酵母双杂交技术筛选NECL1胞内区的结合蛋白

目的 筛选人源膜表面黏附分子NECL1蛋白胞内区相互作用蛋白。 方法 构建含人NECL1蛋白胞内区氨基酸编码序列的诱饵质粒pGBKT7-NECL1C,对人胎脑cDNA文库进行酵母双杂交筛选。用GST pull down实验进行体外蛋白相互作用的验证。结果 酵母双杂交阳性克隆测序后显示共存在9段不同序列（存在重复克隆）。比对氨基酸序列得到5个可能相互作用蛋白。通过GST pull down 实验验证了其中两个蛋白与NECL1胞内区的相互作用。结论 应用酵母双杂交系统,获得了一些候选的NECL1胞内区相互作用蛋白。     

STCH减弱Aβ_(25-35)对SH-SY5Y培养细胞神经毒性作用的初步研究

目的:研究应激和分子伴侣蛋白(stress and chaperon,STCH)拮抗β淀粉样蛋白(beta-amyloid25-35,Aβ25-35)在体外对人神经母细胞瘤细胞系SH-SY5Y毒性的机制。方法:用Aβ25-35(20μmol/L)在不同时间点分别处理SH-SY5Y培养细胞,然后采用逆转录-聚合酶链式反应(RT-PCR)和蛋白印记(Western Blot)法检测内质网应激相关分子的表达和c-Jun amino-terminal kinase(JNK)的磷酸化,并采用负载钙荧光探针(acetoxymethyl ester ofFura-2,Fura-2AM)钙成像技术检测过表达STCH对100nmol/L星孢霉素(staurosporin)引起的钙超载的情况。结果:Aβ25-35处理SH-SY5Y细胞后引起STCHmRNA表达上调,在3h开始升高,12h达到高峰,然后下降;而葡萄糖调节蛋白94(glucose-regulated protein94,Grp94)则于处理后6h表达上调,9h达到高峰,然后快速下降。Aβ25-35处理SH-SY5Y细胞能够激活JNK通路,致其磷酸化增加,过表达STCH,能够抑制JNK的磷酸化。过表达的STCH能够显著拮抗stauroposrine引起的游离钙增加。结论:STCH可能通过拮抗游离钙增加,抑制JNK通路而发挥保护神经元的作用,这将为我们寻找神经保护的策略提供有益的线索。     

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。     

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。     

细菌双杂交系统筛选与tumstatin45-132相互作用蛋白质

目的筛选与新生血管抑制剂肿瘤抑素功能性片段tumstatin45-132相互作用的蛋白质分子。方法构建诱饵蛋白载体pBT-tumstatin45-132,通过细菌双杂交系统筛选人肝细胞cDNA文库;克隆表达MBP-tumstatin45-132及候选蛋白His-MMP-2,利用MBP pull-down assay和蛋白质印迹方法验证pBT-tumstatin45-132与候选蛋白MMP-2间的相互作用。结果pBT-tumstatin45-132无自身转录激活活性,筛选人肝细胞cDNA文库,获得25个双阳性克隆,序列分析和同源检索表明所获其中之一候选蛋白为基质金属蛋白酶MMP-2;MBP pull-down assay和蛋白质印迹方法验证了pBT-tumstatin45-132与候选蛋白MMP-2间确实存在相互作用。结论　这些结果为进一步研究tumstatin45-132新的功能和机制创造了条件。     

新基因HSPCSET的蛋白结构分析和功能初探

目的 研究人脐血CD34+造血干/祖细胞来源的新基因HSPCSET的结构和生物学功能.方法 采用生物信息学技术对HSPCSET蛋白进行结构分析和同源性比较.酵母双杂交实验筛选与HSPCSET蛋白相互作用的蛋白分子,测序后筛选出功能较明确的阳性克隆进一步回复性杂交,采用β-gal实验和GST拉下试验验证.免疫荧光共定位方法 分析HSPCSET和相关基因细胞内定位情况.结果 蛋白结构分析和同源性比对揭示HSPCSET是古老的SET基因家族的成员,可能具有非常保守而重要的生物学功能.应用酵母双杂交技术筛选能与之相互作用的蛋白,获得13个阳性克隆,分别涉及信号传导、转录调控、细胞凋亡、肿瘤发生、发育等途径.与细胞增殖、分化、凋亡及肿瘤发生密切相关.采用GST拉下试验进一步在体外水平验证了其中4个基因:GADD34(凋亡相关分子)、SIVA(凋亡相关分子)、DNAJ(分子伴侣)和PHF](转录相关因子)与HSPCSET蛋白确实存在相互作用.免疫荧光共定位方法 显示HSPCSET和GADD34共转染细胞株后共同定位于细胞核内,两者之间存在强烈的相互作用.结论 新基因HSPCSET可能参与凋亡途径、具有非常保守而重要的生物学功能.本研究结果 为进一步研究HSPCSET在造血调控中的作用及研究肿瘤发生奠定基础.     

应激和分子伴侣对鱼藤酮诱导的SH-SY5Y细胞毒性的保护作用

目的:本文探讨应激和分子伴侣(stress and chaperone,STCH)对鱼藤酮的神经毒性之于SH-SY5Y细胞的保护作用及其可能的作用机制。方法:采用不同浓度鱼藤酮处理SH-SY5Y-pcDNA 3.1和SH-SY5Y-pcDNA3.1-STCH稳定细胞株,并观察细胞形态;采用MTT法和Western蛋白印迹法检测MAPK通路相关的信号分子和凋亡相关蛋白变化。结果:与未处理组相比,鱼藤酮均能引起细胞活力显著下降(P<0.05);STCH对鱼藤酮诱导的SH-SY5Y细胞毒性具有明显的保护作用(0.1μmol/L-5μmol/L),但10μmol/L浓度组与转染空载体组无显著差异。激活型caspase-3蛋白在鱼藤酮毒素处理后表达明显,p38磷酸化增强,过表达STCH在鱼藤酮处理后p38磷酸化受到明显抑制,裂解的caspase-3活性形式减少。结论:STCH对鱼藤酮诱导神经毒性的保护作用可能通过抑制p38磷酸化而实现。     

Cyclin G2与LDHA相互作用的鉴定分析

目的分析细胞周期素G2(cyclin G2)与乳酸脱氢酶A(LDHA)之间的相互作用及其意义。方法将LDHA全长cDNA片段分别克隆到pGEX-5X-1和p3×FLAG-CMV-14载体上,构建pGEX-LDHA原核重组蛋白表达载体和p3×FLAG-CMV-LDHA真核重组蛋白表达载体,诱导表达并纯化GST-LDHA融合蛋白,通过GST pull-down及western blot技术分析cyclin G2与LDHA的相互作用。分别转染p3×FLAG-CMV-LDHA、pEGFP-FALGCCNG2质粒到HEK293细胞使其表达,利用带有抗FLAG蛋白标签抗体的免疫沉淀凝胶进行Co-IP实验,进一步验证cyclin G2与LDHA的相互作用。结果成功构建了pGEX-LDHA原核重组蛋白表达载体和p3×FLAG-CMVLDHA真核重组蛋白表达载体。GST pull-down实验证实了cyclin G2与LDHA在细胞外的相互作用。Co-IP实验证实了cyclin G2和LDHA在细胞内的相互作用。结论 cyclin G2与LDHA在细胞内和细胞外能发生相互作用,为研究cyclin G2生物学功能奠定实验基础。     

NSPc1相互作用蛋白的筛查及其与组蛋白乙酰化酶HBO1相互作用的验证

目的 寻找多梳家族(polycomb group)蛋白NSPc1的体内可能的相互作用蛋白.方法 通过酵母双杂交实验筛选与NSPc1相互作用的蛋白.使用Pull-down实验,Co-IP实验以及细胞内荧光共定位实验进一步证实双杂交中发现的相互作用.结果 分别以NSPc1蛋白的全长、N端和C端作为诱饵筛查人3月胎脑cDNA文库,发现N端诱饵可以与组蛋白乙酰化转移酶HBO1相互作用,该相互作用在体内外实验中都得到了验证.结论 组蛋白乙酰化酶HBO1是NSPc1的相互作用蛋白之一,该相互作用可能参与NSPc1对靶基因转录的表观抑制活性的发挥.     

应用酵母双杂交系统筛选与乙型流感病毒BM2相互作用的蛋白质

目的　筛选与乙型流感病毒BM2蛋白相互作用的蛋白质。方法　应用酵母双杂交系统,以BM2 (2 6 10 9)插入载体pGBKT7作为诱铒,在四缺培养基上筛选人KidneyMATCHMAKERcDNA文库,寻找与BM2蛋白相互作用的宿主蛋白。结果　筛选得到6个阳性AD 文库质粒,并用酵母双杂交实验验证了阳性AD 文库质粒与BM2的相互作用。将阳性AD 文库质粒测序并对测序结果做BLAST分析,发现它们分别是N 乙酰神经氨酸酶丙酮酸裂解酶,Angiopoietin 3、锌指蛋白2 5 1、核糖体蛋白S2 0、蛋白精氨酸N 甲基转移酶1(PRMT)、转录因子样1(TCFL1)。结论　BM2能与这些转录翻译功能相关的蛋白质相互作用,表明BM2蛋白在病毒生活周期中发挥重要作用。     

STCH对多巴胺能神经元保护作用的初步研究

本文通过不同手段探讨了STCH(stress and chaperone)对多巴胺能神经元的作用。采用RT-PCR检测STCH的组织表达模式;采用免疫-激光捕获显微切割技术获得单一多巴胺能神经元,采用RT-PCR检测STCH在中脑不同亚区多巴胺能神经元的表达差异;分别构建STCH过表达pEGFP-N2载体和pSuper-EGFP干扰载体,转染HEK293和SH-SY5Y细胞株,检测细胞对MPP+毒性的反应。结果显示:STCH在海马表达最低,肝脏最高;在中央灰质区的多巴胺能神经元可检测到表达,在黑质区检测不到;将STCH干扰片段转染至HEK293细胞,细胞死亡明显;转染至SH-SY5Y细胞,对细胞生长无明显影响,但细胞形态发生改变;与对照组相比过表达STCH的SH-SY5Y细胞对MPP+毒性有抵抗作用,并能抑制MPP+处理细胞中caspase-12的表达。这些结果提示:STCH对多巴胺能神经元具有潜在的保护作用,其机制可能是通过抑制内质网应激诱导的凋亡途径而实现。该结果为寻找Parkinson病的治疗靶向提供了有益的线索。     

Up-regulation of SUMO E3 ligases during lung schistosomula and adult worm stages

Small ubiquitin-like modifier (SUMO) conjugation of proteins occurs through a concert action of enzymes using a similar ubiquitination mechanism. After a C-terminal peptide is cleaved from the SUMO precursor by a protease to reveal a di-glycine motif, SUMO is activated by an E1 enzyme (Aos1/Uba2) and conjugated to target proteins by the sole E2 enzyme (Ubc9) guided to the appropriate substrates by the SUMO E3 ligase. Previous reports from our group showed that Schistosoma mansoni has two paralogs of SUMO: one E2 conjugation Ubc9 and two SUMO-specific proteases (SENPs). The differential gene expression profile observed for SUMO pathway genes throughout the S. mansoni life cycle attests for the distinct patterns of SUMO conjugates observed during parasite development particularly during the cercariae to schistosomula transition. To continue this investigation, we here analysed the repertoire of SUMO E3 ligases and their expression profiles during cercariae/schistosomula transition. In silico analysis through S. mansoni databases showed two conserved SUMO E3 ligases: protein inhibitor of activated STAT (PIAS) and Ran-binding protein 2 (RanBP2). Furthermore, expression levels of the SUMO E3 ligases were measured by qRT-PCR using total RNA from cercariae, adult worms and mechanically transformed schistosomula. Our data showed an up-regulation of expression in lung schistosomula and adult worm stages. In conclusion, the differential expression of SmPIAS and SmRanBP2 during schistosomula development was similar to the expression levels of all genes related to SUMO conjugation, thereby suggesting that the control of protein activity, localisation or stability during cercariae to schistosomula transition is SUMO-dependent.     

Subcellular distribution of importins correlates with germ cell maturation.

Importin proteins regulate access to the nucleus by recognizing and transporting distinct cargo proteins. Building on studies in Drosophila and Caenorhabditis elegans, we hypothesized that regulated expression and subcellular localization of specific importins may be linked to mammalian gonadal differentiation. We identified distinct developmental and cellular localization patterns for importins beta1, alpha3, alpha4 and RanBP5 (importin beta3) in fetal and postnatal murine testes using Western blotting and immunohistochemistry. Importin beta1 protein is detected in selected germ and somatic cells in fetal gonads, with a striking perinuclear staining evident from embryonic day (E) 14.5 within testicular gonocytes. RanBP5 exhibits age- and gender-specific subcellular localization within fetal gonads. At E12.5, RanBP5 protein is cytoplasmic in gonocytes but predominantly nuclear in oogonia, but by E14.5 RanBP5 appears nuclear in gonocytes and cytoplasmic in oogonia. In postnatal testes, importin alpha3 and alpha4 in spermatocytes, spermatids, and Sertoli cells display cytoplasmic and nuclear localization, respectively.     

Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants.

Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell extracts and dithiothreitol-, Zymolyase-, or beta-mercaptoethanol-solubilized cell extracts were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). From dithiothreitol-solubilized cell extracts, proteins with molecular masses of 55 to 60, 80 to 84, 115, and 165 kDa were observed from wt (A9) cells but were highly reduced in amount or absent from A9V2 cells. Western blot profiles of Zymolyase-solubilized extracts from both A9 and A9V2 were similar in appearance, while 55-, 80- to 84-, 115-, and 165-kDa proteins were observed only in A9 cells extracted with beta-mercaptoethanol. Strain A9V4, also selected by reduced agglutination but which adhered as well as strain A9, lacked the 80- to 84-kDa and 115-kDa proteins but otherwise was similar to strain A9. These results indicate that the 55- to 60- and 165-kDa proteins may be related to an adhesin function in C. albicans. The differences observed in the protein profiles of the wt adhering strain and its derived nonadhering mutant are similar to those described for another matched pair of C. albicans strains.     

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase.     

Expression of human papillomavirus proteins in yeast Saccharomyces cerevisiae.

The L1 and L2 proteins of human papillomavirus (HPV) types 1, 6, and 16 and the E6 and E7 proteins of HPV 16 were expressed in Saccharomyces cerevisiae. The yeast expressed proteins were readily detected by immune blotting and were generally intact. The HPV 1 L1 and L2 proteins expressed in yeast were indistinguishable from the major and minor capsid proteins purified from HPV 1 virions as judged by gel electrophoresis and immunoblotting. The HPV 6 and HPV 16 L2 proteins and HPV 16 E7 proteins were secreted from yeast by fusion to the yeast pre-pro-alpha-factor leader sequence. Following secretion of the HPV 16 E7 protein a rapid method of purification was developed. The yeast expressed proteins were used as antigen targets to study the human immune response in Western blot assay, ELISA, and immune precipitation. One human serum reacted with intact, but not denatured HPV 16 L2 proteins, suggesting that the yeast expressed proteins will be useful to detect antibodies reactive with conformational epitopes.     

Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests

BACKGROUND: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. OBJECTIVE: The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. METHODS: An autoantigen involved in Wegener's disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. Results: In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. CONCLUSION: Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.