Characterization of a novel ER membrane protein SPOC

描述(由申请人提供)：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与一个新的基因相对应的基因，该基因编码一种可能的跨膜蛋白，我们将其命名为SPOC(Survival Promoter Over Exposed in癌症)。我们的初步结果表明，与其匹配的正常组织相比，SPOC mRNA在原发性结肠肿瘤中高度表达。有趣的是，SPOC mRNA的表达在被检查的肿瘤转移中进一步增加。外源GFP标记的SPOC与ER特异性蛋白标记物共定位，表明SPOC似乎驻留在ER中。已知thapsigargin和Sulindac硫化物通过促进内质网钙池耗竭而扰乱细胞内钙稳态。两种药物均可诱导HCT116人结肠癌细胞发生凋亡，同时下调SPOC mRNA的表达。有趣的是，外源性SPOC在HCT116细胞中的结构性表达抵消了这些药物的凋亡效应，表明SPOC似乎促进了细胞的存活。我们现在正在提议进一步研究SPOC在结肠癌等消化系统疾病中的作用。我们将分析更大的新鲜冷冻和石蜡包埋的组织样本，以评估SPOC在mRNA和蛋白质水平的表达。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依赖的生长和生存信号通路的影响。我们还将研究SPOC是否调节细胞内钙稳态和调节钙依赖信号通路(S)以促进细胞存活。为了确定内质网跨膜定位是否对其促存活功能起重要作用，我们将突变和/或删除其N端ER检索基序和跨膜区，并研究突变型SPOC的亚细胞定位及其对细胞存活的影响。这些是探索性/发育性研究，完成后将产生足够的新数据和试剂，为未来深入研究这种新型ER跨膜蛋白在调节细胞存活特别是消化系统疾病中的分子机制(S)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.