Peptide Hormone Sorting to the Secretory/Storage Granule

描述（由申请人提供）：肽激素，胰岛素，调节代谢，将血糖水平维持在一个狭窄的生理范围内。在胰腺细胞中，胰岛素被制造并以高浓度储存在分泌颗粒中。胰岛素分泌的生理刺激（每天多次）需要非常活跃地合成新的胰岛素来补充分泌颗粒储备。胰岛素的合成开始于胰岛素前原的翻译，以传递到内质网（ER）的管腔。因此，胰岛素原必须正确折叠，这比听起来容易：胰岛素原是一种“二硫挑战”蛋白质。此外，当β细胞被迫合成更高水平的胰岛素原时，它们会面临胰岛素原错误折叠/二硫错配的风险，从而导致分泌途径压力。这个新的资助周期的目标是更好地理解胰岛素原折叠和从急诊室输出。我们假设内质网中胰岛素原分子的一个亚部分折叠错误可以阻断来自“旁观者”胰岛素原分子的其他亚部分的胰岛素产生，使内质网中的蛋白质滞留，并驱动内质网应激、β细胞衰竭、胰腺胰岛素含量损失和糖尿病。我们提出了四个具体目标：1)阐明新描述的胰岛素前原编码序列点突变导致新生儿和成人糖尿病的分子机制。2)表征细胞内质网氧化还原酶。3)开发一种新的基于细胞培养的系统来解剖导致β细胞死亡的步骤。4)开展糖尿病患者胰腺胰岛素生成的体内分析。公共卫生相关性：胰岛素调节代谢以维持正常血糖水平。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，超过20个新的胰岛素原突变被发现与新生儿发病糖尿病有关。有证据表明，这些突变的原胰岛素是作为蛋白质产生的，但尚不清楚它们是如何引起糖尿病的。每个病人也有另一个完全正常的胰岛素原的等位基因，它应该有足够的胰岛素原合成来满足身体对胰岛素的需要。这个新的资助周期提出了更好地理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.