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Ca~(2+)：1)消化酶分泌的调节；2)与囊泡运输蛋白Annexin VI的相互作用；以及3)通过钙/钙调蛋白依赖的蛋白激酶II的丝氨酸磷酸化，触发膜相关复合体释放CRHSP-28。这一建议的主要目的是验证CRHSP-28作为腺泡内钙传感器的假设，以促进和稳定关键蛋白质的相互作用，这些蛋白质是膜运输和消化酶分泌所必需的。在具体目标1中，将进行实验，以解决CRHSP-28与Annexin VI相互作用的概念，即CRHSP-28与Annexin VI的相互作用将CRHSP-28与顶端细胞质中新形成的内体联系起来，这是支持腺泡细胞分泌功能所必需的。针对CRHSP-28上膜联蛋白VI结合域的定点突变将在腺泡中表达，并确定它们对酶原分泌和膜转运的影响。特殊目的2将利用CRHSP-28突变体改变主要的CaM激酶II磷酸化位点、丝氨酸100和磷酸特异性抗体来测试磷酸化通过将CRHSP-28从膜结合状态置换而抑制其功能的理论。在特定的目标中，3个实验将解决这一假设，即CRHSP-28存在于不同于酶原颗粒的顶膜运输途径中，并将重要的调节分子穿梭到顶膜上，这是腺泡分泌所必需的。阐明CRHSP-28调节胰腺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，这对于制定治疗胰腺外分泌疾病的策略是至关重要的。

DESCRIPTION (provided by the 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 via Ca2+/calmodulin-dependent protein kinase II, 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 in acini to promote and stabilize key protein interactions that are necessary for membrane trafficking and digestive enzyme secretion. In Specific Aim 1 experiments will be conducted to address the concept that the interaction of CRHSP-28 with annexin VI functions to direct CRHSP- 28 association to newly formed endosomes in the apical cytoplasm that are necessary to support acinar cell secretory function. Site specific mutants targeting the annexin VI binding domain on CRHSP-28 will be expressed in acini and their effects on zymogen secretion and membrane trafficking determined. Specific Aim 2 will utilize CRHSP-28 mutants that alter the major CaM kinase II phosphorylation site, serine 100, and phospho-specific antibodies to test the theory that phosphorylation acts to inhibit CRHSP-28 function by displacing it from a membrane-bound state. In Specific Aim 3 experiments will address the hypothesis that CRHSP-28 is present in an apical membrane trafficking pathway that is distinct from zymogen granules and acts to shuttle important regulatory molecules to the apical membrane that are necessary for acinar secretion. 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.