Role of Molecular Chaperones in Ig Biosynthesis

 描述(申请人提供)：注定要在细胞表面分泌或表达的蛋白质是在内质网(ER)中合成的，在那里它们获得了生物活性结构。当这些蛋白质进入内质网时，它们会遇到各种分子伴侣，包括两个Hsp70及其Dna J辅助因子，这些分子伴侣直接与新生蛋白质结合，帮助它们折叠和组装成多亚单位复合体，或者如果它们不能正常成熟，就会靶向降解。内质网如何为蛋白质沿分泌途径运输、保留在内质网中供进一步折叠尝试或靶向降解设定阈值，这是一个基本的生物学问题，目前还没有很好的理解。我们假设，ER的各种伴侣对特定序列的识别可能是这种质量控制区分的关键方面，尽管ER伴侣的体内特异性从未被确定。为了解决这一重要的知识空白，我们开发了一个体内表达系统/文库，由一系列25个氨基酸的重叠多肽组成，覆盖了免疫球蛋白重链和完整的非分泌型Ig轻链的质量控制关键区域，这两条链都直接与ER HSP70伴侣及其辅助因子结合。这些多肽通过与内质网靶向的、折叠良好的Ig结构域的融合在哺乳动物细胞的内质网中表达，重要的是，该结构域不结合任何这些伴侣蛋白。对这些文库的鉴定表明，Bip和ERdj3在两个客户端上都结合了多个肽，而Grp170、ERdj4和ERdj5结合位点要罕见得多，位置也更受限制。目标1将定义Bip和Grp170的结合位点，并使用该信息来确定抑制底物与这两个伴侣相互作用的生物学后果以及功能关系 两个ER Hsp70之间的关系。在目标2中，在描绘了我们的模型蛋白上三个ERdj辅助因子的结合序列后，建议进行研究，以了解ERdj蛋白和Hsp70伴侣之间发生的机制相互作用，从而为客户带来不同的结果。未能通过内质网质量控制的蛋白质从内质网中提取出来，并由26S蛋白酶体在细胞质中降解；这一过程依赖于它们对逆转录转位和泛素化的靶向。使用我们的一系列肽结构，在AIM中 3我们将在我们的多肽文库的基础上，建立一种用于质谱分析的体外泛素化分析方法，以鉴定为内质网质量控制目的而修饰的氨基酸。将使用比较方法来阐明ERAD机制的非糖基化蛋白质的组成部分，目前对这些组成部分的定义很差。从这些研究的组合中获得的数据将为ER质量控制的分子决定因素和机制提供关键的见解。

 DESCRIPTION (provided by applicant): Proteins destined to be secreted or expressed on the cell surface are synthesized in the endoplasmic reticulum (ER) where they acquire their biologically active structure. As these proteins enter the ER, they encounter a variety of molecular chaperones, including two Hsp70s and their DnaJ co-factors, which bind directly to the nascent proteins and either aid their folding and assembly into multi-subunit complexes or target them for degradation if they fail to mature properly. How the ER sets the threshold for proteins to be transported along the secretory pathway, retained in the ER for further folding attempts, or targeted for degradation is an essential biological question that is not well understood. We hypothesize that recognition of specific sequences by the various chaperones of the ER is likely to provide a critical aspect of this quality control discrimination, although te in vivo specificity of ER chaperones has never been determined. To address this important gap of knowledge, we have developed an in vivo expression system/library composed of a series of 25-amino acid, overlapping peptides covering critical regions for quality control of an immunoglobulin heavy chain and a complete non-secreted Ig light chain, both of which bind directly to the ER Hsp70 chaperones and their co-factors. The peptides are expressed in the ER of mammalian cells via their fusion to an ER-targeted, well-folded Ig domain, which importantly does not bind any of these chaperones. Characterization of these libraries reveals that BiP and ERdj3 bind multiple peptides throughout both clients, whereas Grp170, ERdj4, and ERdj5 binding sites are much rarer and more restricted in their locations. Aim 1 will define binding site for BiP and Grp170 and use this information to determine the biological consequences of inhibiting substrate interactions with these two chaperones, as well as the functional relationship of the two ER Hsp70s to each other. In Aim 2, after delineating binding sequences on our model proteins for the three ERdj co-factors, studies are proposed to understand the mechanistic interactions occurring between the ERdj proteins and with the Hsp70 chaperones that lead to disparate outcomes for clients. Proteins that fail ER quality control are extracted from the ER and degraded in the cytoplasm by the 26S proteasome; processes that are dependent on their targeting to the retrotranslocon and ubiquitination. Using our series of peptide constructs, in Aim 3 we will develop an in vitro ubiquitination assay for mass spectrometric analyses base on our peptide libraries to identify amino acids that are modified for the purpose of ER quality control. comparative approach will be used to elucidate components of the ERAD machinery for non-glycosylated proteins, which are currently poorly defined. The data obtained from the combination of these studies will provide critical insights into the molecular determinants and mechanisms that underlie ER quality control.