相互作用结构域SH3和WW的蛋白质组学研究

在过去十年的研究中,发现许多蛋白质相互作用是通过相互作用结构域(如SH2,WW,SH3等)与其识别的另一个蛋白质中一段短肽序列的结合完成的,蛋白质相互作用结构域在蛋白质亚细胞定位、信号传导和蛋白复合体的组装等方面发挥重要功能。随着蛋白质组学技术的发展,高通量技术用于结构域结合特性及相互作用网络的研究。本文通过概述SH3和WW结构域的蛋白质组学研究策略的最新进展,总结结构域的几种现有研究策略。     

Treg细胞中关键转录因子Foxp3相互作用蛋白的鉴定与分析

目的鉴定Treg细胞中Foxp3相互作用蛋白。方法构建包含SBP和Protein G结合肽的Tags的Foxp3 knock-in转基因小鼠,分选Treg细胞,用亲和磁珠法纯化Foxp3相互作用蛋白复合体,质谱测序鉴定复合体蛋白组分并进行蛋白质组学分析和蛋白相互作用的验证。结果流式细胞分选的Treg纯度超过90%;经过亲和磁珠法纯化和质谱分析显示Foxp3有100个以上候选相互作用蛋白伙伴;免疫共沉淀方法初步鉴定部分伙伴蛋白间可相互结合。结论本研究在小鼠体内Treg细胞中初步获得Foxp3候选相互作用蛋白并证实部分蛋白间可相互作用,为这些蛋白对Treg细胞的功能鉴定提供基础。     

上皮钙粘附素-连环素复合体功能调控的研究进展

上皮钙粘附素-连环素复合体及其与细胞骨架蛋白的相互作用是维持上皮细胞极性和完整性的重要功能单位,并在炎症、组织损伤后的修复及恶性肿瘤的浸润、扩散等过程中发挥重要作用。     

肠出血性大肠埃希菌O157:H7 TCCP蛋白与宿主细胞IRTKS蛋白的相互作用

目的通过体外结合实验验证EHEC O157:H7内膜素受体偶联细胞骨架蛋白重复片段与人肠上皮细胞IRTKS蛋白SH3结构域之间的相互作用,并制备二者蛋白复合体,为利用晶体学方法研究其相互作用奠定基础。方法将IRTKS蛋白的SH3结构域cDNA序列克隆至pET30a表达质粒,转化BL21(DE3),诱导表达纯化SH3(IRTKS)蛋白;制备TCCP重复片段TC-CP(3R)蛋白;将SH3(IRTKS)与TCCP(3R)按照不同比例混合后,非变性丙烯酰胺凝胶电泳鉴定两者相互结合后产物;利用分子筛层析收集标准蛋白、TCCP(3R)、SH3(IRTKS)和TCCP(3R)-SH3(IRTKS)复合体的出峰体积,回归计算复合物的分子量,分析蛋白相互结合的比例。结果成功克隆表达了SH3(IRTKS)重组质粒;并制备了高纯度的SH3(IRTKS)和TCCP(3R)蛋白;非变性丙烯酰胺凝胶电泳结果证实TCCP(3R)与SH3(IRTKS)发生相互作用形成了蛋白质复合体;复合体的分子量约为33 000,二者相互结合的比例约为1:1。结论本研究为TCCP重复片段与SH3(IRTKS)相互作用提供了直接证据,同时复合体的制备,为利用晶体学研究两者的相互作用奠定了基础。     

组蛋白乙酰化酶调控MSCs经5-azaC诱导后的细胞周期和增殖特性

目的 阐述组蛋白乙酰化酶GCN5在间充质干细胞经5-氮杂胞苷诱导过程中的作用及其转录调控机制。方法 MTT法、流式细胞术检测细胞周期和细胞增殖;定量PCR检测P21基因表达;CHIP技术验证GCN5募集蛋白复合体与P21基因的相互作用及P21基因启动子区域的乙酰化水平;Co-IP技术分离、串联质谱技术鉴定GCN5募集蛋白复合体组成。结果 MSCs经5-azaC诱导3 d后G0/G1期细胞比例、P21基因表达量最高,此后逐渐降低,细胞增殖指数及G2/S期细胞比例与上述结果相反;筛选出GCN5募集蛋白复合体为：依赖ATP的染色质重塑复合体成分、转录起始复合体成分、转录因子和锌指结构蛋白;诱导组GCN5与P21基因启动子区域结合能力及P21基因启动子区域组蛋白H3乙酰化水平高于未诱导组。结论 GCN5募集蛋白复合体通过结合于P21基因启动子区域参与调控MSCs经5-azaC诱导体外分化过程中细胞周期G0/G1期和细胞增殖特性的调控。     

Argonaute蛋白家族在RNAi中的作用

RNA干扰是一种双链RNA诱导的基因沉默现象。Argonaute蛋白组成了一个高度保守的家族,该蛋白家族包括许多成员,它们组成了RNA诱导的沉默复合体的核心元件,是RNA干扰所必须的。Argo-naute蛋白选择性地与miRNA和siRNA结合,并与Dicer酶相互作用,其PIWI盒子与Dicer的RNaseⅢ结构域直接相互作用,PIWI与Dicer之间的相互作用可能会促进miRNA/siRNA的释放。Argonaute蛋白很可能是RNA干扰中核酸内切酶活性的执行者。     

RORγt蛋白复合体在Th17细胞分化中的“阴-阳”调节

Th17细胞是诱导组织炎症反应的辅助性T细胞,而维甲酸相关孤核受体γt(RORγt)是Th17细胞的主要转录因子(TF)。目前普遍认为转录因子不会单独发挥功能,因此RORγt会形成介导Th17细胞分化的蛋白复合体。鉴于RORγt及其潜在复合体在Th17细胞发育和功能中的重要性,本综述总结了近期有关RORγt和潜在伙伴蛋白之间相互作用的进展,特别讨论了导致Th17细胞分化的机制及其炎症反应疾病中的潜在临床干预靶点。     

端粒蛋白shelterin复合体影响肿瘤细胞基因组稳定性的研究进展

端粒是真核生物线性染色体末端的帽状保护性结构, 由双链DNA区和富含G碱基的3′突出端(单链DNA区)及端粒结合蛋白共同构成, 是维持基因组稳定性的重要结构。端粒结合蛋白的核心成分, 被称为shelterin复合体, 可帮助端粒成帽, 同时将端粒DNA隐藏于蛋白复合体间, 免受DNA修复系统的识别, 从而在形成端粒结构、保护染色体末端、维持基因组稳定等方面发挥重要作用。shelterin复合体组分的异常会导致端粒功能障碍及染色体末端融合或缺失, 严重威胁基因组的稳定性, 进而促进肿瘤的发生发展。作为基因组不稳定性的重要体现形式, 双微体(double minute chromosomes, DMs)的形成与端粒的结构功能异常也存在相关性。本文总结了肿瘤细胞中shelterin复合体异常与基因组不稳定性的关联, 试图阐明端粒蛋白参与的肿瘤恶性进展机制, 为临床上改善恶性肿瘤预后提供新的靶点和思路。     

RNA干扰技术在生物医学领域中的应用

RNA干扰(RNAi)是由双链RNA(dsRNA)启动的序列特异的转录后基因沉默过程。在这一过程中,dsRNA被核酸内切酶切割成小干扰RNA(siRNA),siRNA与相关的蛋白结合成RNA诱导沉默复合体,这一复合体可识别并降解mRNA,导致基因沉默。它是一种比反义寡聚核苷酸技术更为有效的方法,具有更高的特异性,在生物医学领域有广阔的应用前景。文章就RNAi技术在功能基因组学、基因治疗(如人类免疫缺陷病毒、肝炎病毒、肿瘤)、神经性疾病及其药物设计等领域的实验及其应用研究及存在问题作一综述。     

人巨细胞病毒皮层蛋白pUL23与宿主蛋白IGFBP4相互作用的鉴定

目的:利用蛋白质相互作用的技术筛选与pUL23相互作用宿主蛋白,为研究pUL23蛋白对人巨细胞病毒繁殖的影响提供线索。方法:通过酵母双杂交系统从人胚肾cDNA文库筛选与pUL23相互作用的宿主蛋白;通过GST-pu ll-down技术研究二者体外物理性相互作用;免疫共沉淀技术进一步研究二者在胞内相互作用。结果:Pu ll-down技术、免疫共沉淀技术确定了宿主蛋白IGFBP4与pUL23具有相互作用。结论:上述结果为研究pUL23蛋白调节病毒自身繁殖功能提供重要依据。     

固有免疫调控蛋白PKR串联亲和纯化系统的建立及应用

目的 构建固有免疫调控蛋白--双链RNA(dsRNA)调控的蛋白激酶(PKR)的串联亲和纯化系统(TAP),初步研究PKR蛋白功能,以用于与PKR相互作用的新蛋白的鉴定与功能分析.方法 通过PCR扩增PKR目的基因,克隆至真核表达载体pcTAP-A中.将重组质粒pcTAP-PKR通过脂质体法转染PKR稳定沉默(PKRk...     

Systematic identification of functional orthologs based on protein network comparison

Annotating protein function across species is an important task that is often complicated by the presence of large paralogous gene families. Here, we report a novel strategy for identifying functionally related proteins that supplements sequence-based comparisons with information on conserved protein-protein interactions. First, the protein interaction networks of two species are aligned by assigning proteins to sequence homology clusters using the Inparanoid algorithm. Next, probabilistic inference is performed on the aligned networks to identify pairs of proteins, one from each species, that are likely to retain the same function based on conservation of their interacting partners. Applying this method to Drosophila melanogaster and Saccharomyces cerevisiae, we analyze 121 cases for which functional orthology assignment is ambiguous when sequence similarity is used alone. In 61 of these cases, the network supports a different protein pair than that favored by sequence comparisons. These results suggest that network analysis can be used to provide a key source of information for refining sequence-based homology searches.     

HepG2细胞中RORγ相互作用蛋白的筛选及鉴定

目的筛选HepG2细胞中转录因子RORγ(the retinoid-related orphan nuclear receptor gamma)相互作用蛋白,并对这些蛋白进行初步鉴定,为阐述RORγ介导调节HepG2细胞中各种生理学进程提供理论依据。方法从HepG2细胞中克隆RORγ基因并连接入载体pCeMM CTAP(SG)中形成RORγ-CTAP(SG)融合基因,再将其亚克隆入含嘌呤霉素抗性基因的载体质粒中,构建pMSCVpuro RORγ-CTAP(SG)质粒;稳定转染HepG2细胞并对RORγ-CTAP(SG)融合基因的表达和定位进行检测后,进行大规模扩增培养;用TAP(串联亲和纯化)方法捕获RORγ相互作用蛋白,通过银染找出差异性蛋白条带,质谱鉴定得到候选RORγ相互作用蛋白;用免疫共沉淀方法对候选蛋白RORγ相互作用蛋白进行验证鉴定。结果成功构建了质粒pMSCVpuro RORγ-CTAP(SG)并得到稳定转染HepG2细胞株,同时RORγ-CTAP(SG)融合基因能定位表达于细胞核内;通过TAP方法获得了RORγ蛋白复合物,然后通过串联质谱分析和数据库搜索从银染差异性显著蛋白条带中找到7个候选RORγ相互作用蛋白;用RORγ蛋白进行免疫共沉淀,对纯化出的7个RORγ相互作用蛋白分别检测,证实了RIP140,HSP90和RORγ在HepG2中有相互作用关系。结论筛选并鉴定了HepG2细胞中蛋白RORγ的相互作用蛋白RIP140和HSP90,这些研究发现支持了RORγ是共调解蛋白依赖的转录因子且以复合物形式行使功能的假说。其中,HSP90可能扮演分子伴侣角色,而RIP140在HepG2细胞中则可能充当RORγ蛋白的转录调控伙伴蛋白,具体机制有待进一步研究。     

Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function

The Rev protein of HIV-1 is essential for HIV-1 proliferation due to its role in exporting viral RNA from the nucleus. We used a modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human cells and discovered a prominent interaction between Rev and nucleosome assembly protein 1 (Nap1). This interaction was also observed by specific retention of Nap1 from human cell lysates on a Rev affinity column. Nap1 was found to bind Rev through the Rev arginine-rich domain and altered the oligomerization state of Rev in vitro. Overexpression of Nap1 stimulated the ability of Rev to export RNA, reduced the nucleolar localization of Rev, and affected Rev nuclear import rates. The results suggest that Nap-1 may influence Rev function by increasing the availability of Rev.     

The envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated

To analyze the proteins interacting with the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein, a SARS-CoV was engineered including two tags associated to the E protein. Using this virus, complexes of SARS-CoV E and other proteins were purified using a tandem affinity purification system. Several viral and cell proteins including spike, membrane, non-structural protein 3 (nsp3), dynein heavy chain, fatty acid synthase and transmembrane protein 43 bound E protein. In the present work, we focused on the binding of E protein to nsp3 in infected cells and cell-free systems. This interaction was mediated by the N-terminal acidic domain of nsp3. Moreover, nsp3 and E protein colocalized during the infection. It was shown that E protein was ubiquitinated in vitro and in cell culture, suggesting that the interaction between nsp3 and E protein may play a role in the E protein ubiquitination status and therefore on its turnover.     

新型GATA-1相互作用蛋白质及其在造血调控中的作用

GATA-1在造血干细胞的谱系分化中起关键的调控作用,为红系和巨核系发育成熟必不可少,对肥大细胞系及嗜酸性粒细胞系发育也有一定的调控作用.GATA-1的转录活性受到多层次的精确调节,其调控不同谱系分化的功能大多通过与不同的蛋白质相互作用来实施.近年来,多种新的GTAT-1相互作用蛋白质被确定,特别是GATA-1复合体的研究,揭示了GATA-1转录活性及其调控造血分化的新机制.     

红系分化相关基因相互作用蛋白质的分离与鉴定

目的制备红系分化相关基因(erythroid differentiation-associated gene,EDAG)蛋白的单克隆抗体,利用免疫沉淀联合质谱技术对EDAG相互作用蛋白质进行分离与鉴定。方法构建EDAG原核表达载体,通过诱导、表达、纯化获得EDAG融合蛋白,杂交瘤技术建立分泌EDAG单克隆抗体的杂交瘤细胞株,利用Western印迹筛选阳性杂交瘤细胞,免疫小鼠得腹水,最后用免疫共沉淀与质谱联合鉴定EDAG相互作用蛋白。结果纯化获得EDAG重组蛋白,筛选出4株分泌EDAG单克隆抗体的杂交瘤细胞株,并制备了腹水。该抗体均可用于内源EDAG蛋白的检测,其中两种抗体可用于免疫共沉淀实验,运用质谱技术筛选获得EDAG候选相互作用蛋白。结论利用EDAG单克隆抗体,筛选到EDAG候选相互作用蛋白质13种,涉及细胞增殖及转录等过程,为EDAG的功能研究及其分子机制的阐明提供了新的线索。     

Three-step purification of a fragment of the large immunophilin FKBP52

PPIases catalyze the interconversion of cis and trans isomers of peptidyl-prolyl (Xaa-Pro) bonds in peptide and protein substrates. The PPIase family comprises three subfamilies, two of which interact with immunosuppressant drugs and are therefore termed immunophilins. One subgroup of the immunophilins are the FK506 binding proteins (FKBPs). FKBPs of a relative molecular mass higher than 40 000 also display chaperone activity and are part of the multichaperone complex that Hsp90 forms with substrate proteins. Their function in this chaperone complex is still enigmatic. To further characterize the function of FKBP52 we want to analyze constructs of FKBP52-fragments. Here we describe a fast and effective three-step purification procedure for a fragment of FKBP52 with a relative molecular mass of 48000, termed FKBP52-123, consisting of affinity chromatography, anion-exchange column and gel-permeation chromatography. A yield of 1 mg pure protein per gram of cells was achieved.     

Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.     

端粒相关蛋白TIN2

TIN2(TRF1相互作用核蛋白2)是一重要的端粒相关蛋白。人TIN2蛋白包括N端,TRF1交互作用区(TRF1-Int)和C端3个结构域。它在TRF1复合物、TRF2复合物和Shelterin的功能中扮演关键角色,协同其它端粒蛋白维持端粒长度、结构和功能。TIN2与个体发育、细胞分化和肿瘤发生密切相关。