Molecular Mechanisims of Amylin Trafficking and Toxicity in Human Pancreatic Isle

描述(由申请人提供)： 人胰淀素是一种胰多肽，是胰腺淀粉样沉积的主要成分，是2型糖尿病(T2 DM)的标志。人胰淀素聚集成小的可溶低聚物和富含�片状的纤维与β(�)和其他胰岛细胞功能障碍以及T2 DM的发病有关。虽然在了解胰淀素寡聚体和聚集体的作用方面已经取得了很大的进展，但导致胰淀素寡聚、聚集和胰腺毒性的细胞事件和因素仍然很大程度上是未知的。这项建议的首要目标是阐明调节胰淀素寡聚和聚集的因素，并确定胰淀素介导的胰岛细胞死亡的潜在机制。我们已发表的初步结果表明，质膜(PM)胆固醇和内吞作用在人类胰岛细胞的淀粉素周转和毒性中起着关键作用。在拟议的研究中，我们将检验一种假设，即胰淀素受体(AM-R)、网织蛋白和v-SNARE/Rab内吞蛋白介导溶酶体中胰淀素的摄取和降解，其损伤刺激胰淀素寡聚和聚集，氧化应激，从而促进人胰岛细胞的凋亡。为了验证这一想法，将结合原子力显微镜(AFM)、共聚焦显微镜、光谱和生化方法来探索培养的人类胰岛细胞中胰淀素的运输、降解和毒性途径。该建议的目标是：(1)阐明胰淀素在人胰岛细胞中的转运和降解途径，并确定它们在多大程度上促进有毒胰淀素寡聚体和聚集体的形成和积累；(2)确定淀粉素周转与人胰岛细胞毒性之间的因果关系；以及(3)确定淀粉素寡聚体和/或聚集体刺激人胰岛细胞氧化应激从而激活氧化还原敏感型应激蛋白激酶、凋亡信号调节激酶1(ASK-1)和c-Jun N末端激酶(JNK)的机制。这些研究应该能更好地了解导致淀粉样蛋白聚集体和可溶性有毒淀粉低聚物形成的分子和生化事件，这些知识对于开发治疗T2 DM的新疗法至关重要。

DESCRIPTION (provided by applicant): Human amylin, a pancreatic peptide hormone, is the principal constituent of amyloid deposits in the pancreas, a hallmark of type-2 Diabetes Mellitus (T2DM). Aggregation of human amylin into small soluble oligomers and �-sheet-enriched fibrils is linked to dysfunctions of beta (�)- and other islet cells and pathogenesis of T2DM. Although much progress has been made in understanding the actions of amylin oligomers and aggregates, the cellular events and factors that contribute to amylin oligomerization, aggregation and toxicity in the pancreas remain largely unknown. The overarching goal of this proposal is to elucidate factors regulating amylin oligomerization and aggregation in the pancreatic human islet cells, and to determine the underlying mechanisms of amylin-mediated islet cell death. Our published and preliminary results suggest that plasma membrane (PM) cholesterol and endocytosis play a key role in amylin turnover and toxicity in the human islet cells. In the proposed studies, we will test the hypothesis that the amylin receptor (AM-R), clathrin and v- SNARE/Rab endocytotic proteins mediate amylin uptake and degradation in lysosomes, the impairment of which stimulates amylin oligomerization and aggregation, oxidative stress and consequently apoptosis of human islet cells. To test this idea, amylin trafficking, degradation and toxicity pathways in cultured human islet cells will be explored by combinations of atomic force microscopy (AFM), confocal microscopy, spectroscopy and biochemical approaches. The objectives of the proposal are to: (1) elucidate amylin trafficking and degradation pathways in human islet cells, and determine the extent to which they contribute to formation and accumulation of toxic amylin oligomers and aggregates, (2) determine the causal relationship between amylin turnover and toxicity in human islet cells, and (3) determine the mechanisms by which amylin oligomers and/or aggregates stimulate oxidative stress in human islet cells and thereby activate redox-sensitive stress kinases, apoptosis signal-regulating kinase 1 (ASK-1) and c-Jun N-terminal kinase (JNK). These studies should provide a better understanding of the molecular and biochemical events leading to formation of amyloid aggregates and soluble toxic amylin oligomers, knowledge which is critical for the development of novel therapeutics for the treatment of T2DM.