MOLECULAR CHAPERONES AND IG BIOSYNTHESIS

描述（改编自申请人的摘要）： 蛋白质折叠、装配和从内质网转运 (ER)对于细胞表面事件的保真度至关重要， 淋巴细胞功能和分化。 近年来几 已经鉴定出常驻ER蛋白，其似乎有助于或 监测蛋白质折叠和组装，虽然这些确切的作用， 蛋白质成熟中的“分子伴侣”尚不清楚。 是 该项目的中心假设是，各种ER分子伴侣发挥 在新生IG分子成熟中的独特和重要作用 并因此在B细胞发育和分化中起作用。 IG的失效 与ER伴侣机制的组分相互作用的分子可以 允许异常IG分子的分泌或表面沉积，因此 绕过这个受控过程。 最近， 发现在免疫球蛋白沉积中发现的淀粉样蛋白沉积 疾病是由突变的IG蛋白引起的。 很有可能 这些IG蛋白的突变干扰了它们与 ER伴侣，并允许它们绕过控制机制， 从分泌途径中去除缺陷分子。 实验 具体目标1的目的是提供一个更清晰的理解， 在IG重链和轻链的折叠和组装中事件的分子序列 轻链 在具体目标2中，各种ER分子伴侣在以下方面的作用： 控制IG蛋白的成熟和转运将是 阐明。 为了实现这些目标，一组BiP ATP酶突变体， 在体内结合IG蛋白，但不能释放它们， “伴侣陷阱”已经产生并被充分表征。 两 用于体内表达这些BiP的瞬时和诱导系统 已经开发了突变体，并且已经开发了在所述突变体中缺陷的细胞系。 已经产生了各种ER分子伴侣的合成。 此外，本发明还提供了一种方法， 已经获得了IG重链和轻链的cDNA克隆， 以鉴定IG轻链的体内折叠中间体。 产生了对各种ER分子伴侣特异性的抗血清，并将cDNA 获得了grp94和ERp72的克隆，这将允许 伴侣蛋白缺陷细胞系的重建。 所有这些 试剂将允许描绘IG折叠的中间体 和组装途径，鉴定那些直接 帮助这些过程以及监测反应的过程， 最后确定保留和降解的机制 失败的IG分子。 这些信息可能对 了解免疫病理学，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.