Faresse Nourdine, Debonneville Anne, Staub Olivier
Institute of Anatomy, University of Zurich, Zurich, Switzerland.
Cell Physiol Biochem. 2013;31(2-3):462-72. doi: 10.1159/000343382. Epub 2013 Mar 19.
BACKGROUND/AIMS: Ligand activation of the mineralocorticoid receptor (MR) induces several post-translational modifications (PTMs). Among the different PTMs, MR is known to be dynamically ubiquitylated with impact on its stability and transcriptional activity. Previously, we have shown that MR is monoubiquitylated at the basal state and that aldosterone stimulation induces monoubiquitylation removal prompting polyubiquitin-dependent destabilization of the receptor and proteasomal degradation. This study investigated the role of the aldosterone induced ubiquitin-specific protease USP2-45 on the ubiquitylation state of MR.
Renal epithelial cells M1 were co-transfected with MR with or without wild-type or inactive USP2-45. The association of MR with USP2-45 or TSG101 as well as MR ubiquitylation state were determined by immunoprecipitation and immunoblotting. MR transcriptional activity was assessed via a luciferase reporter gene.
We show that USP2-45 is able to bind MR and, similarly to aldosterone, induce MR monoubiquitylation removal, disruption of MR/TSG101 association and destabilization of MR at protein level.
This study provides a novel role for USP2-45 by playing a pivotal role in the regulation of the ubiquitylation state of MR and reveals the existence of a negative feedback loop for limiting the aldosterone induced response.
背景/目的：盐皮质激素受体（MR）的配体激活可诱导多种翻译后修饰（PTM）。在不同的PTM中，已知MR会动态泛素化，这会影响其稳定性和转录活性。此前，我们已经表明，MR在基础状态下会发生单泛素化，醛固酮刺激会促使单泛素化去除，从而导致受体的多聚泛素依赖性不稳定和蛋白酶体降解。本研究调查了醛固酮诱导的泛素特异性蛋白酶USP2-45对MR泛素化状态的作用。
将肾上皮细胞M1与有或没有野生型或无活性USP2-45的MR共转染。通过免疫沉淀和免疫印迹法测定MR与USP2-45或TSG101的结合以及MR的泛素化状态。通过荧光素酶报告基因评估MR的转录活性。
我们发现USP2-45能够结合MR，并且与醛固酮类似，可诱导MR单泛素化去除、破坏MR/TSG101的结合以及在蛋白质水平上使MR不稳定。
本研究揭示了USP2-45在调节MR泛素化状态中起关键作用的新功能，并揭示了存在一个负反馈回路来限制醛固酮诱导的反应。
