Degradation of Mislocalized Secretory and Membrane Proteins

错误定位的分泌蛋白和膜蛋白的降解

• 批准号: 8351235
• 负责人: Ramanujan S Hegde
• 金额: $ 24.88万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351235
• 关键词: Affinity Chromatography; Degradation Pathway; Elements; Enzymes; Event; Fractionation; Goals; Homeostasis; In Vitro; Membrane Proteins; Microsomes; Molecular Chaperones; Pathway interactions; Preparation; Proteins; Reaction; System; Ubiquitination; high risk; in vivo; reconstitution; secretory protein

The selective recognition and degradation of mislocalized secretory and membrane proteins has been reconstituted in vitro. In this system, secretory and membrane proteins are synthesized in their non-translocated state simply by omitting ER-derived rough microsomes from the reaction or, if they are included, by inhibiting translocation using cotransin. With either of these manipulations, non-translocated proteins are rapidly ubiquitinated in preparation for their degradation. We are employing both classical fractionation and affinity purification approaches to identify the machinery for the selective recognition and degradation of these non-translocated proteins. In the past year, we have successfully reconstituted many of these events in vitro and have used this system to identify factors. We have identified the E2 enzyme involved, and a key chaperone that facilitates degradation. Our current aim is to identify the remaining factors of this pathway and reconstitute the events with purified components. This will pave the way for understanding mechanistically how the pathway operates. In parallel, we are moving into in vivo systems to understand the importance of this degradation pathway in maintaining cellular protein homeostasis.

错误定位的分泌蛋白和膜蛋白的选择性识别和降解已在体外重建。在该系统中，分泌蛋白和膜蛋白以非易位状态合成，只需从反应中省略内质网衍生的粗糙微粒体，或者如果包含的话，通过使用共转蛋白抑制易位。通过这些操作中的任何一种，非易位蛋白质都会迅速泛素化，为它们的降解做好准备。我们采用经典的分级分离和亲和纯化方法来确定选择性识别和降解这些非易位蛋白质的机制。在过去的一年中，我们成功地在体外重建了许多此类事件，并使用该系统来识别因素。我们已经确定了所涉及的 E2 酶，以及促进降解的关键分子伴侣。我们当前的目标是确定该途径的其余因素并用纯化的成分重建事件。这将为从机制上理解该途径如何运作铺平道路。与此同时，我们正在进入体内系统，以了解这种降解途径在维持细胞蛋白质稳态中的重要性。