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.
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.
AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that ...the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.
The LKB1 tumour suppressor kinase phosphorylates and activates a number of protein kinases belonging to the AMP-activated protein kinase (AMPK) subfamily. We have used a modified tandem affinity ...purification strategy to identify proteins that interact with AMPKalpha, as well as the twelve AMPK-related kinases that are activated by LKB1. The AMPK{szligbeta} and AMPKgamma regulatory subunits were associated with AMPKalpha, but not with any of the AMPK-related kinases, explaining why AMP does not influence the activity of these enzymes. In addition, we identified novel binding partners that interacted with one or more of the AMPK subfamily enzymes, including fat facets/ubiquitin specific protease-9 (USP9), AAA-ATPase-p97, adenine nucleotide translocase, protein phosphatase 2A holoenzyme and isoforms of the phospho-protein binding adaptor 14-3-3. Interestingly, the 14-3-3 isoforms bound directly to the T-loop Thr residue of QSK and SIK, after these were phosphorylated by LKB1. Consistent with this, the 14-3-3 isoforms failed to interact with non-phosphorylated QSK and SIK, in LKB1 knockout muscle or in HeLa cells in which LKB1 is not expressed. Moreover, mutation of the T-loop Thr phosphorylated by LKB1, prevented QSK and SIK from interacting with 14-3-3 in vitro. Binding of 14-3-3 to QSK and SIK, enhanced catalytic activity towards the TORC2 protein and the AMARA peptide, and was required for the cytoplasmic localization of SIK and for localization of QSK to punctate structures within the cytoplasm. To our knowledge, this study provides the first example of 14-3-3 binding directly to the T-loop of a protein kinase and influencing its catalytic activity and cellular localization.
Patients with relapsed/refractory multiple myeloma (RRMM) have a high unmet treatment need. Belantamab mafodotin (belamaf), a first-in-class, B-cell maturation antigen-binding antibody-drug ...conjugate, eliminates myeloma cells through direct cell killing and an anti-myeloma immune response.
DREAMM-2 (NCT03525678) was a phase 2, two-arm, open-label trial in patients with heavily pretreated RRMM who had three or more prior therapies, were refractory to an immunomodulatory agent and a proteasome inhibitor, and refractory or intolerant to an anti-CD38 monoclonal antibody. Belamaf was given at 2.5 or 3.4 mg/kg every 3 weeks. The primary end point was overall response rate (ORR); secondary end points included progression-free survival (PFS), overall survival (OS), safety, ocular symptoms, and health-related quality of life (HRQOL).
This final analysis (cutoff date, March 31, 2022), N = 223, with median follow-up of 12.5 and 13.8 months, demonstrated an ORR of 32% and 35%, median PFS of 2.8 and 3.9 months, and median OS of 15.3 and 14.0 months in the 2.5 mg/kg and 3.4 mg/kg cohorts, respectively. Median duration of response was 12.5 and 6.2 months. No new safety signals were observed; the most common Grade 3 and 4 adverse events were keratopathy (29% vs. 25%), thrombocytopenia (22% vs. 29%), and anemia (21% vs. 28%). HRQOL outcomes suggest that overall global health status/quality of life, physical and role functioning, and overall disease symptoms were maintained or improved during treatment.
This final analysis of DREAMM-2 confirms that in patients with triple-class refractory RRMM, single-agent belamaf results in durable and clinically meaningful responses with a manageable safety profile.
Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates ...in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.
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► Loss of MMS22L or TONSL results in spontaneous DNA double-strand breaks ► MMS22L forms an interdependent complex with TONSL ► MMS22L and TONSL accumulate at lesions containing RPA-bound single-stranded DNA ► MMS22L-TONSL promotes RAD51 nucleofilament formation and homologous recombination
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.
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