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，钙离子在调节胰腺腺泡细胞功能中的关键作用已被充分证实，然而，对升高的钙离子的反应发生的分子变化在很大程度上是未知的。这项建议提出了一种新的机制，通过细胞内钙离子调节磷脂和相关的调节蛋白在腺泡细胞的分泌和内吞途径中的运输。钙反应热稳定蛋白(CRHSP-28)是腺泡细胞分泌途径中的关键调控分子。CRHSP-28受细胞内Ca~(2+)变化的高度调控，它依赖于1)消化酶分泌的调节，2)与囊泡运输蛋白Annin VI的相互作用，以及3)丝氨酸磷酸化，从而触发CRHSP-28从膜相关复合体中释放。这一建议的主要目的是验证CRHSP-28作为钙感受器促进和稳定腺泡细胞膜运输所需的关键蛋白质相互作用的假设。在特定的目标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小体的联系这一概念。针对膜联蛋白VI结合域的定点突变体将在腺泡中表达，并确定对酶原分泌和膜转运的影响。特殊目的2将利用改变CRHSP-28主要磷酸化位点丝氨酸136的CRHSP-28突变体来测试磷酸化通过将CRHSP-28从膜结合状态转移来抑制CRHSP-28功能的理论。具体目标3将解决这一假设，即CRHSP-28调节不同于酶原颗粒的顶膜运输途径，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有助于深入了解腺泡细胞分泌途径的钙依赖特性，这对于开发针对胰腺外分泌疾病的治疗策略是至关重要的。本研究旨在了解细胞内钙离子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目标。

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.