Acinar Cell Biology and Pancreatic Disease

描述（由申请人提供）：在正常和病理生理条件下，Ca2+在调节胰腺腺泡细胞功能中的关键作用是公认的，然而，响应Ca2+升高而发生的分子变化在很大程度上是未知的。本研究提出了一种新的机制，即胞质Ca2+在腺泡细胞的分泌和内吞途径中调节磷脂和相关调节蛋白的运输。钙反应性热稳定蛋白（CRHSP-28）是腺泡细胞分泌途径的关键调控分子。CRHSP-28受到细胞Ca2+变化的高度调节，其Ca2+依赖于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，以及3)丝氨酸磷酸化，丝氨酸磷酸化触发CRHSP-28从膜相关复合物释放。本提案的主要目的是验证CRHSP- 28作为Ca2+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Specific Aim 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核内体结合的概念。靶向膜联蛋白VI结合域的位点特异性突变体将在腺泡中表达，并确定其对酶原分泌和膜运输的影响。Specific Aim 2将利用改变CRHSP-28主要磷酸化位点丝氨酸136的CRHSP-28突变体来验证磷酸化通过将其从膜结合状态取代来抑制CRHSP-28功能的理论。特异性目标3将解决CRHSP-28调节不同于酶原颗粒的顶膜运输途径的假设，并将重要的调节分子运送到顶膜。阐明CRHSP-28调节外分泌功能的分子机制，将为我们深入了解这些疾病提供有价值的见解。腺泡细胞分泌途径的Ca2+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至关重要。本研究旨在了解细胞Ca2+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用于治疗胰腺疾病的药物发现和治疗策略的潜在靶点。

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.