人类ZAKβ与YWHAZ蛋白相互作用的初步研究

目的研究ZAKβ与YWHAZ蛋白之间的相互作用。方法以ZAKβ为诱饵蛋白,利用酵母双杂交筛选人类肝脏cDNA文库,经GST Pull-down、免疫共沉淀和细胞共定位实验分别验证ZAKβ与猎物蛋白在体外和细胞内的相互作用,构建ZAKβ的截短体以确定相互作用的区域,用磷酸化抗体检测ZAKβ在相互作用中的磷酸化作用,并通过流式细胞仪检测这对蛋白相互作用对细胞周期的影响。结果利用酵母双杂交筛选到一个能与ZAKβ相互作用的蛋白YWHAZ,并在体外和细胞内都验证了这二者的相互作用,发现这两个蛋白都能共定位于293T细胞的胞质中,确定了ZAKβ的N端1~250氨基酸为与YWHAZ蛋白相互作用的区域。体外实验发现了这二者的相互作用涉及到磷酸化反应,发现这对蛋白的相互作用能提高293T细胞的G2期水平。结论首次发现并证实了ZAKβ和YWHAZ之间的相互作用,发现该相互作用涉及到磷酸化过程并能够对细胞周期产生影响。     

原癌基因LMO3相互作用蛋白的筛选及鉴定

目的 通过筛选LMO3的相互作用蛋白,进一步了解LMO3的作用及可能机制.方法:酵母双杂交方法 筛选LMO3相互作用蛋白,并通过酵母结合试验、免疫共沉淀及荧光共定位等进行验证.结果:在初步获得相互作用蛋白CIB的基础上,在酵母中证实了LMO3与CIB的相互作用,并通过酵母结合试验确定了CIB与LMO3的相互作用位点,发...     

与霍乱毒素A1亚基突变体相互作用蛋白的筛选

目的 寻找新的与霍乱毒素A1亚基（CTA1）突变体（S63F）相互作用的蛋白。来探讨CT佐剂活性的分子机理。方法 应用酵母双杂交技术，以CTA1（S63F）为诱饵蛋白筛选人脾细胞cDNA文库，并通过共转染，免疫共沉淀和Western杂交在哺乳动物细胞COS-7中确证诱饵蛋白和候选蛋白之间的相互作用。结果 筛选获得一个与HLAⅠ类分子α亚基高度同源的蛋白。这个蛋白在酵母核内以及COS-7细胞中都显示出与CTA1（S63F0的相互作用，结论 CTA1（S63F）与HLAⅠ类分子α亚基内以及COS-7细胞中都显示出与CTA1（S63F）的相互作用。结论 CTA1（S63F）与HLAⅠ类分子α亚基的相互作用可能导致HLAⅠ类分子进入“膜筏”，引起膜筏不断聚集增大，聚集到一定程度后筏相关酪氨酸蛋白激酶活化，启动胞内信号的级联传递，增强免疫反应。     

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。     

应用酵母双杂交系统筛选人胃黏膜上皮组织SHIP2的相互作用蛋白

目的通过酵母双杂交系统筛选人胃黏膜上皮组织标准均一化cDNA文库,寻找与含Src同源蛋白2肌醇-5-磷酸酶2(SHIP2)相互作用的蛋白。方法利用酵母双杂交系统,以SHIP2的P1(SH2+5-Ptase)和P2(PRD+SAM)段作为诱饵蛋白,筛选出人胃黏膜上皮组织均一化cDNA文库中与SHIP2相互作用的蛋白,并通过免疫共沉淀法进行验证。结果挑选出39个阳性克隆,经测序比对分析,回复性杂交,免疫共沉淀试验验证,最终确定一个与SHIP2相互作用的蛋白抗增殖蛋白1(prohibitin1/PHB)。结论酵母双杂交系统筛选人胃黏膜上皮组织SHIP2的相互作用蛋白PHB。     

NALP3富含亮氨酸重复序列结构域识别人细胞周期蛋白H和禽冠状病毒蛋白

 目的：寻找与NALP3富含亮氨酸重复序列（leucine-rich repeat, LRR）结构域相互作用的蛋白质分子。方法：克隆NALP3富含亮氨酸重复序列（NALP3-LRR）结构域的DNA序列并经测序检验。应用酵母双杂交技术,构建以NALP3-LRR结构域为诱饵基因的酵母双杂交载体,对人胚肺cDNA文库进行杂交筛选,经酵母回交实验验证蛋白质在酵母细胞内的相互作用并对阳性克隆的DNA进行序列测定和生物信息学分析。将筛选到的阳性克隆进一步用免疫共沉淀实验验证NALP3-LRR结构域与阳性蛋白之间相互作用的可靠性。结果：成功克隆NALP3-LRR结构域的DNA序列并经测序检验正确。用酵母双杂交技术对人胚肺cDNA文库进行酵母杂交筛选共获得4个阳性克隆。免疫共沉淀实验证实能与NALP3-LRR结构域发生相互作用的阳性蛋白是人细胞周期蛋白H和禽传染性支气管炎病毒株Cal99的ORF1a。结论：NALP3的富含亮氨酸重复序列结构域能与人细胞周期蛋白H和禽冠状病毒蛋白发生相互作用。     

酵母双杂交筛选与单剪接型2.2 kb乙型肝炎病毒剪接特异性新蛋白相互作用的肝细胞蛋白

目的 筛选与单剪接型2.2 kb乙型肝炎病毒(HBV)剪接特异性新蛋白相互作用的肝细胞蛋白.方法 PCR扩增单剪接型2.2 kb HBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7,在证实TPss蛋白不具有自激活作用的前提下,以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白,进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用.结果 构建酵母双杂交诱饵载体pGBKT7-TPss,Western blot显示其在酵母中表达TPss蛋白.酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用,即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链.结论 TPss可与多种肝细胞蛋白相互作用.     

人gp130结合分子与TLE1分子通过同源的Q结构域相互作用

目的：确定人gp130结合分子（GAM）与transducin lide enhancer of split 1(TLE1)分子间是否存在相互作用及相互作用的结构基础，以深入研究这两种分子的功能。方法：扩增编码GAM与TLE1分子不同结构域的cDNA，构建含有这些不同结构域的酵母双杂交载体，通过酵母双杂交系统分析和免疫共沉淀试验，研究这两种分子间的相互作用。结果：DNA序列测定证实成功构建了含GAM与TLE1分子不同结构域的酵母双杂交载体，酵母双杂交分析表明GAM与TLE1分子通过氨基端的Q结构域相互结合，免疫共沉淀试验证实了这一发现。结论：证实GAM与TLE1分子通过氨基端的Q结构域相互结合。     

与14-3-3ζ相互作用的蛋白Polo样激酶1的筛选与鉴定

目的:应用酵母双杂交系统筛选与14-3-3ζ相互作用的蛋白,进一步鉴定其与Polo样激酶1(Plk1)相互作用。方法:构建pGBKT7-14-3-3ζ诱饵表达载体,筛选HeLa细胞cDNA文库中与14-3-3ζ相互作用蛋白,进一步通过共转酵母、免疫荧光以及外源性和内源性的细胞免疫共沉淀实验验证两者的相互作用。结果:通过酵母双杂交系统筛选出的阳性相互作用蛋白中包括Plk1,进一步通过共转酵母,外源性和内源性的细胞免疫共沉淀实验证实两者的相互作用,免疫荧光实验证实两者共定位于有丝分裂过程中胞质分裂期的中体。结论:Plk1是高度保守的丝氨酸/苏氨酸蛋白激酶,在中体的成熟,有丝分裂期染色体的分离,胞质分裂以及DNA的损伤应答等环节发挥重要作用,其与14-3-3ζ的相互作用为14-3-3蛋白家族参与有丝分裂(M期)的调控提供了直接证据。     

The PH domain adaptor protein Bam32/DAPP1 functions in mast cells to restrain FcɛRI-induced calcium flux and granule release

Mast cell activation triggered by IgE binding to its high affinity receptor Fc?RI is highly dependent on signaling via phosphoinositde 3-kinases (PI3K). The phosphoinositide phosphatase SHIP controls mast cell activation by regulating accumulation of D3 phosphoinositide second messengers generated by PI3K. The PH domain adaptor protein Bam32/DAPP1 binds specifically to the D3 phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 (the substrate and product of SHIP respectively). In B cells, Bam32 is phosphorylated by Src family kinases including Lyn, and is required for antigen receptor-induced activation; however the function of Bam32 in mast cells is unknown. Here we report that Bam32 is expressed in mast cells, is recruited to the plasma membrane upon stimulation and functions in Fc?RI signaling. Examination of bone marrow-derived mast cells (BMMC) isolated from Bam32-deficient mice revealed enhanced Fc?RI-induced degranulation and IL-6 production, indicating that Bam32 may function to restrain signaling via Fc?RI. These enhanced degranulation responses were PI3K-dependent, as indicated by blockade with PI3K inhibitors wortmannin or IC87114. While Bam32-deficient BMMC showed reduced Fc?RI-induced activation of mitogen-activated protein kinases ERK and JNK, Fc?RI-induced calcium flux and phosphorylation of PLCγ1 and Akt were increased. Bam32-deficient BMMC showed significantly reduced phosphorylation of Lyn and SHIP, indicating reduced activity of inhibitory signaling pathways. Together our results identify Bam32 as a novel regulator of mast cell activation, potentially functioning in membrane-proximal integration of positive and negative signaling pathways.     

Alzheimer's disease-associated presenilin 2 interacts with DRAL, an LIM-domain protein

Using the yeast two-hybrid system, we screened for proteins interacting with presenilin 2 (PS2) and cloned DRAL. DRAL is an LIM-only protein containing four LIM domains and an N-terminal half LIM domain. Previously DRAL has been cloned as a co-activator of the androgen receptor and as a protein interacting with a DNA replication regulatory protein, hCDC47. Our yeast two-hybrid assay showed that DRAL interacted with a hydrophilic loop region (amino acids 269-298) in the endoproteolytic N-terminal fragment of PS2, but not that of PS1, although the region 269-298 of PS2 and the corresponding PS1 sequence differ by only three amino acids. Each point mutation within this region, R275A, T280A, Q282A, R284A, N285A, P287T, I288L, F289A and S296A, in PS2 abolished the binding. This suggests that DRAL recognizes the PS2 structure specifically. The in vitro interaction was confirmed by affinity column assay and the physiological interactions between endogenous PS2 and DRAL by co-immunoprecipitation from human lung fibroblast MRC5 cells. Furthermore, in PS2-overexpressing HEK293 cells, we found an increase in the amount of DRAL in the membrane fraction and an increase in the amount of DRAL that was co-immunoprecipitated with PS2. The potential role of DRAL in the cellular signaling suggests that DRAL functions as an adaptor protein that links PS2 to an intracellular signaling.     

KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis

Mutations in KRIT1, a protein initially identified based on a yeast two-hybrid interaction with the RAS-family GTPase RAP1A, are responsible for the development of the inherited vascular disorder cerebral cavernous malformations (CCM1). As the function of the KRIT1 protein and its role in CCM pathogenesis remain unknown, we performed yeast two-hybrid screens to identify additional protein binding partners. A fragment containing the N-terminal 272 amino acid residues of KRIT1, a region lacking similarity to any known protein upon database searches, was used as bait. From parallel screens of human fetal brain and HeLa cDNA libraries, we obtained multiple independent isolates of human integrin cytoplasmic domain-associated protein-1 (ICAP-1) as interacting clones. The interaction of KRIT1 and ICAP-1 was confirmed by GST-KRIT1 trapping of endogenous ICAP-1 from 293T cells. The alpha isoform of ICAP-1 is a 200 amino acid serine/threonine-rich phosphoprotein which binds the cytoplasmic tail of beta1 integrins. We show that mutagenesis of the N-terminal KRIT1 NPXY amino acid sequence, a motif critical for ICAP-1 binding to beta1 integrin molecules, completely abrogates the KRIT1/ICAP-1 interaction. The interaction between ICAP-1 and KRIT1, and the presence of a FERM domain in the latter, suggest that KRIT1 might be involved in the bidirectional signaling between integrin molecules and the cytoskeleton. Furthermore, these data suggest that KRIT1 might affect cell adhesion processes via integrin signaling in CCM1 pathogenesis.     

Klotho:与FPC蛋白相互作用的蛋白质分子

目的:利用酵母双杂交技术筛选与纤囊素相互作用的蛋白质,为进一步探讨纤囊素(FPC)在常染色体隐性遗传多囊肾病(ARPKD)发生、发展中的作用机制提供依据。方法:利用酵母双杂交系统以质粒pG-BKT7-FPC为"诱饵",在人类胚肾cDNA文库中筛选与FPC蛋白C末端相互作用宿主蛋白的基因,再通过一对一回交试验验证两者之间的相互作用。结果:酵母双杂交筛选得到相互作用的蛋白分子Klotho(后简称KL),回交试验再次确认KL能够与FPC蛋白相互作用。结论:FPC的C末端能够与KL相互作用,它们之间的相互作用可能为研究FPC在ARPKD发病中的功能及作用机制提供新途径。     

Identification of cyclophilin A as a CD99-binding protein by yeast two-hybrid screening

CD99 is a 32kDa surface glycoprotein, which is involved in the migration of leukocytes and the transport of ganglioside GM1 and transmembrane proteins. To identify signaling mechanisms triggered by CD99 engagement, a LexA-based yeast two-hybrid system was utilized to identify proteins interacting with the cytoplasmic domains of CD99. In seven positive clones, we attempted to ascertain whether cyclophilin A (CypA) was involved in CD99-mediated signaling, since CypA had been implicated as a signaling regulator for kinases and phosphatases. The interaction between CD99 and CypA was confirmed by co-immunoprecipitation and confocal immunofluorescence studies. Interestingly, the amounts of CypA associated with CD99 increased upon CD99 engagement. We prepared an expression plasmid by inserting CypA cDNA into pEGFP, in order to visualize cellular CypA. In HeLa or HEK 293T cells transfected with the pEGFP-CypA plasmid, GFP-tagged CypA was diffusely present in the cytoplasm of untreated cells. However, CypA-GFP moved to the cell periphery and membrane blebbing, and became colocalized with CD99 upon CD99 engagement. These results suggest that CypA may be either a signaling mediator or a signaling regulator for CD99.     

Src kinase Lyn is crucial for Pseudomonas aeruginosa internalization into lung cells

Lyn is an important B cell signaling kinase of the Src tyrosine kinase family with a broad range of functions from cytoskeletal changes to induction of apoptosis. However, the role of Lyn in infectious diseases is not clear. Here, we demonstrate that Lyn activation by phosphorylation significantly impacted invasion of an alveolar epithelial cell line, primary lung cells, and rat lungs by Pseudomonas aeruginosa (PA), a common opportunistic lung pathogen affecting individuals with deficient lung immunity. Our results indicate that activation of Lyn and its interaction with rafts and TLR2, played an important role in the initial stages of PA interaction with host cells. The role of Lyn was further evaluated using the pharmacologic Src-specific inhibitor PP2, a dominant negative mutant, and finally confirmed with Lyn-deficient (Lyn(-/-)) bone marrow-derived mast cells. Inhibition of Lyn's function by above approaches prevented PA internalization. Moreover, blocking of Lyn also affected downstream events: induction of inflammatory cytokines and apoptosis. This report brings out a new role of Lyn in infectious diseases and indicates potential new targets for prevention and treatment of infections.     

Bam32: a novel mediator of Erk activation in T cells

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.     

Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins

The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways, but the role of NS5A in HCV pathogenesis has not been firmly established. To further characterize this multifunctional protein, we instigated the studies of the subcellular localization of NS5A in a hepatoma cell line. NS5A was localized to the perinuclear membrane structures, including the endoplasmic reticulum (ER) and the Golgi apparatus, by immunofluorescence staining and confocal microscopy. In addition, it was also associated with the surface of cytoplasmic globular structures when expressed alone or as a part of the NS3-5B polyprotein. Oil red O staining revealed that these globular structures were lipid droplets, where the HCV core protein was also localized. The association of NS5A with intracellular membrane was further confirmed by membrane flotation analysis. To determine whether NS5A interacts with any cellular lipid-binding protein, we performed yeast two-hybrid screening in both HepG2 and human liver cDNA libraries. Apolipoprotein A1 (apoA1), one of the protein components of high-density lipoprotein (HDL) particles, was identified by two independent screening processes. The interaction between NS5A and apoA1 was confirmed by both in vitro pull-down and in vivo coimmunoprecipitation experiments. Immunofluorescence staining revealed a significant colocalization of NS5A and apoA1 in the Golgi apparatus. Our results established an association of NS5A with lipid droplets and apoA1, suggesting that NS5A, together with the core protein, may play a role in the pathogenesis of the derangement of lipid metabolism, contributing to liver steatosis commonly observed in hepatitis C.     

ATPase 抑制因子1是与HCMV pUL23蛋白相作用的宿主蛋白分子-酵母双杂交技术筛选与鉴定

目的： 利用酵母双杂交技术筛选与人巨细胞病毒相互作用的宿主蛋白分子,为探讨人巨细胞病毒pUL23蛋白在HCMV生活周期中的作用机制提供依据。方法: 利用GAL4酵母双杂交系统筛选人胚肾cDNA文库,以获得与人巨细胞病毒pUL23蛋白相互作用的宿主蛋白分子,再通过回交试验和体外GST-pulldown试验验证两者之间的相互作用。结果: 酵母双杂交筛选得到宿主蛋白分子ATPase inhibitory factor 1（ATIF1）,回交试验和体外GST-pulldown试验再次确认ATIF1能够与人巨细胞病毒pUL23蛋白相互作用。结论: pUL23确实能够与ATIF1相互作用,它们之间的相互作用可能为研究pUL23在病毒生活周期发挥的功能提供依据。