BackgroundIn complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage.MethodsWe have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage.ResultsA non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420–520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420–520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency.ConclusionsUSP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420–520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0311-7) contains supplementary material, which is available to authorized users.

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

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The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.

Ubiquitin-specific protease (USP)1 is a member of the USP family of deubiquitinating enzymes. Efficient USP1 activity requires binding to its cofactor USP1-associated factor 1 (UAF1), and the USP1-UAF1 deubiquitinase complex has important roles in regulating DNA damage-related processes. USPs show common folding of their catalytic domain, with three subdomains termed Thumb, Palm, and Fingers. The Fingers subdomain appears to be the primary site for ubiquitin binding. In USP1, the Fingers subdomain also mediates its interaction with UAF1, and thus represents a crucial, but poorly characterized, motif in USP1. To explore the role of USP1-UAF1 in ubiquitin-dependent nuclear processes, we tested the effect of modulating USP1-UAF1 activity on the level and/or localization of conjugated ubiquitin and the DNA damage-related proteins phosphorylated histone H2AX, Lys56-acetylated histone H3, and p53-binding protein 1 (53BP1). Small interfering RNA-mediated USP1 knockdown or treatment with the novel USP1-UAF1 inhibitor ML323 increased the recruitment of conjugated ubiquitin and 53BP1 into nuclear foci. Strikingly, ectopic coexpression of USP1 and UAF1 depleted conjugated ubiquitin in the nucleus and blocked the recruitment of 53BP1 to DNA damage foci. In a direct comparison with other overexpressed USPs, USP1-UAF1 behaved as a relatively promiscuous deubiquitinase. Experimental and cancer-related mutations in the USP1 The Fingers subdomain abrogated substrate deubiquitination without interfering with other USP1 activities, such as UAF1 binding or autocleavage. These results provide new insights into the function and regulation of the USP1-UAF1 complex.