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。     

与接头蛋白Bam32相互作用的蛋白分子的鉴定

目的:研究接头蛋白(adaptorprotein)Bam32在B细胞抗原受体(Bcellantigenreceptor,BCR)信号转导级联反应中的作用。方法:以Bam32全序列为诱饵,应用酵母双杂交技术筛选能与Bam32相互作用的蛋白分子,并用293T细胞共转染和免疫共沉淀法加以证实。结果:应用酵母双杂交技术筛选出能与Bam32相互作用的蛋白分子,其中1个出现强阳性反应的克隆编码酪氨酸激酶Lyn。用293T细胞共转染和特异性免疫共沉淀法证实,Bam32可在哺乳动物细胞中与Lyn共沉淀。应用抗磷酸化酪氨酸抗体检测显示,Bam32与Lyn相互作用可导致Bam32磷酸化。结论:Bam32可同Lyn相互作用,导致Bam32磷酸化,这在激活下游产物的级联反应中可能起重要作用。     

Tdrd3 is a novel stress granule-associated protein interacting with the Fragile-X syndrome protein FMRP

Tudor domains are widespread among proteins involved in RNA metabolism, but only in a few cases their cellular function has been analyzed in detail. Here, we report on the characterization of the ubiquitously expressed Tudor domain containing protein Tdrd3. Apart from its Tudor domain, we show that Tdrd3 possesses an oligosaccharide/nucleotide binding fold (OB-fold) and an ubiquitin associated domain capable of binding tetra-ubiquitin. A set of biochemical experiments revealed an interaction of Tdrd3 with FMRP, the product of the gene affected in Fragile X syndrome, and its autosomal homologs FXR1 and FXR2. FMRP has been implicated in the translational regulation of target mRNAs and shown to be a component of stress granules (SG). We demonstrate that overexpression of Tdrd3 in cells induces the formation of SGs and as a result leads to its co-localization with endogenous FMRP in these structures. Interestingly, the disease-associated FMRP missense mutation I304N identified in a Fragile X patient severely impairs the interaction with Tdrd3 in biochemical experiments. We propose a contribution of Tdrd3 to FMRP-mediated translational repression and suggest that the loss of the FMRP-Tdrd3 interaction caused by the I304N mutation might contribute to the pathogenesis of Fragile X syndrome.     

Cloning and characterization of a novel gene which encodes a protein interacting with the mitosis-associated kinase-like protein NTKL

NTKL is an evolutionarily conserved kinase-like protein. The cell-cycle-dependent centrosomal localization of NTKL suggested that it was involved in centrosome-related cellular function. The mouse NTKL protein is highly homologous with human NTKL. A novel mouse protein was identified as an NTKL-binding protein (NTKL-BP1) by yeast two-hybrid screening, and the full-length cDNA was amplified based on the result of a sequence data analysis cloning strategy. The full-length cDNA sequence of the NTKL-BP1 gene consists of 2,537 bp, which encode 368 amino acids. A database search revealed that homologues of NTKL-BP1 exist in different organisms, including Arabidopsis thaliana, Drosophila melanogaster, Plasmodium falciparum, Geobacter metallireducens, Anopheles gambiae and human. It suggests that NTKL-BP1 is an evolutionarily conserved protein. The expression of NTKL-BP1 was observed in multiple normal mouse tissues. The interaction of the two proteins was confirmed by co-immunoprecipitation. Moreover, immunofluorescent staining indicated that NTKL and NTKL-BP1 were all localized in the cytoplasm.     

Five novel RPGR mutations in families with X-linked retinitis pigmentosa

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe because of their early onset, often leading to significant visual impairment before the fourth decade. RP3, genetically localized at Xp21.1, accounts for 70% of XLRP in different populations. The RPGR (Retinitis Pigmentosa GTPase Regulator) gene that was isolated from the RP3 region is mutated in 20% of North American families with XLRP. From mutation analysis of 27 independent XLRP families, we have identified five novel RPGR mutations in 5 of the families (160delA, 789 A>T, IVS8+1 G>C, 1147insT and 1366 G>A). One of these mutations was detected in a family from Chile. Hum Mutat 17:151, 2001.     

蛋白激酶R与丙型肝炎病毒核心蛋白相互作用区域定位

目的： 观察丙型肝炎病毒（HCV）核心蛋白（CP）对蛋白激酶R(PKR)表达的影响；定位PKR与CP直接结合的区域。方法: 对Huh-7、转染表达CP的Huh-7及含有全长HCV复制子(replicon) Huh-7细胞株的PKR表达水平及干扰素（IFN）诱导前后replicon Huh-7细胞中HCV结构蛋白和非结构蛋白表达水平作比较；对CP与PKR进行免疫共沉淀试验、谷胱苷肽S转移酶（GST）结合试验。结果: Replicon Huh-7中PKR表达水平高于Huh-7及转染表达CP的Huh-7；IFN诱导后PKR表达增加，且明显抑制HCV结构和非结构蛋白的表达；PKR能与CP直接结合，依赖于PKR的N端1-180氨基酸（aa）。结论: CP能直接作用于PKR N端1-180 aa，导致PKR组成性激活，从而干扰PKR介导的相关信号转导通路。CP与PKR的相互作用是HCV病毒蛋白与细胞蛋白相互作用又一新的模式，在HCV持续感染及肝癌2者发病机制方面可能起重要作用。     

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 a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed.     

一个X-连锁视网膜色素变性中国家系的RPGR基因的新突变

目的 对中国人X-连锁视网膜色素变性一家系进行分子遗传学检测，报告RPGR基因突变。方法 首先对该家系X染色体进行致病基因的连锁分析，然后用单链构象多态性技术和直接DNA测序方法进行基因突变分析。结果 连锁分析在多态性微卫星遗传标记DXSS012和DXS8025产生正的Lod值分别为2．41（Zmax=2．40，θ=0）和1．26。进一步单倍型分析确定该家系致病基因位于Xp21．1，与RP3连锁。用RPGR基因突变分析，在外显子ORF15＋483－484发现GA缺失，引起阅读框架的改变，该基因缺失突变在家系中共分离。结论 报告了中国人X-连锁视网膜色素变性RPGR基因外显子ORF15＋483-484的GA缺失突变，丰富了中国人RPGR基闪突变谱，为今后研究X-连锁视网膜色素变性的基因奠定基础。     

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。     

乙型肝炎病毒E抗原肝细胞作用蛋白AK026018表达谱芯片的研究

目的 应用基因芯片技术,筛选能被乙型肝炎病毒E抗原（HBeAg）肝细胞作用蛋白AK026018反式调节的靶基因,初步研究该蛋白的生物学功能.方法 应用反转录聚合酶链反应（RT-PCR）技术,从HepG2细胞中扩增编码AK026018蛋白的全基因,构建真核表达载体,转染肝母细胞瘤系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1的HepG2细胞进行DNA芯片分析并比较.结果 经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确.在8464个基因表达谱的筛选中,发现有122个基因有差异表达,其中78种基因表达水平显著下调,45种基因表达水平显著上调.结论 成功地应用DNA芯片技术筛选出HBeAg结合蛋白新基因AK026018的反式调节蛋白,证明该基因对于肝细胞基因表达谱有显著影响.     

A novel mutation of RPGR gene in an X-linked Chinese family with retinitis pigmentosa

PurposeTo localize and identify the gene and mutations causing an X-linked Chinese family with retinitis pigmentosa.MethodsAn XLRP Chinese family was ascertained and patients underwent ophthalmological examinations. Blood samples were collected and genomic DNA was extracted. Linkage scan was performed on genomic DNA from affected and unaffected family members using microsatellite markers flanking 17 known autosomal dominant loci and markers covering the entire X chromosome. Mutation screening of RPGR gene was carried out by direct DNA sequence analysis.ResultsA genome wide scan yielded a lod score of 2.7 at θ = 0 with DXS1068 and 3.29 at θ = 0 with DXS993. This region harbors the RPGR gene. Direct DNA sequence analysis reveals one base pair deletion, gORF15 + 556delA, in all affected individuals. The deletion results in the frameshift change of RPGR gene and produces a truncated protein.ConclusionsWe identified a novel mutation, gORF15 + 556delA (p.Lys184fs), in a Han Chinese family with retinitis pigmentosa. This mutation expands the mutation spectrum of RPGR and helps to study molecular pathogenesis of RP further.     

Identification of a novel tobacco DnaJ-like protein that interacts with the movement protein of tobacco mosaic virus

The movement protein (MP) of tobacco mosaic virus (TMV) mediates the transport of viral RNA from infected cells to neighboring uninfected cells via plasmodesmata by interacting with putative host factors. To find such host factors, we screened tobacco proteins using the yeast two-hybrid system. NtMPIP1, a novel subset of DnaJ-like proteins, was identified from a tobacco cDNA library, and its specific interaction with TMV MP was confirmed with an in vitro filter-binding assay. In a deletion analysis, using a series of truncated TMV MPs and NtMPIP1s, at least two regions of TMV MP, amino acid residues 65–86 and 120–185, conferred the ability to interact with the C-terminal domain of NtMPIP1, which is thought to be involved in substrate binding. Virus-induced gene silencing of NtMPIP1 significantly inhibited the spread of TMV. Therefore, it is reasonable to consider that endogenous NtMPIP1 is a host factor involved in virus cell-to-cell spread by interacting with TMV MP. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB092334.     

Identification of small subunit of serine palmitoyltransferase a as a lysophosphatidylinositol acyltransferase 1‐interacting protein

Lysophosphatidylinositol acyltransferase 1 (LPIAT1), also known as MBOAT7, is a phospholipid acyltransferase that selectively incorporates arachidonic acid (AA) into the sn‐2 position of phosphatidylinositol (PI). We previously demonstrated that LPIAT1 regulates AA content in PI and plays a crucial role in brain development in mice. However, how LPIAT1 is regulated and which proteins function cooperatively with LPIAT1 are unknown. In this study, using a split‐ubiquitin membrane yeast two‐hybrid system, we identified the small subunit of serine palmitoyltransferase a (ssSPTa) as an LPIAT1‐interacting protein. ssSPTa co‐immunoprecipitated and colocalized with LPIAT1 in cultured mammalian cells. Knockdown of ssSPTa decreased the LPIAT1‐dependent incorporation of exogenous AA into PI but did not affect the in vitro enzyme activity of LPIAT1 in the microsomal fraction. Interestingly, knockdown of ssSPTa decreased the protein level of LPIAT1 in the crude mitochondrial fraction but not in total homogenate or the microsomal fraction. LPIAT1 was localized to the mitochondria‐associated membrane (MAM), where AA‐selective acyl‐CoA synthetase is enriched. These results suggest that ssSPTa plays a role in fatty acid remodeling of PI, probably by facilitating the MAM localization of LPIAT1.