Studies on genes involved in thrombois on endothelial cells

高半胱氨酸水平升高与动脉硬化和血栓形成有关。同型半胱氨酸可能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诱导的人脐静脉内皮细胞基因表达的变化。我们发现了6个上调和1个下调的基因。一个上调的基因是GRP 78，一种应激蛋白，表明由于同型半胱氨酸引起的氧化还原电位变化，未折叠的蛋白质将在内质网（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ER驻留蛋白）的cDNA克隆。RTP由394个氨基酸组成。RTPmRNA的表达不仅受同型半胱氨酸的诱导，也受2-巯基乙醇和tumicamycin的诱导。免疫组化结果表明，RTP是一种胞浆蛋白，存在于肾皮质近端小管和小肠微绒毛中，是一种磷酸化蛋白，有8个磷酸化位点。Herp由391种氨基酸组成。估计其具有跨膜结构域。免疫学分析表明，它是一个ER常驻蛋白。还原剂和tumicamycin也能诱导Herp mRNA的表达。酵母双杂交系统研究表明，Herp可以与一种新的蛋白质--Herp相互作用蛋白（HIP）结合。HIP由236个氨基酸组成。HIP显示了对ER驻留蛋白，神经内分泌特异性蛋白的序列同源性。因此，我们推测，疱疹病毒是目前在ER与一个复杂的ER常驻蛋白HIP。

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.