Studies on genes involved in endothelial dysfunction involved in thrombosis and atherosclerosis

同型半胱氨酸水平升高与动脉硬化和血栓形成有关。同型半胱氨酸可能促进血管疾病的机制尚未阐明。我们以前分离到了两个新的cDNA克隆，RTP和Herp，这两个克隆都被同型半胱氨酸上调。在本研究中，我们制备了针对重组蛋白的多克隆抗血清。Western印迹分析表明，RTP主要定位于细胞质中。RTP在7个或更多的位点部分磷酸化。它的磷酸化被Forsklin处理增强，并被蛋白激酶A抑制剂和钙调蛋白激酶抑制剂抑制。蛋白激酶A在体外直接磷酸化重组RTP。在对数生长期早期，细胞中的磷酸化形式丰富，然后随着细胞密度的增加而减少。免疫组织化学结果显示，RTP在小鼠肾近端小管和肠绒毛中大量表达。这些结果表明，RTP在肾近端小管和肠绒毛中均有大量表达。这些数据表明，RTP是细胞质中的磷酸蛋白，其磷酸化可能与细胞生长有关。另一方面，HERP的Western印迹分析表明，它是一种与内质网(ER)相关的54 kDa蛋白，并受到干扰的强烈诱导，导致未折叠蛋白在内质网中积累。有趣的是，HERP的N端和C端都位于内质网的细胞质一侧。人类疱疹病毒基因由8个外显子组成，全长12kb，定位于染色体16q12.2-13。在其启动子区域，有一个19个碱基的序列对应于内质网胁迫反应顺式作用元件ERSE。HERP可能在细胞对应激的生存反应中扮演一个未知的角色。

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. We previously isolated two novel cDNA clones, RTP and Herp, both of which have been up-regulated by homocysteine. In the present study, we raised polyclonal anti-serum against recombinant proteins. Western blot analysis showed that RTP was mainly located in the cytoplasm. RTP was partially phosphorylated at seven or more sites. Its phosphorylation was enhanced by the forskolin treatment and inhibited by a protein kinase A inhibitor and a calmodulin kinase inhibitor. Protein kinase A directly phosphorylated recombinant RTP in vitro. The phosphorylated forms were abundant in the cells at the early log phase and then decreased in relation to increased cell density. Immunohistochemistry of mouse tissues demonstrated that RTP was present abundantly in proximal tubule of kidney and in villi of intestine. These data demonstrated that RTP was present abundantly in proximal tubule of kidney and in villi of intestine. These data demonstrated that RTP is the cytoplasmic phosphoprotein and its phosphorylation may be related to cell growth. On the other hand, Western blot analysis of Herp revealed that it was an endoplasmic reticulum (ER)-associated 54-kDa protein, and was strongly induced by perturbation that lead to accumulation of unfolded proteins in the ER. Interestingly, both the N and C termini of Herp were present on the cytoplasmic side of the ER. The human Herp gene consisted of 8 exons spanning 12 kb and localized to chromosome 16q12.2-13. In its promoter region, there was a single 19-bp sequence corresponding to the ER-stress responsive cis-acting element, ERSE. Herp may play an unknown role in the cellular survival response to stress.