| Proteasome system as a multiplex protein degradation system plays an important role in protein degradation in vivo. It is regulated by many complex factors in protein degradation. Previous studies have revealed that the proteasome is mainly composed of the 20S central catalytic cavity and the regulatory subunits on both sides. The 20S central catalytic cavity was a cylindrical core structure, and was composed of twoα-helices in the outer ring and twoβ-helices in the inner ring, and each ring consists of seven subunits. Theα-helice formed a narrow ring axial gated channel and theβ-helice formed a degradation chamber which contains a variety of proteolytic activity sites. Proteasome regulatory particles on both sides are devided into two categories. One is the 19S proteasome activator which is mainly involved in energy- and ubiquitin-dependent proteasome degradation pathway. The other is REG family proteasome activators, including REGα, REGβand REGγ, which are mainly involved in the energy- and ubiquitin-independent protein degradation pathway and further regulate cellular immune response, cell cycle, apoptosis, cell growth, cell differentiation and so on. REGγwas firstly identified in the serum of patients with systemic lupus erythematosus and was named Ki antigen. Subsequent studies revealed that REGγwas a highly conserved nuclear protein in both invertebrates and vertebrates and found in the nuclei of animals, insects and worms, but not in yeast and plant cells. Recent findings identified that REGγmediates the degradation of proteins involved in regulation of the cell cycle, apoptosis, tumorigenesis, and other important cellular processes including p21, p16, p19, and SRC-3 in an energy- and ubiquitin-independent manner.Ubiquitin-proteasome system is a highly selective eukaryotic protein degradation system. It consists of the ubiquitin, the ubiquitin-activating enzyme, the ubiquitin conjugating enzyme, the ubiquitin-protein ligase, the 26S proteasome, and the deubiquitinase. The ubiquitin-protein ligase is the main component in the ubiquitin-proteasome system and determines the substrate bingding specificity. It can be divided into RING (Really interesting new gene) zinc finger- and HECT (Homologous to E6AP C-terminus) domain-types. Smurf1 (Smad ubiquitylation regulatory factor 1) as a member of the NEDD4 family belongs to the HECT domain type and mainly 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 REGγcan 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 REGγcan not degrade CKIP1. REGγinteracts with Smurf1 in vivo and in vitro. RNAi experiment displayed that Smurf1 was increased and its substrate Smad1 was significantly decreased when endogenous REGγwas knocked down. Proteolytic analysis in vitro confirmed that REGγpromoted 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.
|
PDF Full Text Request