Solution Structure of the Human Ubiquitin-specific Protease 15 DUSP Domain

Abstract

Ubiquitin-specific proteases (USPs) can remove covalently attached ubiquitin moieties from target proteins and regulate both the stability and ubiquitin-signaling state of their substrates. All USPs contain a conserved catalytic domain surrounded by one or more subdomains, some of which contribute to target recognition. One such specific subdomain, the DUSP domain (domain present in ubiquitin-specific proteases), is present in at least seven different human USPs that regulate the stability of or interact with the hypoxia-inducible transcription factor HIF1-␣, the Von HippelLindau protein (pVHL), cullin E3 ligases, and BRCA2. We describe the NMR solution structure of the DUSP domain of human USP15, recently implicated in COP9 (constitutive photomorphogenic gene 9) -signalosome regulation. Its tripod-like structure consists of a 3-fold ␣-helical bundle supporting a triple-stranded anti-parallel ␤-sheet. The DUSP domain displays a novel fold, an ␣/␤ tripod (AB3). DUSP domain surface properties and previously described work suggest a potential role in protein/protein interaction or substrate recognition.There are at least 67 human deubiquitinating enzymes, subdivided in ubiquitin-specific proteases (USPs) 4 and ubiquitin carboxyl-terminal hydrolases. By removing covalently attached ubiquitin moieties from target proteins, USPs counteract the activity of a relatively small number of E2 enzymes and hundreds of E3 enzymes responsible for specific protein ubiquitination. The subclass of ubiquitin carboxyl-terminal hydrolase enzymes cleaves ubiquitin from small peptides. Deubiquitination serves a number of roles: it protects Lys 48 -linked polyubiquitinated proteins from targeting to the proteasome and subsequent degradation, but can also control the signaling state characterized by monoubiquitination or Lys 63 -linked polyubiquitination (1). Human USP7 was shown to stabilize p53 and, as a consequence, affect cell viability and apoptosis. Initial observations suggested that USP7 stabilizes p53 by direct binding and deubiquitination of p53 (2, 3). Subsequently, USP7 was also demonstrated to deubiquitinate the murine double minute 2 homolog protein, the E3 ubiquitin ligase that targets p53 for proteasomal degradation. The total absence of USP7 destabilized murine double minute 2 homolog, causing an indirect stimulation of p53 protein stability (4, 5). The balance in deubiquitination of these two targets might determine the net outcome of both opposing actions. If the flexibility in target recognition by USP7 is exemplary for the whole USP protein family, unraveling USP-regulated ubiquitin signaling and USP target specificity will prove a challenging task.A first structural view on the action of USPs was provided by the crystal structure of the USP7 catalytic domain in complex with a covalently attached ubiquitin aldehyde that specifically inhibits USP enzymes (3). The ubiquitin moiety is recognized by an extended protein surface, and its carboxyl-terminal region contacts a narrow groove containing the conserved catalytic t...

The Regulations of Deubiquitinase USP15 and Its Pathophysiological Mechanisms in Diseases

“…One study demonstrated that the Asp/Asn residue is required for polarization of His residue to stabilize the catalytic activity of DUBs [14]. In USP15, delineation of amino acid sequence of the catalytic domain reveals the existence of six conserved boxes and the catalytic triad composed of Cys, His, and Asp residues, which are localized at Box 1, Box 5, and Box 6, respectively [11]. The subsequent study further revealed that the presence of two Cys-X-X-Cys motifs within Box 3 and Box 4 is critical for formation of a zinc-binding motif, which is important for conformational stability [15].…”

“…Ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), ubiquitin C-terminal hydrolases (UCHs), and Machado-Joseph disease proteases (MJDs) are categorized into papain-like cysteine proteases, while JAB1/MPN/Mov34 proteases (JAMMs) belong to zinc-dependent metalloproteases [10]. In this review, we focus on the ubiquitin carboxyl-terminal hydrolase 15 (also known as ubiquitin-specific-processing protease 15, USP15), which features two consensus sequences harboring a classical catalytic triad (Cys-His-Asp) [11]. The amplification of USP15 gene has been previously identified in glioblastoma, breast cancer and ovarian cancer.…”

Structure and Catalytic Regulatory Function of Ubiquitin Specific Protease 11 N-Terminal and Ubiquitin-like Domains

“…Loop 2 (L2) comprising residues Gly 76 -Asp 111 in hUSP11 (Figure 1B) is tethered to the DUSP surface by interactions from residues Pro 85 and Ile 88 with Trp 66 from α-helix 2. These residues form part of the signature PGPI sequence identified by bioinformatic analysis as being shared by DUSP domains from seven distinct USPs, 36 but the second proline is substituted by Cys 87 in hUSP11DU. Prior to strand S2 there is a turn spanning residues Glu 108 -Asp 111 that contains several acidic residues with the sequence EGED.…”

Alternative exon skipping biases substrate preference of the deubiquitylase USP15 for mysterin/RNF213, the moyamoya disease susceptibility factor

“…The C-region harbors the enzymatic core responsible for deubiquitylation activity1213, so the C-cluster presumably recognizes the ubiquitin moiety of the ubiquitylated mysterin. Previous structural studies demonstrated that the DUSP domain of USP15 exposes multiple hydrophobic patches on its molecular surface, which are predicted to mediate protein–protein interactions via hydrophobic interactions2930. Indeed, USP15 specifically binds to SART3 (also known as Tip110) via DUSP domain and removes ubiquitin from both SART3 and its binding partner, histone H2B92728.…”