Abdallah Al-Hakim

The RIDDLE Syndrome Protein Mediates a Ubiquitin-Dependent Signaling Cascade at Sites of DNA Damage

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.

The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1

DNA double-strand breaks (DSBs) pose a potent threat to genome integrity. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DSBs elicit a signalling cascade that modifies the chromatin surrounding the break, first by ATM-dependent phosphorylation and then by RNF8-, RNF168- and BRCA1-dependent regulatory ubiquitination. Here we report that OTUB1, a deubiquitinating enzyme, is an inhibitor of DSB-induced chromatin ubiquitination. Surprisingly, we found that OTUB1 suppresses RNF168-dependent poly-ubiquitination independently of its catalytic activity. OTUB1 does so by binding to and inhibiting UBC13 (also known as UBE2N), the cognate E2 enzyme for RNF168. This unusual mode of regulation is unlikely to be limited to UBC13 because analysis of OTUB1-associated proteins revealed that OTUB1 binds to E2s of the UBE2D and UBE2E subfamilies. Finally, OTUB1 depletion mitigates the DSB repair defect associated with defective ATM signalling, indicating that pharmacological targeting of the OTUB1–UBC13 interaction might enhance the DNA damage response.

The ubiquitous role of ubiquitin in the DNA damage response.

Abstract Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response. In this review, we discuss how DNA repair and checkpoint pathways utilize the diversity offered by the ubiquitin conjugation system to modulate the response to genotoxic lesions in space and time. In particular, we will highlight recent work done on the regulation of DNA double-strand breaks signalling and repair by the RNF8/RNF168 E3 ubiquitin ligases, the Fanconi anemia pathway and the role of protein degradation in the enforcement and termination of checkpoint signalling. We also discuss the various functions of deubiquitylating enzymes in these processes along with potential avenues for exploiting the ubiquitin conjugation/deconjugation system for therapeutic purposes.

An Allosteric Inhibitor of the Human Cdc34 Ubiquitin-Conjugating Enzyme

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Interaction Proteomics Identify NEURL4 and the HECT E3 Ligase HERC2 as Novel Modulators of Centrosome Architecture

Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.

Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4

Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT-PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line.

Expression of adenylyl cyclase-4 (AC-4) in Y1 and forskolin-resistant adrenal cells.

Forskolin-resistant mutants of a mouse adrenocortical cell line present a complex phenotype in which adenylyl cyclase (AC) is resistant to activation by forskolin and by ACTH. ACTH-resistance results from a defect affecting transcription of the ACTH receptor and can be overcome by transfecting mutant cells with expression vectors encoding G beta/gamma. Forskolin-resistance results from an AC-4 deficiency. We now demonstrate that the AC-4 deficiency in forskolin-resistant mutants results from a transcription defect affecting the promoter activity of the AC-4 gene. Furthermore, the underlying defect leading to AC-4 deficiency and forskolin-resistance can be overcome by transfection of mutant clones with expression vectors encoding G beta/gamma. These data support our hypothesis that AC-4 is a preferred target of forskolin action in Y1 cells, demonstrate novel roles for G beta/gamma in gene expression and indicate that a common underlying defect, suppressible by G beta/gamma, accounts for both the resistance to ACTH and to forskolin.