Cysteine String Protein and Acute Pancreatitis

半胱氨酸串蛋白与急性胰腺炎

• 批准号: 6558604
• 负责人: GUY E GROBLEWSKI
• 金额: $ 14.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6558604
• 关键词: acinar cell; adenosinetriphosphatase; antisense nucleic acid; cysteine; cytotoxicity; enzyme activity; gene induction /repression; heat shock proteins; laboratory rat; lysozyme; membrane activity; molecular chaperones; pancreatitis; protein engineering; protein structure function; proteolysis; secretion; tissue /cell culture; zymogens

DESCRIPTION (provided by applicant): Acute pancreatitis is an inflammatory disease that is triggered by the premature activation of proteolytic zymogens in acinar cells. As a protective mechanism, zymogens are synthesized as inactive pro-enzymes and packaged in specialized secretory granules destined for exocytosis into the pancreatic duct. During pancreatitis, proteolytic zymogens are prematurely activated in acinar cells by aberrantly mixing with hydrolases present in lysosomes. The mixing of lysosomal hydrolases with zymogens occurs in large cytoplasmic vacuoles as a result of pathogenic alterations in acinar cell membrane trafficking. It is well established that the stress-induced expression of heat shock protein-70 (HSP70) in acini provides a natural protective effect against the pathogenic mixing of lysosomal and secretory granule membrane compartments. This proposal is designed to elucidate the mechanism by which HSP70 inhibits the intra-acinar cell activation of zymogens in preventing the on-set of acute pancreatitis. HSP70 is a molecular chaperone in the cytosol that requires a co-chaperone protein to stimulate ATPase activation and substrate binding. We have 1) identified an HSP70 co-chaperone protein called cysteine string protein (CSP) in acinar cells, 2) localized this membrane associated molecule throughout the secretory pathway and 3) developed the TAT-fusion protein system to manipulate CSP expression in acinar cells and thereby study its function. We hypothesize that CSP mediates the protective effects of HSP70 against pancreatitis by targeting HSPT0 chaperone activity from the cytosol to the secretory pathway. In Specific Aim 1, mutant forms of CSP that no longer anchor to secretory granule membranes will be overexpressed acinar cells to determine the importance of CSP in targeting HSP70 to these organelles during pancreatitis. In Specific Aim II, short interference RNAs will be used to inhibit CSP expression in pancreatic Iobules and the protective effects of HSP70 on secretagogue-induced acinar cell damage will be evaluated. These studies will allow us to directly evaluate the role of CSP in the secretory pathway of acini under physiological and pathophysiological conditions.

描述（由申请人提供）：急性胰腺炎是一种由腺泡细胞中蛋白水解酶原过早激活引发的炎症性疾病。作为一种保护机制，酶原被合成为无活性的酶原，并包装在专门的分泌颗粒中，最终通过胞吐作用进入胰管。在胰腺炎期间，腺泡细胞中的蛋白水解酶原通过与溶酶体中存在的水解酶异常混合而过早激活。由于腺泡细胞膜运输的致病性改变，溶酶体水解酶与酶原的混合发生在大细胞质液泡中。众所周知，腺泡中应激诱导的热休克蛋白 70 (HSP70) 表达提供了针对溶酶体和分泌颗粒膜区室的致病性混合的天然保护作用。该提案旨在阐明HSP70抑制腺泡细胞内酶原激活以预防急性胰腺炎发病的机制。 HSP70 是细胞质中的分子伴侣，需要辅助伴侣蛋白来刺激 ATP 酶激活和底物结合。我们 1) 在腺泡细胞中鉴定了一种称为半胱氨酸串蛋白 (CSP) 的 HSP70 共伴侣蛋白，2) 将这种膜相关分子定位在整个分泌途径中，3) 开发了 TAT 融合蛋白系统来操纵腺泡细胞中的 CSP 表达并由此研究其功能。我们假设 CSP 通过靶向从细胞质到分泌途径的 HSPT0 伴侣活性来介导 HSP70 对胰腺炎的保护作用。在具体目标 1 中，不再锚定于分泌颗粒膜的 CSP 突变形式将在腺泡细胞中过度表达，以确定 CSP 在胰腺炎期间将 HSP70 靶向这些细胞器的重要性。在Specific Aim II中，将使用短干扰RNA抑制胰腺小球中的CSP表达，并评估HSP70对促分泌素诱导的腺泡细胞损伤的保护作用。这些研究将使我们能够直接评估生理和病理生理条件下 CSP 在腺泡分泌途径中的作用。