IDENTIFYING PROTEINS INVOLVED IN DEGRADATION OF UNSTABLE PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 先前的工作已经表明，小的FKBP衍生的蛋白质可以被工程化以表现出小分子配体依赖性的蛋白质稳定性拯救。 当与其他感兴趣的蛋白质（POI）融合并在细胞中表达时，这些“去稳定化结构域”（DD）导致融合蛋白在不存在配体的情况下快速降解。 细胞可渗透配体的加入稳定了结构域，并允许融合蛋白发挥其生物学效应。 我们的初步工作表明DD降解是蛋白酶体依赖性的，泛素也参与了降解过程。 我们的假设是，在没有配体的情况下，细胞蛋白质质量控制机制特异性靶向降解的DD。 由于各种成功靶向的POI融合的DD，我们进一步假设，参与DD识别和降解的蛋白质可能是一般的质量控制和/或蛋白质折叠机制的“看门人”，其成员已难以通过其他生物化学技术确定。 通过使用一个有效的DD捕获技术和质谱鉴定共纯化蛋白质，我们希望确定参与识别和处理降解DD的蛋白质。 蛋白质质量控制的一般细胞机制的知识可以帮助我们制定策略，以打击蛋白质错误折叠的疾病，如阿尔茨海默氏症，帕金森氏症和朊病毒疾病。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous work has shown that a small FKBP-derived protein can be engineered to exhibit small molecule ligand-dependent rescue of protein stability. When fused to other proteins of interest (POI) and expressed in cells, these "destabilization domains" (DDs) cause the fusion protein to be rapidly degraded in the absence of the ligand. Addition of the cell-permeable ligand stabilizes the domain and allows the fusion protein to exert its biological effect. Our preliminary work shows DD degradation to be proteasome-dependent, with ubiquitin also implicated in the degradation process. Our hypothesis is that in the absence of ligand, the DDs are specifically targeted by cellular protein quality control machinery for degradation. Because of the variety of successfully targeted POIs fused to the DDs, we further hypothesize that proteins involved in DD recognition and degradation could be general "gatekeepers" of quality control and/or protein folding machinery, members of which have been difficult to identify by other biochemical techniques. By using an efficient DD capture technique and mass spectrometry to identify co-purifying proteins, we hope to identify proteins involved in the recognition and processing of DDs for degradation. Knowledge of general cellular mechanisms for protein quality control may help us devise strategies to combat diseases of protein misfolding, such as Alzheimer's, Parkinson's and prion diseases.