Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability

Abstract

SummaryDeubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.

Profiling DUBs and Ubl-specific proteases with activity-based probes

“…Nearly a hundred genes encoding DUBs have been identified in the human genome, which can be classified in seven distinct families. The subfamilies of ubiquitin-specific proteases (USP), ubiquitin C-terminal hydrolases (UCH), Ovarian TUmor domain proteases (OTU), Machado-Joseph disease proteases (MJD), Motif interacting with ubiquitin-containing novel DUB family (MINDY), and Zinc finger with UFM1-specific peptidase domain protein (ZUFSP) are cysteine proteases, whereas JAB1/MPN/ MOV34 proteases (JAMMs) are zinc-dependent metalloproteases (Abdul Rehman et al, 2016;Komander, Clague, & Urbe, 2009;Kwasna et al, 2018;Nijman et al, 2005;Reyes-Turcu, Ventii, & Wilkinson, 2009). As distinct Ub linkages result in distinct biological signals (Komander & Rape, 2012;Yau & Rape, 2016), the determination of the linkage specificities of DUBs gives fundamental insights into the biological pathways they are involved in.…”

Evaluating enzyme activities and structures of DUBs

“…Expanding upon this concept, diUb-based ABPs have also been developed that report on added levels of specificity (Haj-Yahya et al, 2014;Iph€ ofer et al, 2012;Li, Liang, Gong, Tencer, & Zhuang, 2014;McGouran, Gaertner, Altun, Kramer, & Kessler, 2013;Mulder, El Oualid, ter Beek, & Ovaa, 2014;Weber et al, 2017) (discussed in more detail below). A Ub-ABP can also be coupled with a reporter (e.g., an epitope tag or fluorescent group) at the amino-terminus to allow for measurement of DUB activity in cellular lysates following, e.g., inhibitor treatment (Turnbull et al, 2017), as well as enrichment and identification of novel DUBs (e.g., Abdul Rehman et al, 2016;Hewings et al, 2018;Kwasna et al, 2018;Misaghi et al, 2006). The same approach can also be applied to UBL modifiers, and thus Ub/UBL ABP reactivity can immediately report on substrate specificity.…”

“…Eraser enzymes, also known as deubiquitinases (DUBs), are key regulators of the Ub system. Humans encode approximately 100 DUB genes belonging to seven protease families (Haahr et al, 2018;Hermanns et al, 2018;Hewings et al, 2018;Kwasna et al, 2018;Mevissen & Komander, 2017). Additional proteases are specific toward UBL modifiers; herein we collectively refer to all Ub/UBL proteases as DUBs for simplicity.…”

OTUB1 is a key regulator of RIG-I dependent immune signalling and is targeted for proteasomal degradation by influenza A NS1

“…To isolate DUBs from IAV-infected cells, we followed an experimental set-up as illustrated in the schematic (Figure 1B) (Jahan et al, 2016;Zhang et al, 2018). We and others have previously employed similar approaches with ubiquitin C-terminal electrophiles to isolate cellular DUBs (Jahan et al, 2016;Kwasna et al, 2018).…”