RENAL CELL RESPONSE TO STRESS

肾上皮细胞，特别是内髓质细胞， 经常暴露于不利的压力，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 进化上保守的胁迫基因的过表达（例如，热 休克蛋白），其作为分子伴侣协助 蛋白质合成和折叠。最近，我们的实验室克隆和 对一种新的cDNA进行了测序，称为渗透应激蛋白，94 kDa（Osp 94） 它是分子伴侣/热休克超家族的成员 proteins. Osp 94与Hsp 110和Hsp 70 RY的同源性最高， 新发现的Hsp 110亚家族。编码的蛋白质 Osp 94、Hsp 110和Hsp 70 RY具有N-末端ATP结合结构域， C-末端肽结合结构域的分子特征 监护人这些基因普遍表达，并且可以被极大地 上调高渗NaCl和其他压力。整体 该项目的目标是描述法规的特点， 这三个基因的生物学功能。使用mIMCD 3细胞，我们将 1）在mRNA水平上定义应答， Osp 94、Hsp 110和Hsp 70 RY对高渗NaCl胁迫的敏感性， 热休克重金属中毒和ATP耗竭。2)定义在 蛋白质水平的反应，这三个基因的压力，创造 用于Western和免疫组织化学分析的特异性抗体。第三章 仅对于Osp 94，确定胁迫诱导的基因表达涉及 增加转录并定义基因启动子元件， 都有责任在核试验分析之后， 荧光素酶报告基因测定，使用来自 我们已经分离的Osp 94的5 ′侧翼区。四、 通过以下方式表征Osp 94、Hsp 110和Hsp 70 RY的伴侣蛋白性质： 使用结合分子的未折叠蛋白质亲和柱 以ATP依赖的方式与伴侣蛋白结合。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 深入了解它们在生理和病理生理中的作用 states.

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.