Acinar Cell Biology and Pancreatic Disease

描述（由申请方提供）：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受到细胞Ca 2+变化的高度调节，如其Ca 2+依赖性所示：1）调节消化酶分泌; 2）与囊泡运输蛋白膜联蛋白VI相互作用;和3）通过Ca 2 +/钙调蛋白依赖性蛋白激酶II的丝氨酸磷酸化，其触发CRHSP-28从膜相关复合物释放。该提案的主要目的是检验CRHSP-28作为腺泡中的Ca 2+传感器以促进和稳定膜运输和消化酶分泌所必需的关键蛋白质相互作用的假设。在具体目标1中，将进行实验以解决CRHSP-28与膜联蛋白VI的相互作用的概念，其功能是指导CRHSP-28与顶端细胞质中新形成的内体的结合，所述内体是支持腺泡细胞分泌功能所必需的。靶向CRHSP-28上的膜联蛋白VI结合结构域的位点特异性突变体将在腺泡中表达，并确定它们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CaM激酶II磷酸化位点，丝氨酸100，和磷酸特异性抗体，以测试磷酸化作用，以抑制CRHSP-28功能的理论，通过取代它从膜结合状态。在特定目标3实验中，将解决CRHSP-28存在于顶膜运输途径中的假设，该途径与酶原颗粒不同，并用于将重要的调节分子穿梭至腺泡分泌所必需的顶膜。CRHSP-28调节外分泌功能的分子机制的阐明，应提供有价值的洞察腺泡细胞中的分泌途径的Ca 2+依赖性的性质，这是必不可少的治疗策略，旨在治疗外分泌胰腺疾病的发展。

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.