RENAL CELL RESPONSE TO STRESS

肾上皮细胞，特别是内髓细胞 经常暴露在不利的压力下，包括高渗透压、低氧 紧张和肾毒素。这种不利刺激的生存需要 进化上保守的应激基因(如热)的过度表达 休克蛋白)作为分子伴侣来协助 蛋白质合成和折叠。最近，我们实验室克隆了 克隆了一个新的cDNA94 kDa(Osp94)，称为渗透胁迫蛋白 它是分子伴侣/热休克超家族的成员 蛋白质。Osp94与Hsp110和Hsp70RY成员的同源性最高 属于新发现的Hsp110亚家族。编码的蛋白质 Osp94、Hsp110和Hsp70RY具有N-末端的ATP结合结构域和 一个C端肽结合域的分子特征 监护人。这些基因无处不在地表达，可以在很大程度上 被高渗的氯化钠和其他胁迫上调。整体而言 本项目的目标是描述法规和 这三个基因的生物学功能。使用mIMCD3细胞，我们将 解决你的具体目标：L)在信使核糖核酸水平上定义反应 Osp94、Hsp110和Hsp70RY对诸如高渗氯化钠的胁迫， 热休克、重金属毒性和ATP耗竭。2)在 蛋白质水平这三个基因对压力的反应是通过创造 用于蛋白质和免疫组织化学分析的特异性抗体。3) 仅对于Osp94，确定应激诱导的基因表达涉及 增加转录并定义基因启动子元件(S)， 是有责任的。在核运行分析之后，我们将执行 荧光素酶报告基因分析使用来自 我们已经分离到的Osp94的5‘侧翼区域。4) 通过以下方式表征Osp94、Hsp110和Hsp70RY的伴侣属性 使用结合分子的未折叠蛋白质亲和柱 以依赖于三磷酸腺苷的方式。通过比较和对比 这些基因的调节和伴侣功能，我们将获得更大的 洞察它们在生理和病理生理学中的作用 各州。

Renal epithelial cells, particularly cells of the inner medulla, are often exposed to adverse stress including hyperosmolality, low oxygen tension and nephrotoxins. Survival of such adverse stimuli requires over-expression of evolutionarily conserved stress genes (e.g., heat shock proteins) that function as molecular chaperones to assist in protein synthesis and folding. Recently our laboratory cloned and sequenced a new cDNA, known as Osmotic Stress Protein, 94 kDa (Osp94) which is a member of the superfamily of molecular chaperone/heat shock proteins. Osp94 shows greatest homology to Hsp110 and Hsp70RY, members of the newly discovered Hsp110 subfamily. The proteins encoded by Osp94, Hsp110, and Hsp70RY possess an N-terminal ATP-binding domain and a C-terminal peptide-binding domain characteristic of molecular chaperones. These genes are ubiquitously expressed and can be greatly up-regulated by hyperosmolar NaCl and other stresses. The overall objective of this project is to characterize the regulation and biological function of these three genes. Using mIMCD3 cells we will address Jour Specific Aims: l) Define at the mRNA level the responses of Osp94, Hsp110, and Hsp70RY to stresses such as hyperosmolar NaCl, heat shock, heavy metal toxicity, and ATP depletion. 2) Define at the protein level the responses of these three genes to stress by creating specific antibodies for western and immunohistochemical analyses. 3) For Osp94 only, establish that stress-induced gene expression involves increased transcription and define the gene promoter element(s) that are responsible. Following nuclear run-on analysis we will perform luciferase reporter assays using deletion/truncation constructs from the 5' flanking region of Osp94 which we have already isolated. 4) Characterize the chaperone properties of Osp94, Hsp110, and Hsp70RY by using an unfolded protein affinity column that binds molecular chaperones in an ATP-dependent manner. By comparing and contrasting the regulation and chaperone function of these genes we will gain greater insight into their roles in physiological and pathophysiological states.