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Studies on genes involved in thrombois on endothelial cells

内皮细胞血栓形成相关基因的研究

基本信息

批准号：
08044333
负责人：
MIYATA Toshiyuki
金额：
$ 4.67万
依托单位：
National Cardiovascular Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

An elevated level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not been elucidated yet. In the present study, we evaluated changes of gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells. We identified six up-regulated and one down-regulated gene. One up-regulated gene was GRP78, a stress protein, suggesting that unfolded proteins would accumulate in the endoplasmic reticulum (ER) because of redox potential changes caused by homocysteine. We isolated cDNA clones of two novel up-regulated genes, RTP (reducing agents and tumicamycin-responsive protein) and Herp (homocysteine inducible ER resident protein). RTP consisted of 394 amino acids. RTP mRNA was induced by not only homocysteine but also 2-mercaptoethanol and tumicamycin. We raised the polyclonal antibody against the recombinant RTP.Immunological analysis revealed that it was a cytosolic protein and was present in the proximal tubule of renal cortex and the microvilli of intestine.RTP was a phosphorylated protein with 8 phosphorylation sites. Herp consisted of 391 amino acids. It was estimated to have a transmembrane domain. Immunological analysis revealed that it was an ER-resident protein. Herp mRNA also induced by reducing agents and tumicamycin. Yeast two-hybrid system revealed that Herp can bind with a novel protein, Herp interacting protein (HIP). HIP was 236 amino acids. HIP showed a sequence homology against an ER-resident protein, neuroendocrine specific protein. Therefore, we speculated that Herp is present in the ER with a complex with an ERresident protein HIP.

同型半胱氨酸水平升高与动脉硬化和血栓形成有关。同型半胱氨酸促进血管疾病的机制尚未阐明。在本研究中，我们评估了同型半胱氨酸处理对培养的人脐静脉内皮细胞基因表达的影响。我们发现了6个上调基因和1个下调基因。其中一个上调的基因是应激蛋白GRP78，这表明未折叠的蛋白会由于同型半胱氨酸引起的氧化还原电位变化而在内质网（ER）中积累。我们分离了两个新的上调基因，RTP（还原剂和tumicamycin反应蛋白）和Herp（同型半胱氨酸诱导内质网驻留蛋白）的cDNA克隆。RTP由394个氨基酸组成。除同型半胱氨酸外，2-巯基乙醇和tumicamycin均可诱导RTP mRNA表达。我们建立了针对重组RTP的多克隆抗体。免疫学分析表明，它是一种细胞质蛋白，存在于肾皮质近端小管和肠微绒毛中。RTP是一个磷酸化蛋白，有8个磷酸化位点。疱疹病毒由391个氨基酸组成。据估计它具有跨膜结构域。免疫学分析显示它是一种er驻留蛋白。Herp mRNA也可被还原剂和tumicamycin诱导。酵母双杂交系统表明，Herp可以与一种新的蛋白结合，即Herp相互作用蛋白（HIP）。HIP为236个氨基酸。HIP与er驻留蛋白、神经内分泌特异性蛋白具有序列同源性。因此，我们推测Herp存在于内质网中，并与ERresident蛋白HIP形成复合物。