Kim Hyoung Tae, Goldberg Alfred L
From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
J Biol Chem. 2017 Jun 9;292(23):9830-9839. doi: 10.1074/jbc.M116.763128. Epub 2017 Apr 17.
The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'--(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins ( Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.
蛋白酶体相关去泛素化酶Usp14/Ubp6通过催化底物去泛素化以及尚未完全了解的变构作用来抑制蛋白质降解。然而，在结合泛素链后，Usp14通过刺激ATP和肽的降解来增强蛋白酶体的降解作用。开展这些研究是为了阐明Usp14这些看似相反的调节作用及其重要性。为了了解26S蛋白酶体上Usp14的存在如何影响其不同活性，我们比较了从野生型小鼠胚胎成纤维细胞中纯化的26S蛋白酶体与缺乏Usp14的26S蛋白酶体的酶活性和调节特性。缺乏Usp14的蛋白酶体比野生型26S具有更高的基础肽酶活性，并且与野生型颗粒相比，5'-（硫代三磷酸）腺苷（ATPγS）对该活性的刺激作用更大。即使在野生型小鼠胚胎成纤维细胞中，Usp14仅存在于26S的一小部分（30-40%）中，这些差异仍然很明显。将纯化的Usp14添加到野生型和缺乏Usp14的蛋白酶体中，会降低它们的基础肽酶活性以及ATPγS的刺激作用。Usp14通过变构抑制这些过程，因为催化无活性的Usp14突变体也能抑制它们。缺乏Usp14的蛋白酶体与野生型颗粒相比，还表现出更高的Rpn11去泛素化活性和更高的基础ATP酶活性。如果野生型蛋白酶体结合了一个折叠松散的泛素化蛋白质，其ATP水解会被激活。令人惊讶的是，缺乏Usp14的蛋白酶体即使不存在泛素链，也能被此类蛋白质激活。此外，缺乏Usp14的蛋白酶体在降解非泛素化蛋白质（Sic1）方面比野生型颗粒活跃得多。因此，在不存在泛素化底物的情况下，Usp14会抑制多种蛋白酶体活性，尤其是基础ATP消耗和非泛素化蛋白质的降解。这些变构效应因此减少了无活性蛋白酶体的ATP水解和非特异性蛋白水解，并增强了蛋白酶体对泛素化蛋白质的特异性。
