Characterization of a novel ER membrane protein SPOC

描述（申请人提供）：最近的研究表明，某些内质网（ER）膜蛋白通过控制钙从内质网向线粒体的迁移来调节细胞存活。我们已经鉴定并鉴定了一个cDNA，该cDNA对应于一个新基因，该基因编码一种假定的跨膜蛋白，我们将其命名为SPOC（癌症中过度表达的生存启动子）。我们的初步结果表明，与与之匹配的正常组织相比，SPOC mRNA在原发性结肠肿瘤中高表达。有趣的是，SPOC mRNA的表达在肿瘤转移中进一步升高。外源性gfp标记的SPOC与ER特异性蛋白标记共定位，表明SPOC似乎存在于ER中。已知thapsigarin和sulindac硫化物通过促进ER Ca2+池耗竭来扰乱细胞内Ca2+稳态。两种药物通过下调SPOC mRNA表达诱导HCT116人结肠癌细胞凋亡。有趣的是，外源性SPOC在HCT116细胞中的组成性表达抵消了这些药物的凋亡作用，表明SPOC似乎可以促进细胞存活。我们现在正在提议进一步研究SPOC在消化道疾病如结肠癌中的作用。我们将分析大量新鲜冷冻和石蜡包埋的组织标本，以评估SPOC在mRNA和蛋白水平上的表达。为了探索SPOC促进细胞存活的分子机制，我们将研究其对PI3-K/ akt依赖性生长和存活信号通路的影响。我们还将研究SPOC是否调节细胞内Ca2+稳态并调节Ca2+依赖的信号通路以促进细胞存活。为了确定内质网跨膜定位对其促存活功能是否重要，我们将突变和/或删除其假定的n端内质网检索基序以及跨膜区域，并研究突变型SPOC的亚细胞定位及其对细胞存活的影响。这些都是探索性/发育性研究，一旦完成，将产生足够的新数据和试剂，为未来深入研究这种新型内质网跨膜蛋白在一般情况下，特别是消化系统疾病中调节细胞存活的分子机制奠定基础。

DESCRIPTION (provided by applicant): Recent studies have shown that certain endoplasmic reticulum (ER) membrane proteins modulate cell survival by controlling the migration of calcium from ER to mitochondria. We have identified and characterized a cDNA that corresponds to a novel gene which encodes a putative transmembrane protein that we have named SPOC (Survival Promoter Overexpressed in Cancers). Our preliminary results indicate that the SPOC mRNA is highly expressed in primary colon tumors when compared to their matching normal tissues. Interestingly, SPOC mRNA expression is further increased in tumor metastases examined. Exogenous GFP-tagged SPOC co-localizes with ER-specific protein markers suggesting that SPOC appears to reside in the ER. Thapsigargin and sulindac sulfide are known to perturb intracellular Ca2+ homeostasis by promoting ER Ca2+ pool depletion. Both agents induced apoptosis in HCT116 human colon cancer cells that was coupled with down-regulation of SPOC mRNA expression. Interestingly, constitutive expression of exogenous SPOC in HCT116 cells countered the apoptotic effects of these agents suggesting that SPOC appears to promote cell survival. We are now proposing studies to further investigate the role of SPOC in digestive diseases such as colon cancer. We will analyze a larger pool of fresh-frozen and paraffin-embedded tissue specimens to evaluate the expression of SPOC at mRNA and protein levels. To explore the molecular mechanisms by which SPOC promotes cell survival, we will study its effect on PI3-K/Akt-dependent growth and survival signaling pathways. We will also investigate whether SPOC regulates intracellular Ca2+ homeostasis and modulates the Ca2+-dependent signaling pathway(s) to promote cell survival. To determine whether ER transmembrane localization is important for its survival promoting function, we will mutate and/or delete its putative N-terminal ER-retrieval motif as well as the transmembrane regions and study the subcellular localization of mutant SPOC and its effect on cell survival. These are exploratory/developmental studies that, upon completion, will generate sufficient new data and reagents that will form the basis of future in-depth studies investigating the molecular mechanism(s) of action of this novel ER transmembrane protein in regulation of cell survival in general and digestive diseases in particular.