Molecular Mechanisims of Amylin Trafficking and Toxicity in Human Pancreatic Isle

描述（由申请人提供）： 人胰淀素是一种胰肽激素，是胰腺中淀粉样蛋白沉积物的主要成分，是2型糖尿病（T2 DM）的标志。人胰淀素聚集成小的可溶性低聚物和富含β-片的原纤维与β-和其他胰岛细胞的功能障碍和T2 DM的发病机制有关。虽然在理解胰淀素寡聚体和聚集体的作用方面已经取得了很大进展，但是导致胰腺中胰淀素寡聚化、聚集和毒性的细胞事件和因素仍然在很大程度上未知。这项建议的首要目标是阐明调节胰岛细胞中胰淀素寡聚化和聚集的因素，并确定胰淀素介导的胰岛细胞死亡的潜在机制。我们发表的初步结果表明，质膜（PM）胆固醇和内吞作用在人胰岛细胞胰淀素周转和毒性中起关键作用。在所提出的研究中，我们将检验以下假设：胰淀素受体（AM-R）、网格蛋白和v-SNARE/Rab内吞蛋白介导胰淀素在溶酶体中的摄取和降解，其损伤刺激胰淀素寡聚化和聚集、氧化应激以及因此的人胰岛细胞的凋亡。为了测试这一想法，胰淀素的运输，降解和毒性途径在培养的人胰岛细胞将探索原子力显微镜（AFM），共聚焦显微镜，光谱和生化方法的组合。该提案的目标是：（1）阐明人胰岛细胞中胰淀素运输和降解途径，并确定它们对毒性胰淀素寡聚体和聚集体的形成和积累的贡献程度，（2）确定人胰岛细胞中胰淀素周转和毒性之间的因果关系，和（3）确定胰淀素寡聚体和/或聚集体刺激人胰岛细胞中的氧化应激并由此激活氧化还原敏感性应激激酶的机制，凋亡信号调节激酶1（ASK-1）和c-Jun N-末端激酶（JNK）。这些研究应提供对导致淀粉样蛋白聚集体和可溶性毒性胰淀素寡聚体形成的分子和生物化学事件的更好理解，这些知识对于开发治疗T2 DM的新型疗法至关重要。

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.