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

Grp94分子伴侣折叠胰岛素样生长因子的机制

• 批准号: 8937336
• 负责人: Timothy Oliver Street
• 金额: $ 31.51万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8937336
• 关键词: Aging; C-Peptide; Cell model; Cells; Characteristics; Disease; Disulfides; Economics; Endoplasmic Reticulum; Family; Goals; Heat-Shock Proteins 90; Homeostasis; IGF1 gene; IGF2 gene; In Vitro; Individual; Insulin; Intervention; Learning; Life; Macromolecular Complexes; Malignant Neoplasms; Measures; Mediating; Metabolic; Methods; Molecular; Molecular Chaperones; Mutation; Organism; Pathway interactions; Process; Proinsulin; Proteins; Recording of previous events; Resistance; Rewards; Role; Somatomedins; Stress; Structure of beta Cell of islet; Testing; Work; Yeasts; analog; cost; improved; in vivo; large scale production; member; prevent; protein activation; protein folding; protein misfolding; protein transport; public health relevance; research study

 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.

 描述（由适用提供）：生命可以在很大程度上由于分子链酮而生存。除了蛋白质折叠之外，链酮还代表了干预癌症，代谢和衰老疾病的关键点，因为这些疾病通常取决于需要链酮的蛋白质。尽管它们无处不在，但大多数伴侣机制还是很了解。该提案的目的是阐明内质网中钥匙链的机理，GRP94。 GRP94是HSP90分子伴侣家族的成员，在后生动物中至关重要，但其机制知之甚少。 GRP94是胰岛素样生长因子（IGF1，IGF2）胰岛素的折叠所必需的，但由于与整个IGF家族的遗传遗传的错误折叠ND聚集遗传，因此胰岛素的大规模折叠效率不高。相比之下，只有几克胰腺β细胞可以在一个人的一生中产生胰岛素。这种差异表明细胞中IGF折叠的机制与体外折叠根本不同。揭示了GRP94介导的IGF折叠的机理需要了解IGF如何以及为什么错误折叠。 AIM 1A的目的是定义IGF2的实验确定的折叠和错误折叠的能量景观。为了实现这一目标，我将利用IGF家族的独特折叠特征。简而言之，IGF重新折叠导致二硫键捕获的折叠和错误折叠中间体。早就知道，这些中间体可以在色谱上分离，因此，可以研究IGF折叠能量景观上的许多状态。 IGF折叠和错误折叠的能量景观将用于AIM 1B，以评估GRP94如何改变IGFS折叠的过程。如AIM 2中所述，可以立即通过已建立的细胞模型在体内测试这种体外方法的发现。一起，这些实验将显示如何借助于分子伴侣，在细胞中如何有效地折叠IGF蛋白。胰岛素的大规模折叠无效，其经济和环境成本。大规模生产胰岛素的一种方法是通过酵母分泌。但是，由于未知原因，本地促硫素不是分泌的，但是可以分泌具有缩短C肽的胰岛素类似物。由于酵母不会表达GRP94，因此AIM 3的目标是测试GRP94是否可以使酵母秘密蛋白酶蛋白。