The Differential Modulation of USP Activity by Internal Regulatory Domains, Interactors and Eight Ubiquitin Chain Types

Division of Biochemistry and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Chemistry & biology (Impact Factor: 6.65). 12/2011; 18(12):1550-61. DOI: 10.1016/j.chembiol.2011.10.017
Source: PubMed


Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.

Download full-text


Available from: Dharjath Ahamed Shahul Hameed, Aug 04, 2014
  • Source
    • "In contrast, loss of USP8 had no impact on, for example, mitochondrial dynamics, depolarization or PINK1 accumulation, further supporting that USP8 controls parkin activity via deubiquitination of the E3 ligase. A key finding of the paper is that USP8 selectively removed K6-linked ubiquitin chains from parkin, although it shows no preference for this linkage type when processing unattached ubiquitin dimers in vitro (Faesen et al, 2011). Expression of an ubiquitin mutant that cannot assemble K6-linked ubiquitin conjugates (Ub K6R ) in USP8- depleted cells was able to rescue delayed parkin recruitment to damaged mitochondria and impaired mitophagy as compared to wildtype ubiquitin. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Parkinson's disease-associated ubiquitin E3 ligase parkin impacts various cellular processes including the autophagic clearance of defective mitochondria. In this issue of The EMBO Journal, Durcan et al (2014) reveal a novel control mechanism of parkin-mediated mitophagy via the selective removal of atypical K6-linked ubiquitin chains from parkin by the deubiquitinase USP8. Together with recent studies on USP15 and USP30, this establishes a functional role for deubiquitination in mitophagy regulation.
    Full-text · Article · Sep 2014 · The EMBO Journal
  • Source
    • "In contrast to the characterized USPs, which have promiscuous linkage preferences (Faesen et al., 2011), the H. sapiens OTU DUBs have more strict linkage specificities (Mevissen et al., 2013). The distinct linkage specificities associated with OTU DUBs could be exploited in restriction analyses to determine the linkage types of the ubiquitin chains conjugated on endogenous substrates (Fiil et al., 2013; Hospenthal et al., 2013; Mevissen et al., 2013), to purify ubiquitylated substrates with specific linkage types, and to assemble UB chains with specific linkage(s) (Bremm et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1=K48>K63), OTU3/4/7/10 (K63>K48>M1), OTU2/9 (K48=K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the encoded proteins have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.
    Full-text · Article · Mar 2014 · Frontiers in Plant Science
  • Source
    • "Similar to other posttranslational modifications, ubiquitination is a reversible process, and there is a family of enzymes, termed deubiquitinases (DUBs), that act on ubiquitinated substrates to catalyze the removal of ubiquitin moieties [10]. In vitro studies have shown that, while some DUBs preferentially cleave specific ubiquitin linkages [11,12], others show a notable promiscuity with respect to the type of ubiquitin linkage they can hydrolyze [13]. Importantly, a DUB that specifically cleaves linear ubiquitin chains has recently been identified [14,15]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.
    Full-text · Article · Aug 2013 · Molecular Cancer
Show more