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

 描述(申请人提供)：生命能够在很大程度上由于分子伴侣而在环境压力下生存下来。除了蛋白质折叠，伴侣还代表着癌症、代谢和衰老疾病的关键干预点，因为这些疾病通常依赖于需要伴侣的蛋白质。尽管它们无处不在的重要性，但大多数陪伴机制却鲜为人知。这项建议的目的是阐明内质网中的一个关键伴侣Grp94的机制。Grp94是Hsp90分子伴侣家族的成员之一，分子伴侣在后生动物中是必不可少的，但其机制尚不清楚。胰岛素样生长因子(IGF1、IGF2)的折叠需要Grp94。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，大规模的胰岛素折叠效率低下。相比之下，仅仅几克的胰岛β细胞就可以在人的一生中产生胰岛素。这种差异表明IGF在细胞内折叠的机制与体外折叠有根本的不同。要揭示Grp94介导的IGF折叠的机制，需要了解IGFS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图景。为了实现这一点，我将利用IGF家族的独特折叠特征。简而言之，IGF复性导致二硫化物陷阱折叠和错误折叠中间产物。人们早就知道，这些中间体可以通过层析进行分离，因此，可以研究IGF折叠能量图景上的许多状态。目标1b将使用IGF折叠和错误折叠的能量图景来评估Grp94如何改变IGFS折叠的过程。这种体外方法的发现可以立即用已经建立的细胞模型在体内进行测试，如目标2中所述。这些实验将共同展示IGF蛋白如何在分子伴侣的帮助下在细胞中高效折叠。胰岛素的大规模折叠效率低，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，天然胰岛素原不能分泌，但可以分泌缩短了C-肽的胰岛素类似物。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的胰岛素原。

 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.