MOLECULAR CHAPERONES AND IG BIOSYNTHESIS

描述(改编自申请人的摘要)：控制 蛋白质的折叠、组装和内质网的运输 (ER)对于调节细胞表面事件的保真度是必不可少的 淋巴细胞功能与分化。近年来，有几个 驻留的ER蛋白已被鉴定为似乎有助于或 监测蛋白质的折叠和组装，尽管这些的确切作用 蛋白质成熟过程中的“分子伴侣”尚不清楚。它是 这个项目的中心假设是不同的内质网伴侣发挥作用 在新生免疫球蛋白分子成熟过程中的独特而重要的作用 从而促进B细胞的发育和分化。免疫球蛋白的失败 与内质网伴侣机制组件相互作用的分子可能 允许异常免疫球蛋白分子的分泌或表面沉积，因此 绕过这一受控过程。最近，它一直是 发现在免疫球蛋白沉积中发现的淀粉样沉积 疾病是由突变的Ig蛋白引起的。很有可能 这些Ig蛋白的突变会干扰它们与 并允许他们绕过控制机制 从分泌途径中去除有缺陷的分子。实验在 具体目标1旨在更清楚地了解 Ig Heavy和Ig Heavy在折叠和组装过程中的分子序列 轻链。在具体目标2中，不同的内质网伴侣在 控制免疫球蛋白的成熟和运输将是 已澄清。为了实现这些目标，一系列Bip ATPase突变体 在体内结合Ig蛋白，但不能释放它们，从而起到 已经产生了“陪伴陷阱”，并对其进行了充分描述。两者都有 这些BiP在体内表达的瞬时和可诱导系统 突变体已经被开发出来，而缺乏这种基因的细胞系 目前已有多种内质网伴侣的合成。此外, 免疫球蛋白重链和轻链的cDNA克隆已获得并应用 鉴定免疫球蛋白轻链的体内折叠中间体。 制备了多种内质网伴侣的抗血清，并获得了 获得了grp94和erp72的克隆，这将允许 伴侣缺失细胞系的重建。加在一起，这些 试剂将允许描绘Ig折叠的中间产物 和组装途径，鉴定那些直接 帮助这些过程以及那些监测反应的过程，以及 最后确定保留和降解的机制 不成功的免疫球蛋白分子。这些信息可能对 了解免疫病理，如轻链沉积病。

DESCRIPTION (Adapted from the Applicant's Abstract): The control of protein folding, assembly, and transport from the endoplasmic reticulum (ER) is essential to the fidelity of cell surface events that regulate lymphocyte function and differentiation. In recent years, several resident ER proteins have been identified that appear to either aid or monitor protein folding and assembly, although the precise roles of these "molecular chaperones" in protein maturation is unclear. It is the central hypothesis of this project that the various ER chaperones play distinct and essential roles in the maturation of nascent Ig molecules and thus in B cell development and differentiation. The failure of Ig molecules to interact with components of the ER chaperon machinery could allow the secretion or surface deposition of aberrant Ig molecules, thus circumventing this controlled process. Most recently, it has been discovered that the amyloid deposits found in immunoglobulin deposition disease are caused by mutant Ig proteins. It is very possible that the mutations in these Ig proteins interfere with their interactions with ER chaperones and allow them to by-pass the control mechanisms for removing defective molecules from the secretory pathway. Experiments in Specific Aim 1 are designed to provide a clearer understanding of the molecular sequence of events in the folding and assembly of Ig heavy and light chains. In Specific Aim 2, the role of various ER chaperones in controlling the maturation and transport of Ig proteins will be elucidated. To achieve these aims, a battery of BiP ATPase mutants that bind Ig proteins in vivo but that cannot release them and thus act as "chaperon traps" have been generated and fully characterized. Both transient and inducible systems for the in vivo expression of these BiP mutants have been developed and cell lines that are deficient in the synthesis of various ER chaperones have been produced. In addition, cDNA clones for Ig heavy and light chains have been obtained and used to identify the in vivo folding intermediates for Ig light chains. Antisera specific for various ER chaperones were produced and cDNA clones for grp94 and ERp72 were obtained, which will permit reconstitution of the chaperon-deficient cell lines. Together, these reagents will allow delineation of the intermediates of the Ig folding and assembly pathways, identification of those proteins that directly aid these processes as well as those that monitor the reactions, and finally determination of the mechanisms for retention and degradation of unsuccessful Ig molecules. This information could be crucial to understanding immune pathologies such as light chain deposition disease.