Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe ubiquitin conjugation machinery regulates almost every process in the eukaryotic cell. Deubiquitinating enzymes (DUBs) are a critical component of the machinery since they can remove ubiquitin adducts and thereby control the level of ubiquitin signals (). In accordance with their important roles, DUBs are frequently deregulated in human pathologies including cancer and neurological disease (), making DUBs potential prime targets for therapeutic intervention.The level of the intrinsic DUB activity is important and requires precise control. For a subset of DUBs, there is emerging evidence that the catalytic activity can be modulated by regulatory proteins or by internal domains (). Notable examples include USP7 activation by its HUBL domain and GMPS (), USP1 activation by UAF1 (), and Ubp8 activation in the SAGA complex (). The most striking example is UCH-L5, for which both activation and inhibition have been observed () by two different proteins, RPN13 (ADRM1) and INO80G (NFRKB), respectively.Understanding the mechanisms of DUB activation is important for interpreting their roles in specific cellular contexts. Mechanistic insight into regulatory mechanisms also can provide vital information for the development of inhibitors or activators. So far, the only available crystal structure of a DUB-activator complex is that of the SAGA DUB module (), but no structure is available for its inactive state. Due to this lack of structural data, detailed mechanisms of DUB regulation are still poorly understood.UCH-L5 (UCH37) is a cysteine protease of the ubiquitin C-terminal hydrolase (UCH) family of DUBs, which also includes UCH-L1, UCH-L3, and BAP1. UCH-L5 is overexpressed in several carcinomas () and knockout of the gene is embryonically lethal in mice (). Functionally, it has been linked to TGF-β signaling, Alzheimer’s disease, and longevity (). UCH-L5 constitutes a component of proteasomes and INO80 chromatin remodeling complexes, where it is activated and inhibited, respectively.As a non-essential component of the proteasome 19S regulatory particle, UCH-L5 catalyzes K48-linked polyubiquitin hydrolysis. This activity requires the RPN13 subunit whose C-terminal domain binds UCH-L5 (). In vitro, RPN13 is able to directly promote UCH-L5 activity against a minimal substrate ().UCH-L5 has a less well-defined role in metazoan INO80 chromatin remodeling complexes. INO80 is an essential determinant of embryonic stem cell identity () and participates in the DNA damage response (), but the function of the metazoan-specific subunits, such as INO80G, is poorly defined. A recent report has implicated UCH-L5 and INO80G as key factors of the DNA double-strand-break response (href="#bib39" rid="bib39" class=" bibr popnode">Nishi et al., 2014). Interestingly, in the context of the INO80 complex, the DUB activity of UCH-L5 is inhibited by the INO80G subunit (href="#bib56" rid="bib56" class=" bibr popnode">Yao et al., 2008). Intriguingly, an artificial shorter version of INO80G was found to activate UCH-L5 in vitro (href="#bib56" rid="bib56" class=" bibr popnode">Yao et al., 2008).The UCH enzymes have a small highly conserved papain-like catalytic domain (CD) characterized by a flexible active site cross-over loop (CL). The CL is thought to select substrates according to leaving group size (href="#bib40" rid="bib40 bib57" class=" bibr popnode">Popp et al., 2009; Zhou et al., 2012). In UCH-L5 and UCH family member BAP1 the CL is relatively large, enabling them to process larger substrates (href="#bib57" rid="bib57" class=" bibr popnode">Zhou et al., 2012).Within the UCH family UCH-L5 and BAP1 are close relatives. BAP1 is a critical tumor suppressor whose regulation is important for proper gene regulation (href="#bib5" rid="bib5 bib22 bib52" class=" bibr popnode">Carbone et al., 2013; Goldstein, 2011; White and Harper, 2012). UCH-L5 and BAP1 share an unusual C-terminal helical extension, called ULD (href="#bib36" rid="bib36" class=" bibr popnode">Misaghi et al., 2009). The ULD domain could mediate protein-protein interactions, including higher-order homo-oligomerization (href="#bib4" rid="bib4 bib25" class=" bibr popnode">Burgie et al., 2012; Jiao et al., 2014), and was proposed to act as an auto-inhibitory module (href="#bib55" rid="bib55" class=" bibr popnode">Yao et al., 2006).Like UCH-L5, BAP1 can be activated by a regulatory protein, in this case ASX, to promote H2A deubiquitination (href="#bib45" rid="bib45" class=" bibr popnode">Scheuermann et al., 2010). Phylogenetic analyses have uncovered a conserved domain within the UCH regulatory proteins RPN13, INO80G, and ASX, which was named the DEUBAD domain (href="#bib44" rid="bib44" class=" bibr popnode">Sanchez-Pulido et al., 2012). As all three proteins affect UCH activity, it was proposed that the DEUBAD domain is responsible for this modulation. The conservation suggests a common mechanism of regulation, but where ASX and RPN13 activate their cognate DUB, INO80G inhibits it. Thus, the DEUBAD domain has shifted from activator to inhibitor mode. The mechanistic details of this dual mode of action of the DEUBAD domains are unclear.Here we present structural and functional analyses that explain how DEUBAD domains can switch UCH-L5 activity and thus provide either positive or negative regulation. We show how the DEUBAD domain in RPN13 activates UCH-L5 by tuning the conformation of structural elements in UCH-L5, and inhibits in INO80G, where it exploits molecular mimicry and UCH-L5 conformational plasticity to prevent ubiquitin docking and catalysis. We also show how the inhibitory domain in INO80G has retained the ability to activate, by its N-terminal INO80Gshort region, and identify the structural elements in the DEUBAD domains that confer the activating or inhibitory effects on UCH-L5 enzymatic activity. Our data show that this remarkable tuning of activity involves large conformational changes and is mediated by precise positioning of both the UCH-L5 C-terminal ULD and active site CL.