蛋白酶体激活因子REGγ与泛素连接酶Smurf1相互作用的初步研究-生物化学与分子生物学专业论文.docxVIP

安徽医科大学 安徽医科大学 2007 级硕士研究生毕业论文 PAGE PAGE 4 mediates protein degradation in BMP signaling pathway in the osteoblast cells. Our previous study revealed that the CKIP-1 (Casein Kinase 2 Interacting Protein-1) specifically targeted the linker between the two WW domains of Smurf1 and enhanced the affinity of the Smurf1 to its substrates Smad1/5, and promoted the ubiquitin-dependent substrate protein degradation. Our yeast two-hybrid screening of human adult brain library using the WW domains and the linker region of Smurf1 and the full length CKIP-1 as baits identified a potential interacting protein REGγ. Overexpression of REGγ can promote the degradation of Smurf1 wild-type and its mutant C699A. By contrast, overexpression of Smurf1 had no effect on the steady-state level of REGγ, and overexpression of REGγ can not degrade CKIP1. REGγ interacts with Smurf1 in vivo and in vitro. RNAi experiment displayed that Smurf1 was increased and its substrate Smad1 was significantly decreased when endogenous REG γwas knocked down. Proteolytic analysis in vitro confirmed that REGγ promoted 20S proteasome to degrade Smurf1 in an energy- and ubiqution-indenpentant pathway. These findings reveal that the REGγ-proteasome targets an ubiquitin ligase for protein degradation. Keywords: Ubiquitin ligase/Smurf1/REGγ/ATP- and ubiquitin-independent degradation 英文缩写 aa Amino acid CKIP-1 Casein Kinase-2 Interacting Protein-1 Co-IP Co-immunoprecipitation DMEM Dulbecco modified Eagle medium FBS Fetal bovine serum GST Glutathione S-transferase IB Immunoblot IP Immunoprecipitation PCR Polymerase chain reaction REGγ 11S proteasome regulator complex gamma subunit Smurf1 Smad ubiquitylation regulatory factor-1 UPS Ubiquitin-proteasome system 前 言 人类细胞中存在两类重要的蛋白质降解途径：溶酶体依赖的蛋白质降解途径 和蛋白酶体依赖的蛋白质降解途径。前者主要降解细胞外蛋白和细胞膜蛋白，而 后者则是一种高效、特异的受多种复杂因素调控的蛋白质降解途径，控制着动植 物细胞内绝大部分蛋白质的降解，通过蛋白酶体降解途径可以消除受损的或者错 误折叠的蛋白质，进而调节基本的细胞生理过程如细胞增殖、细胞周期、细胞转 导、细胞凋亡和免疫应答[1]。 蛋白酶体主要是由 20S 中央核心催化腔和两端的调节亚基构成。其中，20S 中央核心催化腔由外侧的 14 个 α 亚基和内侧