IDENTIFYING PROTEINS INVOLVED IN DEGRADATION OF UNSTABLE PROTEINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 以前的工作表明，可以对FKBP衍生的小蛋白进行改造，使其表现出依赖小分子配体的蛋白质稳定性拯救。当与其他感兴趣的蛋白(POI)融合并在细胞中表达时，这些“不稳定结构域”(DDS)会导致融合蛋白在没有配体的情况下迅速降解。细胞可穿透配体的加入稳定了结构域，并使融合蛋白发挥其生物学效应。我们的初步工作表明，DD的降解是蛋白酶体依赖的，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，DDS是细胞蛋白质质量控制机制专门针对降解的。由于各种成功的靶向POI与DDs融合，我们进一步假设，参与DD识别和降解的蛋白质可能是质量控制和/或蛋白质折叠机制的一般“守门人”，这些成员已经很难通过其他生化技术来识别。通过使用高效的DD捕获技术和质谱学来鉴定共纯化的蛋白质，我们希望能够识别参与识别和处理DDs以供降解的蛋白质。了解蛋白质质量控制的一般细胞机制可能有助于我们设计出对抗蛋白质错误折叠疾病的策略，如阿尔茨海默氏症、帕金森氏症和普里恩病。

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.