Immunoglobulin Structure, Stability and Aging

描述（由申请人提供）：父母补助金仍然有三个目标。首先是阐明分子伴侣BiP和GRP 94如何与细胞中错误折叠的IG反应，以及如何利用这种相互作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是从我们对目标1的研究中发展而来的，表明除了免疫球蛋白外，胰岛素样生长因子的产生还需要GRP 94。这一发现使我们能够设计出IGF依赖性细胞，其存活取决于GRP 94的功能。我们证明了这种方法适用于筛选突变体，在本项目的目标1中，我们将利用它来确定GRP 94的功能残基和活性位点。这些突变将被设计用于精确定位这种必需应激蛋白的客户结合位点和其他已知的生化特征。目标2将基于细胞的测定的使用扩展到人类衰老。由于低IGF生产与较长的寿命，我们假设，在人类GRP 94的序列变异是那些导致部分IGF伴侣活性。已经用GRP 94的中间结构域中的一个SNP获得了概念证明。该假设将通过在Health ABC联盟的大规模人群中具有最低或最高15%全身性IGF-1水平的受试者中对人GRP 94进行基因分型和深度测序来检验。将在存活试验中检测可能改变功能的变体，以将基因型与功能直接相关。 公共卫生相关性：这一竞争性修订利用了分子伴侣与易聚集免疫球蛋白相互作用的发现（根据将授予AG 18001），并将研究范围扩展到与人类衰老高度相关的相关新主题-通过伴侣控制胰岛素样生长因子信号传导。

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.