INTRACELLULAR AMYLOIDOGENESIS AND APOPTOSIS IN NIDDM

NIDDM 中的细胞内淀粉样生成和细胞凋亡

• 批准号: 6350745
• 负责人: NORMAN L EBERHARDT
• 金额: $ 14.53万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350745
• 关键词: BCL2 gene /protein; amylin; amyloid proteins; annexins; antisense nucleic acid; apoptosis; calcium binding protein; calcium ion; cysteine endopeptidases; cytochrome c; endoplasmic reticulum; enzyme activity; intracellular; mitochondria; noninsulin dependent diabetes mellitus; northern blottings; nuclear factor kappa beta; protein biosynthesis; terminal nick end labeling; tissue /cell culture; transfection; western blottings

Non-insulin dependent diabetes (NIDDM) is an aging-related disease associated with amyloid formation in the pancreas and loss of beta cell mass. The mechanism(s) of pancreatic beta cell death in NIDDM are unknown. NIDDM is a major disease that currently affects approximately 7.2 million Americans with annual costs exceeding 100 billion dollars. A mutation (S20G) in the human islet amyloid precursor protein (hIAPP, amylin) has been associated with premature onset NIDDM in Japanese populations, providing a direct linkage of the IAPP gene with NIDDM. IAPP is the major component of islet cell amyloid deposits which are a hallmark of NIDDM, suggesting that islet amyloid may be involved in the pathogenesis of this disease. We demonstrated that hIAPP expression in COS-1 cells results in the accumulation of intracellular amyloid within the endoplasmic reticulum (ER)/Golgi and induction of apoptosis. Accordingly, we have hypothesized that: (i) intracellular amyloidogenesis is a primary cause of beta cell death, and (ii) protective mechanisms that prevent intracellular amyloidogenesis are compromised by factors predisposing to NIDDM. In preliminary studies we have found that the accumulation of amyloid within the ER/Golgi is associated with an early induction of the molecular chaperone Bip/GRP78, a protein that is involved in sealing ER translocon pores and protein folding. The induction of Bip/GRP78 is a characteristic of two ER stress response pathways: unfolded protein response (UPR) and ER overload response (EOR). Both of these pathways are involved in ER quality control. To date our data suggest that intracellular amyloid accumulation and subsequent induction of apoptosis may be mediated by one or both of these pathways. The current studies will: (i) assess the relative apoptogenic activity of the wild-type and mutant IAPPS20G expression on COS-1 and pancreatic betaTC-3 cells, (ii) establish whether the UPR or EOR stress response is activated by intracellular amyloid accumulation and their role(s) in inducing apoptosis, (iii) determine whether changes in ER Ca2+ activate apoptosis, (iv) determine the relative role of mitochondrial proteins in amyloid-induced apoptosis, and (v) define the distal caspase-mediated pathways leading to apoptosis. These studies will increase our understanding of the contribution of intracellular amyloid accumulation to the pathogenesis of NIDDM.

非胰岛素依赖性糖尿病（NIDDM）是与胰腺中淀粉样蛋白形成相关的衰老疾病和β细胞量的损失。 NIDDM中胰腺β细胞死亡的机制尚不清楚。 NIDDM是一种主要疾病，目前影响约720万美国人，每年成本超过1000亿美元。 人胰岛淀粉样蛋白前体蛋白（HIAPP，淀粉蛋白）中的突变（S20G）与日本人群的过早发作NIDDM相关，从而提供了IAPP基因与NIDDM的直接联系。 IAPP是胰岛细胞淀粉样蛋白沉积物的主要组成部分，这是NIDDM的标志，这表明胰岛淀粉样蛋白可能与该疾病的发病机理有关。 我们证明，HIAPP在COS-1细胞中的表达导致内质网（ER）/高尔基体内细胞内淀粉样蛋白的积累和凋亡诱导。 因此，我们假设：（i）细胞内淀粉样生成是β细胞死亡的主要原因，并且（ii）预防细胞内淀粉样蛋白生成的保护机制受到NIDDM偏爱的因素的损害。 在初步研究中，我们发现淀粉样蛋白在ER/高尔基体内的积累与分子伴侣BIP/GRP78的早期诱导有关，这是一种参与密封ER转运孔和蛋白质折叠的蛋白质。 BIP/GRP78的诱导是两个ER应力反应途径的特征：展开的蛋白质反应（UPR）和ER超载反应（EOR）。 这两种途径都参与了ER质量控制。 迄今为止，我们的数据表明，细胞内淀粉样蛋白的积累和随后的凋亡诱导可能是由其中一种或两种途径介导的。 当前的研究将：（i）评估野生型和突变体IAPEN20G在COS-1和胰腺BetATC-3细胞上表达的相对凋亡活性，（ii）确定UPR或EOR应激响应是通过细胞内淀粉样淀粉样蛋白积累及其在诱导凋亡中的作用（III）（III）（III）（III）（III）（III）（III）（III）（III）（III）是否会激活的（III）（III）是否会激活（III）。线粒体蛋白在淀粉样蛋白诱导的细胞凋亡中的作用，并且（V）定义了caspase介导的远端介导的途径，导致凋亡。 这些研究将增加我们对细胞内淀粉样蛋白积累对NIDDM发病机理的贡献的理解。