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ER向线粒体的迁移来调节细胞存活。我们已经鉴定并表征了对应于编码推定的跨膜蛋白的新基因的cDNA，我们将其命名为SPOC（Survival Promoter Overexpressed in Cancers）。我们的初步研究结果表明，SPOC mRNA在原发性结肠肿瘤中的高表达时，与其匹配的正常组织相比。有趣的是，SPOC mRNA表达在检查的肿瘤转移中进一步增加。外源性GFP标记的SPOC与ER特异性蛋白标记物共定位，表明SPOC似乎存在于ER中。已知毒胡萝卜素和舒林酸硫化物通过促进ER Ca 2+池耗竭来扰乱细胞内Ca 2+稳态。两种药物均诱导HCT 116人结肠癌细胞凋亡，并伴有SPOC mRNA表达下调。有趣的是，在HCT 116细胞中外源SPOC的组成型表达抵消了这些试剂的凋亡作用，表明SPOC似乎促进细胞存活。我们现在正在提出研究，以进一步调查SPOC在消化系统疾病，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PI 3-K/Akt依赖的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Ca 2+稳态和调节Ca 2+依赖性信号通路以促进细胞存活。为了确定ER跨膜定位是否对其生存促进功能很重要，我们将突变和/或删除其假定的N-末端ER检索基序以及跨膜区域，并研究突变SPOC的亚细胞定位及其对细胞存活的影响。这些是探索性/开发性研究，完成后将产生足够的新数据和试剂，这些数据和试剂将成为未来深入研究的基础，研究这种新型ER跨膜蛋白在一般细胞存活调节中的作用分子机制，特别是消化系统疾病。

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.