Burr Marian L, Boname Jessica M, Lehner Paul J
Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.
Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2013;960:109-125. doi: 10.1007/978-1-62703-218-6_9.
The covalent attachment of ubiquitin to a protein is one of the most common post-translational modifications and regulates diverse eukaryotic cellular processes. Ubiquitination of MHC class I was first described in the context of viral proteins which target MHC class I for degradation in the endoplasmic reticulum and at the cell surface. Study of viral-induced MHC class I degradation has been extremely instructive in elucidating cellular pathways for degradation of membrane and secretory proteins. More recently, ubiquitination of endogenous MHC class I heavy chains which fail to achieve their native conformation and undergo endoplasmic-reticulum associated degradation has been demonstrated.In this chapter we describe methods for identification of endogenous ubiquitinated MHC class I heavy chains by MHC class I-immunoprecipitation and ubiquitin-specific immunoblot or by metabolic labeling and immunoprecipitation.
泛素与蛋白质的共价连接是最常见的翻译后修饰之一，可调节多种真核细胞过程。MHC I类分子的泛素化最初是在病毒蛋白的背景下被描述的，这些病毒蛋白在内质网和细胞表面靶向MHC I类分子进行降解。对病毒诱导的MHC I类分子降解的研究在阐明膜蛋白和分泌蛋白的细胞降解途径方面极具启发性。最近，已经证明了未能达到其天然构象并经历内质网相关降解的内源性MHC I类重链的泛素化。在本章中，我们描述了通过MHC I类免疫沉淀和泛素特异性免疫印迹或通过代谢标记和免疫沉淀来鉴定内源性泛素化MHC I类重链的方法。
