Peptide Hormone Sorting to the Secretory/Storage Granule

描述(申请人提供)：多肽激素，胰岛素，调节新陈代谢，使血糖水平维持在一个狭窄的生理范围内。在胰腺SS细胞中，胰岛素被制造并以高浓度的方式储存在分泌颗粒中。生理刺激胰岛素分泌(一天多次)需要非常活跃的合成新的胰岛素来补充分泌颗粒储备。胰岛素合成始于胰岛素原的翻译，将其输送到内质网(ER)的管腔内。其中，胰岛素原必须正确折叠，这比听起来容易：胰岛素原是一种“二硫键挑战”的蛋白质。此外，当β细胞被迫合成比它们基因编程所能处理的水平更高的胰岛素原时，它们有可能进一步错误折叠胰岛素原/二硫键错配，这会导致分泌途径压力。这一新的赠款周期的目标是更好地了解胰岛素原折叠和从ER输出。我们假设，内质网中胰岛素原的一个亚组分的错误折叠可以阻止来自另一个“旁观者”胰岛素原分子亚组分的胰岛素产生，使内质网中的蛋白质积压，并导致内质网应激、β细胞衰竭、胰腺胰岛素含量丧失和糖尿病。我们提出四个具体目标：1)阐明新发现的胰岛素原编码序列的点突变导致新生儿和成人糖尿病的分子机制(S)。2)研究ss-cell内质网氧化还原酶的特性。3)开发一种新的基于细胞培养的系统，以剖析导致β细胞死亡的步骤。4)建立糖尿病患者胰腺胰岛素产生的体内分析方法。与公众健康相关：胰岛素调节新陈代谢以维持正常的血糖水平。新胰岛素的合成始于内质网中胰岛素原的翻译。在过去的一年里，发现了20多个新的胰岛素原突变与新生儿发病的糖尿病有关。有证据表明，这些突变的胰岛素原是以蛋白质的形式产生的，但尚不清楚它们是如何导致糖尿病的。每个患者都有另一个完全正常的胰岛素原等位基因，该等位基因的胰岛素原合成应该足以满足人体对胰岛素的需求。这一新的赠款周期提出了更好地了解胰岛素原折叠和输出的实验，以了解内质网中胰岛素原亚部分的错误折叠如何阻止来自“旁观者”胰岛素原分子的胰岛素产生，从而导致内质网应激、β细胞衰竭和糖尿病。

DESCRIPTION (provided by applicant): The peptide hormone, insulin, regulates metabolism to homeostatically maintain blood glucose levels within a narrow physiological range. In pancreatic ss-cells, insulin is made and stored at high concentration within secretory granules. Physiological stimulation of insulin secretion (multiple times per day) requires very active synthesis of new insulin to replenish secretory granule reserves. Insulin synthesis begins with translation of preproinsulin for delivery into the lumen of the endoplasmic reticulum (ER). Therein, proinsulin must properly fold, which is easier than it sounds: proinsulin is a "disulfide-challenged" protein. Moreover, when beta cells are forced to synthesize higher levels of proinsulin than they are genetically-programmed to handle, they risk further proinsulin misfolding/disulfide mispairing, which leads to secretory pathway stress. The objective of this new grant cycle is to better understand proinsulin folding and export from the ER. We hypothesize that misfolding of a subfraction of proinsulin in the ER can block insulin production derived from the other subfracton of "bystander" proinsulin molecules, backlogging the protein in the ER, and driving ER stress, beta cell failure, loss of pancreatic insulin content, and diabetes. We propose four Specific Aims: 1) To elucidate the molecular mechanism(s) by which newly-described point mutations in the coding sequence of preproinsulin lead to human diabetes in neonates and adults. 2) To characterize ss-cell ER oxidoreductases. 3) To develop a new cell culture-based system to dissect steps leading to beta cell death. 4) To develop an in vivo analysis of pancreatic insulin production in diabetes. PUBLIC HEALTH RELEVANCE: Insulin regulates metabolism to maintain normal blood glucose levels. Synthesis of new insulin begins with translation of proinsulin in the endoplasmic reticulum. In the past year, more than 20 new proinsulin mutations have been found to be associated with neonatal onset diabetes. Evidence suggests that these mutant proinsulins are made as proteins but it is not known how they cause diabetes. Each patient also havs another allele of perfectly normal proinsulin which should be more than enough proinsulin synthesis to satisfy the body's need for insulin. This new grant cycle proposes experiments to better understand proinsulin folding and export in order to see how misfolding of a subfraction of proinsulin in the ER can block insulin production derived from "bystander" proinsulin molecules, causing ER stress, beta cell failure, and diabetes.