Acinar Cell Biology and Pancreatic Disease

腺泡细胞生物学和胰腺疾病

• 批准号: 7033181
• 负责人: GUY E GROBLEWSKI
• 金额: $ 28.74万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-08 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033181
• 关键词: acinar cell; annexins; apical membrane; calcium; cell morphology; endocytosis; immunoelectron microscopy; intracellular transport; laboratory rat; pancreas; pancreas disorder; phosphoproteins; phosphorylation; protein protein interaction; protein purification; secretory protein; serine; site directed mutagenesis; western blottings; zymogens

DESCRIPTION (provided by applicant): The pivotal role of Ca2+ in regulating pancreatic acinar cell function under normal and pathophysiological conditions is well established, however, the molecular changes that occur in response to elevated Ca2+ are largely unknown. This proposal addresses a novel mechanism by which cytosolic Ca2+ modulates the trafficking of phospholipids and associated regulatory proteins in the secretory and endocytic pathways in acinar cells. Calcium responsive heat-stable protein (CRHSP-28) is a key regulatory molecule in the secretory pathway of acinar cells. CRHSP-28 is highly modulated by changes in cellular Ca2+ as indicated by its Ca2+-dependent 1) regulation of digestive enzyme secretion, 2) interaction with the vesicle trafficking protein annexin VI, and 3) serine phosphorylation, which triggers the release of CRHSP-28 from a membrane associated complex. The primary objective of this proposal is to test the hypothesis that CRHSP- 28 acts as Ca2+-sensor to promote and stabilize key protein interactions necessary for acinar cell membrane trafficking. In Specific Aim 1 experiments will address the concept that interaction of CRHSP-28 with annexin VI directs CRHSP-28 association with endosomes that are necessary to support secretory function. Site specific mutants targeting the annexin VI binding domain will be expressed in acini and effects on zymogen secretion and membrane trafficking determined. Specific Aim 2 will utilize CRHSP-28 mutants that alter the major CRHSP-28 phosphorylation site, serine 136, to test the theory that phosphorylation inhibits CRHSP-28 function by displacing it from a membrane-bound state. Specific Aim 3 will address the hypothesis that CRHSP-28 regulates an apical membrane trafficking pathway that is distinct from zymogen granules and acts to shuttle important regulatory molecules to the apical membrane. Elucidation of the molecular mechanism by which CRHSP-28 modulates exocrine function should provide valuable insight into the.Ca2+- dependent nature of the secretory pathway in acinar cells, which is essential for the development of therapeutic strategies aimed at the treatment of exocrine pancreatic disease. This proposal is aimed at understanding the biochemical mechanism by which changes in cell Ca2+ regulate pancreatic function in normal and pathological states. As such, these studies will help to identify potential targets for drug discovery and therapeutic strategies aimed at treating pancreatic disease.

描述（由申请人提供）：在正常和病理生理条件下，Ca 2+在调节胰腺腺泡细胞功能中的关键作用已得到充分证实，然而，对Ca 2+升高的反应发生的分子变化在很大程度上尚不清楚。这一建议提出了一种新的机制，胞质钙离子调节运输的磷脂和相关的调节蛋白在腺泡细胞的分泌和内吞途径。钙应答热稳定蛋白（CRHSP-28）是腺泡细胞分泌途径中的关键调节分子。CRHSP-28受细胞Ca 2+变化的高度调节，如其Ca 2+依赖性1）消化酶分泌的调节，2）与囊泡运输蛋白膜联蛋白VI的相互作用，和3）丝氨酸磷酸化，其触发CRHSP-28从膜相关复合物的释放。该提案的主要目的是检验CRHSP- 28作为Ca 2+传感器促进和稳定腺泡细胞膜运输所需的关键蛋白相互作用的假设。在具体目标1中，实验将解决CRHSP-28与膜联蛋白VI的相互作用指导CRHSP-28与支持分泌功能所必需的内体缔合的概念。靶向膜联蛋白VI结合结构域的位点特异性突变体将在腺泡中表达，并确定对酶原分泌和膜运输的影响。具体目标2将利用CRHSP-28突变体，改变主要的CRHSP-28磷酸化位点，丝氨酸136，以测试磷酸化通过将其从膜结合状态置换来抑制CRHSP-28功能的理论。具体目标3将解决CRHSP-28调节顶膜运输途径的假设，该途径与酶原颗粒不同，并用于将重要的调节分子穿梭至顶膜。CRHSP-28调节外分泌功能的分子机制的阐明应该提供有价值的洞察腺泡细胞中分泌途径的Ca 2+依赖性，这对于旨在治疗外分泌胰腺疾病的治疗策略的发展是必不可少的。该建议旨在了解细胞Ca 2+变化在正常和病理状态下调节胰腺功能的生化机制。因此，这些研究将有助于确定旨在治疗胰腺疾病的药物发现和治疗策略的潜在靶点。