Article

A UAF1-Containing Multisubunit Protein Complex Regulates the Fanconi Anemia Pathway

Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Molecular Cell (Impact Factor: 14.02). 01/2008; 28(5):786-97. DOI: 10.1016/j.molcel.2007.09.031
Source: PubMed

ABSTRACT

The deubiquitinating enzyme USP1 controls the cellular levels of the DNA damage response protein Ub-FANCD2, a key protein of the Fanconi anemia DNA repair pathway. Here we report the purification of a USP1 multisubunit protein complex from HeLa cells containing stoichiometric amounts of a WD40 repeat-containing protein, USP1 associated factor 1 (UAF1). In vitro reconstitution of USP1 deubiquitinating enzyme activity, using either ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) or purified monoubiquitinated FANCD2 protein as substrates, demonstrates that UAF1 functions as an activator of USP1. UAF1 binding increases the catalytic turnover (kcat) but does not increase the affinity of the USP1 enzyme for the substrate (KM). Moreover, we show that DNA damage results in an immediate shutoff of transcription of the USP1 gene, leading to a rapid decline in the USP1/UAF1 protein complex. Taken together, our results describe a mechanism of regulation of the deubiquitinating enzyme, USP1, and of DNA repair.

Full-text preview

Available from: sciencedirect.com
  • Source
    • "In those cases, the involvement of active-site proximal palm/thumb surfaces makes it simpler to envision how stimulatory co-factors could influence specific active-site arrangement and thereby affect enzymatic turnover (k cat ) more than substrate binding per se (K M ). Such a k cat -oriented activation mechanism has also been ascribed to WDR48's effects on USP proteins (Cohn et al., 2007;Faesen et al., 2011b;Villamil et al., 2012a). Given the distinct binding mode elucidated here, it is less straightforward to reconcile this structure with direct active-site modulation , yet we propose a mechanism centered on WDR48-depen- dent USP stabilization that is consistent with implications from our observations and k cat modulation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein ubiquitination patterns are an important component of cellular signaling. The WD-repeat protein WDR48 (USP1-associated factor UAF-1) stimulates activity of ubiquitin-specific proteases USP1, USP12, and USP46. To understand how WDR48 exerts its effect on the USP scaffold, we determined structures of the ternary WDR48:USP46:ubiquitin complex. WDR48 interacts with the USP46 fingers subdomain via a relatively small, highly polar surface on the top center of the WDR48 β propeller. In addition, WDR48 has a novel ancillary domain and a C-terminal SUMO-like domain encircling the USP46-bound ubiquitin. Mutation of residues involved in the WDR48:USP46 interaction abrogated both binding and deubiquitinase activity of the complex. An analogous mutation in USP1 similarly blocked WDR48-dependent activation. Our data suggest a possible mechanism of deubiquitinase stimulation via stabilization and prolonged residence time of substrate. The unprecedented mode of interaction between the USP fingers domain and the WD-repeat β propeller serves as a prototypical example for this family of deubiquitinases.
    Full-text · Article · Sep 2015 · Structure
  • Source
    • "The deubiquitinating enzyme USP1 regulates the level of FANCD2-Ub (Nijman et al., 2005). USP1 associates with its activating factor UAF1, and the USP1-UAF1 complex removes monoubiquitin from FANCD2 to complete the repair (Cohn et al., 2007) (Fig. 1D). In addition to its stimulatory role, UAF1 is also necessary for recruiting the FANCD2-FANCI complex to USP1 (Yang et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genome instability, primarily caused by faulty DNA repair mechanisms, drives tumorigenesis. Therapeutic interventions that exploit deregulated DNA repair in cancer have made considerable progress by targeting tumor-specific alterations of DNA repair factors, which either induces synthetic lethality or augments the efficacy of conventional chemotherapy and radiotherapy. The study of Fanconi anemia (FA), a rare inherited blood disorder and cancer predisposition syndrome, has been instrumental in understanding the extent to which DNA repair defects contribute to tumorigenesis. The FA pathway functions to resolve blocked replication forks in response to DNA interstrand cross-links (ICLs), and accumulating knowledge of its activation by the ubiquitin-mediated signaling pathway has provided promising therapeutic opportunities for cancer treatment. Here, we discuss recent advances in our understanding of FA pathway regulation and its potential application for designing tailored therapeutics that take advantage of deregulated DNA ICL repair in cancer.
    Full-text · Article · Jul 2015 · Moleculer Cells
  • Source
    • "The DUB activity of USP46 and USP12 has been proven in vitro (Quesada et al. 2004; Cohn et al. 2009; Kee et al. 2010). In C. elegans, USP46 and USP12 both bind to USP1-associated factor 1 (UAF1/WDR48) and the binding dramatically enhances the activity of USP12 and USP46 (Cohn et al. 2007, 2009; Dahlberg and Juo 2014). Our findings indicate that USP46 function is well conserved among species. "

    Full-text · Article · Jan 2015 · Journal of Neurochemistry
Show more

Similar Publications