Mechanism by which the Grp94 molecular chaperone folds insulin-like growth factors

 描述（由申请人提供）：生命可以在环境压力下生存，主要是由于分子伴侣。除了蛋白质折叠，分子伴侣还代表了癌症、代谢和衰老疾病的干预关键点，因为这些疾病通常依赖于需要分子伴侣的蛋白质。尽管其普遍存在的重要性，大多数分子伴侣机制知之甚少。该提案的目标是阐明内质网中的关键伴侣Grp94的机制。 Grp94是Hsp90分子伴侣家族的成员，其在后生动物中是必需的，但其机制知之甚少。Grp94是胰岛素样生长因子（IGF1，IGF2）折叠所必需的。胰岛素有着悠久的历史，但由于整个IGF家族固有的错误折叠和聚集，胰岛素的大规模折叠效率低下。相比之下，仅几克胰腺β细胞就可以在个体的一生中产生胰岛素。这种差异表明IGF在细胞中折叠的机制与体外折叠根本不同。 揭示Grp94介导的IGF折叠机制需要了解IGF错误折叠的方式和原因。目标1a的目标是定义IGF2的实验确定的折叠和错误折叠能量景观。为了实现这一点，我将利用IGF家族独特的折叠特性。简而言之，IGF重折叠导致二硫键捕获的折叠和错误折叠中间体。人们早就知道，这些中间体可以通过色谱分离，因此，可以研究IGF折叠能量景观上的许多状态。目标1b中将使用IGF折叠和错误折叠能量景观来评估Grp 94如何改变IGF折叠的过程。如目标2所述，可以立即用已经建立的细胞模型在体内测试来自这种体外方法的发现。总之，这些实验将显示IGF蛋白如何在分子伴侣的帮助下在细胞中有效折叠。 胰岛素的大规模折叠是低效的，这具有经济和环境成本。大规模生产胰岛素的一种方式是通过酵母分泌。然而，由于未知的原因，天然胰岛素原不被分泌，但具有缩短的C肽的胰岛素类似物可以被分泌。由于酵母不表达Grp94，目标3的目的是测试Grp94是否能够使酵母分泌天然胰岛素原。

 DESCRIPTION (provided by applicant): Life can survive environmental stress largely due to molecular chaperones. Beyond protein folding, chaperones represent a critical point of intervention in cancer, metabolic, and aging diseases, because these diseases are often dependent on proteins that require chaperones. Despite their ubiquitous importance, most chaperone mechanisms are poorly understood. The goal of this proposal is to elucidate the mechanism of a key chaperone in the endoplasmic reticulum, Grp94. Grp94 is a member of the Hsp90 family of molecular chaperones, which are essential in metazoans but whose mechanism is poorly understood. Grp94 is required for the folding of insulin-like growth factors (IGF1, IGF2) Insulin has a storied history, but large-scale folding of insulin is inefficient due to misfolding nd aggregation inherent to the entire IGF family. In contrast, just a few grams of pancreatic beta-cells can produce insulin for the lifetime of an individual. This discrepancy indicates mechanisms of IGF folding in the cell that are fundamentally different from in-vitro folding. Revealing the mechanism Grp94-mediated IGF folding requires understanding how and why IGFs misfold. The goal of Aim 1a is to define an experimentally determined folding and misfolding energy landscape for IGF2. To achieve this I will take advantage of a unique folding characteristic of the IGF family. Briefly, IGF refolding results in disulfide-trapped folding and misfolding intermediates. It has long been known that these intermediates can be separated chromatographically, and as a result, numerous states on the IGF folding energy landscape can be studied. The IGF folding and misfolding energy landscape will be used in Aim 1b to evaluate how Grp94 alters the process by which IGFs fold. Findings from this in-vitro approach can immediately be tested in-vivo with an already established cell model, as described in Aim 2. Together, these experiments will show how IGF proteins are folded efficiently in the cell with the aid of molecular chaperones. The large-scale folding of insulin is inefficient, which has an economic and environmental cost. One way insulin is produced in large-scale is via yeast secretion. However, for unknown reasons, native proinsulin is not secreted but insulin analogs with shortened C- peptides can be secreted. Since yeasts do not express Grp94, the goal of Aim 3 is to test whether Grp94 can enable yeast to secrete native proinsulin.