【作者】 万兵兵；

【导师】 霍克克； 韩泽广；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 分泌蛋白是一类具有重要生理功能的蛋白,参与了细胞信号传递、形态发生、细胞凋亡、细胞分化、免疫防御等多种过程。由于它们能够适应不同的药物运输机制,可以用作临床治疗药物或治疗靶点。为了寻找新的分泌蛋白并研究其生物学功能,本研究运用生物信息学预测结合实验验证的策略从人类公共蛋白数据库中筛选并鉴定新的与细胞生长密切相关的分泌蛋白基因。在第一部分研究工作中,我们采用上述的策略筛选到了一个新的人类分泌蛋白基因hOLFML1(人Olfactomedin样蛋白1,human Olfactomedin-like protein 1),该基因编码蛋白全长为402个氨基酸,C端含有OLF(Olfactomedin)结构域,是一个N-连接的糖基化分泌蛋白,可以被分泌到培养基中。Northern实验显示hOLFML1 mRNA主要在小肠、肝脏、心脏和肺中表达。通过制备它的抗体,我们检测到了hOLFML1蛋白的内源性分泌,而且发现hOLFML1蛋白的分泌是有血清依赖性的。小肠组织的免疫组化实验显示,hOLFML1蛋白主要在小肠的绒毛边缘表达。此外还发现:hOLFML1的表达能明显地促进细胞的生长,而hOLFML1的RNAi却能抑制细胞的生长。当在培液中加入hOLFML1的抗体后,它能起到部分阻断hOLFML1的生物学功能的作用。在第二部分研究工作中,基于同样的策略,我们识别了一个含信号肽的人类新基因C10ORF58(人第10号染色体的58号开放读码框,Q9BRX8。简称RX8)。它是一个高度保守的基因,在线虫等低等多细胞生物中就已经存在它的直系同源基因。RX8基因的组织表达比较广谱,其中在脑、心、肾、胰腺、肝脏和睾丸中RX8呈现高表达。在HepG2-Teton细胞株中,诱导表达的RX8能明显地抑制细胞的生长和细胞克隆的形成,并使细胞周期中处于合成期的细胞数减少和新生蛋白的翻译量降低。此外,RX8还能选择性地增强BFA(Brefeldin A)等破坏内质网药物的促凋亡作用。细胞定位分离实验显示RX8是一个定位于内质网上的整合膜蛋白。通过酵母双杂交实验,我们找到了RX8的一个互作蛋白CAML(钙离子调控的亲环素配体蛋白,calcium-modulating cyclophilin ligand)。通过细胞的Co-IP实验、体外的GST-Pulldown实验以及免疫荧光实验,我们证实了RX8和CAML蛋白在细胞内存在着相互作用。进一步证明了RX8、CAML和EGFR(表皮生长因子受体)在细胞内能形成一个复合体。此外,我们还发现RX8的过表达能明显抑制EGF信号通路下游分子ERK1/2的活化,使其磷酸化水平下降。而RX8的下调表达却明显地促进ERK1/2的磷酸化,而且这种磷酸化能被EGF信号通路ERK1/2分子上游的MEK特异性的抑制剂PD98059所抑制。根据以上这些实验结果,本文提出了一个初步的分子模型假说:EGFR的正常循环依赖于结合在内质网上的CAML蛋白;而当有RX8蛋白表达时,RX8能破坏或者竞争性地抑制EGFR和CAML之间的结合,从而使EGF信号下游分子ERK1/2的激活受到抑制,进而出现生长明显减慢的细胞学现象。在第三部分中,我们初步描述了基于同样策略筛选到的一个新的人类分泌蛋白基因C12ORF39(人第12号染色体39号开放读码框)。免疫印迹试验表明,在转染的HeLa细胞中带有c-myc标签的C12ORF39蛋白可以被分泌到培养基中。在氨基酸序列上,这个蛋白的中间包含有一个典型的酰胺化的蛋白酶加工处理信号(Gly-Arg-Arg基序,GRR Motif)。但C12ORF39蛋白并没有因而被水解成短肽,而是一个具有全长序列但不包含信号肽的蛋白。本文对这三个人类分泌蛋白新基因的发现和初步研究结果对于进一步深入了解其生物学功能有着重要的理论研究意义和实际应用前景。更多还原

【Abstract】 Secreted proteins play important roles in many fundamental cellular and organism functions,such as signaling pathway,morphogenesis,apoptosis,cell differentiation and immune defense.Moreover,secreted proteins have properties that let themselves to be particularly utilized as therapeutic agents or targets.It is interesting and important to search for novel secreted proteins and study their biological functions.The strategy of bioinformatic prediction combined with experimental confirmation was used to isolate and identify novel potential human secreted protein genes which are associated with cell growth from the public protein database in this work.In the first part,we researched a novel member of human olfactomedin family, called hOLFML1(human Olfactomedin-like protein 1),which encodes 402 aa with an OLF domain in its C-terminus.hOLFML1 protein could be secreted into culture medium and was subjected to the N-linked glycosylational modification.Northern blot analysis indicated that hOLFML1 was mainly expressed in small intestine,liver, lung,and heart.Furthermore,immunohistochemical study on human small intestine demonstrated that it localized preferentially surrounding the intestinal villi. Interestingly,when overexpressed in HeLa cell lines.The recombinant hOLFML1 protein can promote the proliferation of cells and increase the percentage of S phase. Moreover,the rabbit anti-hOLFML1 antibody could block growth and cell cycle progression.Surprisingly,in human hepatocarcinoma Huh7 cells,knockdown of hOLFML1 negatively altered the effect of hOLFML1 on the cell growth and cell cycle profile.Together,our findings suggest that hOLFML1 is primarily expressed as an extracellular glycoprotein which could enhance the growth of cells and facilitate cell cycle progression in vitro.In the second part,we report a novel human gene C100RF58(chromosome10 open-reading framework 58,Q9BRX8,short for RX8),with a signal peptide but cannot secreted into serum.C10ORF58 is a very conserved gene in evolution,and there is a orthologuous gene existing in C.elegans.Realtime PCR assay indicated that RX8 ubiquitously expressed in adult human tissues,especially in brain,heart,kidney, pancreas and testis.In HepG2-Teton cell lines,the induced expression of RX8 inhibited cell growth and the formation of cell colones,and deduced S phase cell number and the de novo protein synthesis.Moreover,RX8 can specifically enhance the apoptosis-stimulating activity of BFA(Brefeldin A).Furthermore,subcellular fractionation assay showed that RX8 is an integrated membrane protein in endoplasmic reticulum.We found the interacting protein of RX8:CAML (calcium-modulating cyclophilin ligand) via yeast two-hybrid experiment.Then,we confirmed the interaction of RX8 and CAML thought co-immunoprecipitation, GST-pulldown and immunofluorescence assay.Importantly,we found that RXS, CAML and EGFR(EGF Receptor) co-existed in a complex.Interestingly,the Overexpression of RX8 significantly reduced the phosphorylation level and inhibited the activation of EGF signaling pathway downstream molecules:ERK1/2.On the other hand,the knockdown of RX8 by RNAi promoted the phosphorylation of ERK1/2,and the promotion could be block by the specific inhibitor of MEK1/2: PD98059.Finally,We provide a hypothetic molecular model.The normal recycling of EGFR depended on CAML which integrated in the endoplasmic reticulum.When the RX8 is overexpressed,RX8 could interfere or competitively inhibit CAML and EGFR interaction,therefore,the activation of ERK1/2 and cell growth were been inhibited.In the third part,we describe a novel secreted protein,named C12ORF39 (chromosome12 open-reading framework 39),in the middle of which contains a typical amidation-proteolytic processing signal(Gly-Arg-Arg motif).Western blotting assay indicated that the c-myc tagged C12ORF39 could be secreted into culture medium in transfected HeLa cells.Interestingly,C12ORF39 protein is not been hydrolysated,but is a full-length sequence protein without signal peptides.Our current research results about the three novel human genes will help to understand more deeply their biological functions and discovery their applied Prospects.更多还原