Welk Vanessa, Coux Olivier, Kleene Vera, Abeza Claire, Trümbach Dietrich, Eickelberg Oliver, Meiners Silke
From the Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University, Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), 81377 Munich, Germany.
the Centre de Recherche de Biochimie Macromoléculaire (CRBM-CNRS UMR 5237), Université de Montpellier, 34293 Montpellier, France, and.
J Biol Chem. 2016 Jun 17;291(25):13147-59. doi: 10.1074/jbc.M116.717652. Epub 2016 Apr 18.
The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28αβ, PA28γ, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28γ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28γ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of β5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28γ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28γ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.
蛋白酶体是一种细胞内蛋白酶复合物，由20S催化核心及其相关调节因子组成，包括19S复合物、PA28αβ、PA28γ、PA200和PI31。蛋白酶体的抑制会诱导20S和26S蛋白酶体复合物的自调节从头形成。然而，迄今为止尚未研究替代蛋白酶体复合物的形成。我们在此表明，催化性蛋白酶体抑制会导致PA28γ和PA200在2-6小时内快速募集到20S和26S蛋白酶体。替代蛋白酶体复合物的快速形成不涉及PA28γ和PA200的转录激活，而是将预先存在的激活剂募集到20S和26S蛋白酶体复合物中。蛋白酶体激活剂的募集取决于蛋白酶体活性位点的抑制程度，β5活性位点的抑制足以诱导募集。此外，通过siRNA介导的19S亚基RPN6敲低对26S蛋白酶体活性进行特异性抑制，仅诱导PA200募集到20S蛋白酶体，而PA28γ未被动员。在此，替代PA200复合物的形成涉及激活剂的转录激活。当细胞在脉冲暴露于蛋白酶体抑制剂后恢复蛋白酶体活性时，替代蛋白酶体复合物仍然存在。PA28γ的敲低使细胞对蛋白酶体抑制剂介导的生长停滞敏感。因此，替代蛋白酶体复合物的形成似乎是细胞对蛋白酶体功能受损和蛋白质稳态改变的反应中一个以前未被认识但不可或缺的部分。
