101 mRNA was highly expressed in stomach and hepatopancreas, and its expression was induced significantly in different tissues (hemocytes, gills and intestine) by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I: C) with various expression patterns. Recombinant TSG101 protein was expressed in Escherichia coli, and a possible protein-protein interaction between TSG101 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was explored by far-western blotting. RNA interference of TSG101 affected the gene expression of members of the Toll pathway. These results suggest that TSG101 is involved in the innate immune responses of P. clarkii.     

Differentially Expressed Genes in Hormone Refractory Prostate Cancer : Association with Chromosomal Regions Involved with Genetic Aberrations

Differential gene expression between the androgen sensitive human prostate cancer cell line LNCaP and an insensitive clonal variant, LNCaP-r, was demonstrated by suppression subtractive hybridization. Twenty-one sequences were identified of which 9 are homologous to known genes, 11 are represented by expressed sequence tags (ESTs), and 1 is novel. We present data for 5 of 7 sequences confirmed to be differentially expressed by Northern blot analysis and semiquantitative RT-PCR. Only one gene, fibronectin (FN), was highly overexpressed (>60-fold) in LNCaP-r cells, consistent with previously reported overexpression of FN in prostate cancer. Four sequences were down-regulated in LNCaP-r cells, including an inactive variant of the E2 ubiquitin conjugating enzyme (UEV-1), a novel metalloproteinase-related collagenase (PM5), and a potential tumor suppressor gene (breast basic conserved gene, BBC1). UEV-1 is multifunctional, regulates the cell cycle via cdk1, has homology to MMS2 and likewise functions as a DNA protection protein, and also has homology to TSG101. Aberrant splice variants of TSG101 occur frequently in both breast and prostate cancer, but its mechanism of action is unknown. FN, BBC1, and UEV-1 localize to regions of chromosomal aberration (2q3.4, 16q24.3, and 20q13.2, respectively) associated with advanced prostate cancer and thus may be highly relevant to disease progression.     

GISP increases neurotransmitter receptor stability by down-regulating ESCRT-mediated lysosomal degradation

GPCR interacting scaffold protein (GISP) is a multi-domain brain-specific scaffold protein that can regulate GABA_B receptor complexes by both enhancing their surface expression and by inhibiting their lysosomal degradation. GISP retards degradation of GABA_B receptors through its interaction with tumour susceptibility gene 101 (TSG101), a member of the endosomal sorting complex required for transport (ESCRT) lysosomal sorting machinery. We show that in addition to GABA_B, GISP exerts a more general role to increase the steady-state levels of several neurotransmitter receptors. Further, GISP delays TSG101-dependent agonist-induced EGFR down-regulation in human embryonic kidney (HEK) 293 cells whereas a mutant GISP lacking the TSG101 binding domain has no effect. These data suggest that GISP acts as a negative regulator of TSG101-dependent lysosomal degradation and plays an important role in determining the availability of neurotransmitter receptors.     

Mutational and expressional analyses of SPOP,a candidate tumor suppressor gene,in prostate,gastric and colorectal cancers

Mounting evidence exists that alterations of ubiquitination processes are involved in cancer pathogenesis. Speckle‐type POZ protein (SPOP) is a key adaptor for Cul3‐based ubiquitination process. Recent studies reported that SPOP may be a tumor suppressor gene (TSG) and somatic mutation of SPOP was detected in prostate cancer (PCA). The aim of this study was to see whether alterations of SPOP protein expression and somatic mutation of SPOP gene are features of cancers. In this study, we analyzed SPOP somatic mutation in 45 gastric (GC), 45 colorectal cancer (CRC) and 45 PCA by single‐strand conformation polymorphism (SSCP). Also, we analyzed SPOP protein expression in 60 GC, 60 CRC and 60 PCA by immunohistochemistry. Overall, we detected three somatic missense mutations of SPOP gene in the coding sequences (p.Ser14Leu, p.Tyr87Cys and p.Phe133Leu). The mutations were observed in two PCA and one CRC. Of note, the p.Phe133Leu was a recurrent mutation reported in an earlier study. In the immunohistochemistry, SPOP protein was expressed in normal gastric, colonic and prostate epithelial cells, whereas it was lost in 30% of GC, 20% of CRC and 37% of PCA. Our data indicate that loss of SPOP expression was common in GC, CRC and PCA, but somatic mutation of SPOP in this study was rare in these tumors. Also, the data provide a possibility that loss of expression of SPOP gene might play a role in cancer pathogenesis by altering TSG functions of SPOP.     

Fluorescent‐based evaluation of chaperone‐mediated autophagy and microautophagy activities in cultured cells

The autophagy‐lysosome protein degradation is further classified into macroautophagy (MA), microautophagy (mA), and chaperone‐mediated autophagy (CMA). While MA is involved in various functions and disease pathogenesis, little is known about CMA and mA because of the absence of easy methods to assess their activities. We have recently established a method to assess CMA activity using glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), a CMA substrate, and HaloTag (HT) system. Another group has recently identified a mammalian mA pathway, in which substrates are delivered to late endosomes in an heat shock cognate protein (Hsc)70‐dependent manner. Because Hsc70 is also involved in CMA, our method would detect both CMA and mA activities. In this study, we attempted to assess CMA and mA activities separately through the siRNA‐mediated knockdown of CMA‐ and mA‐related proteins. Knockdown of LAMP2A, a CMA‐related protein, and TSG101, an mA‐related protein, significantly but only partially decreased the punctate accumulation of GAPDH‐HT in AD293 cells and primary cultured rat cortical neurons. Compounds that activate CMA significantly increased GAPDH‐HT puncta in TSG101‐knockdown cells, but not in LAMP2A‐knockdown cells, suggesting that punctate accumulation of GAPDH‐HT under LAMP2A‐ and TSG101‐knockdown represents mA and CMA activities, respectively. We succeeded in establishing the method to separately evaluate CMA and mA activities by fluorescence observation.