Immunoglobulin Structure, Stability and Aging

描述(申请人提供)：家长助学金继续有三个目标。第一个是阐明分子伴侣Bip和GRP94如何与细胞中错误折叠的Ig反应，以及这种相互作用如何被用来减少导致淀粉样蛋白形成的聚集。目的2是了解聚合反应的结构基础，目的3是建立轻链淀粉样变性的小鼠模型。所要求的竞争性修订从我们在AIM 1上的工作演变而来，表明除了免疫球蛋白外，胰岛素样生长因子的生产还需要GRP94。这一发现使我们能够设计出依赖IGF的细胞，其生存依赖于GRP94的功能。我们证明了这种方法适合于筛选突变体，在本项目的目标1中，我们将利用它来定义GRP94的功能残基和活性位点。这些突变将被设计成精确定位这种基本应激蛋白的客户结合部位和其他已知的生化特征。目标2将扩大基于细胞的检测方法在人类衰老方面的应用。由于低IGF产量与更长的寿命相关，我们假设在人类GRP94的序列变体中，有那些导致部分IGF陪伴活性的序列变体。在GRP94的中间区域有一个SNP获得了概念证明。这一假设将通过对人类GRP94进行基因分型和深度测序来验证，受试者的系统IGF-1水平在健康ABC联盟的大量人群中最低或最高15%。可能改变功能的变种将在存活试验中进行测试，以将基因与功能直接联系起来。 公共卫生相关性：这一竞争性修订利用了关于分子伴侣与易于聚集的免疫球蛋白相互作用的发现(根据Will Grant AG18001做出)，并将研究范围扩展到与人类衰老具有极高相关性的相关新主题--伴侣对胰岛素样生长因子信号的控制。

DESCRIPTION (provided by applicant): The parent grant continues to have three aims. The first is to elucidate how the molecular chaperones BiP and GRP94 react to misfolded Ig in the cell and how this interaction can be employed to reduce the aggregation that leads to amyloid formation. Aim 2 is to understand the structural basis of the polymerization reaction and Aim 3 is to develop a mouse model for light chain amyloidosis. The requested competitive revision evolved from our work on Aim 1, showing that in addition to immunoglobulins, production of insulin-like growth factors requires GRP94. This discovery allowed us to devise IGF-dependent cells whose survival depends on the functionality of GRP94. We demonstrated that this assay is suitable for screening mutants and in Aim 1 of this project we will exploit it to define functional residues and active sites of GRP94. The mutations will be designed to pinpoint the client binding site and other known biochemical features of this essential stress protein. Aim 2 will extend the use of the cell-based assay to human aging. Since low IGF production is associated with longer lifespan, we hypothesize that among the sequence variants in human GRP94 are those that lead to partial IGF chaperoning activity. Proof of concept had been obtained with one SNP in the middle domain of GRP94. This hypothesis will be tested by genotyping and deep sequencing of human GRP94 in subject with the lowest or highest 15% levels of systemic IGF-1 in the large population of the Health ABC consortium. Variants that are likely to change function will be tested in the survival assay, to relate the genotype to function directly. PUBLIC HEALTH RELEVANCE: This competitive revision takes advantage of discoveries about the interactions of molecular chaperones with aggregation-prone immunoglobulins (made under will grant AG18001) and expands the scope of research to a related novel topic with extremely high relevance to human aging - the control of insulin-like growth factor signaling by chaperones.