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Human papillomavirus (HPV) is a significant contributor to the global cancer burden, and its carcinogenic activity is facilitated in part by the HPV early protein 6 (E6), which interacts with the E3-ligase E6AP, also known as UBE3A, to promote degradation of the tumor suppressor, p53. In this study, we present a single-particle cryoEM structure of the full-length E6AP protein in complex with HPV16 E6 (16E6) and p53, determined at a resolution of ~3.3 Å. Our structure reveals extensive protein-protein interactions between 16E6 and E6AP, explaining their picomolar binding affinity. These findings shed light on the molecular basis of the ternary complex, which has been pursued as a potential therapeutic target for HPV-driven cervical, anal, and oropharyngeal cancers over the last two decades. Understanding the structural and mechanistic underpinnings of this complex is crucial for developing effective therapies to combat HPV-induced cancers. Our findings may help to explain why previous attempts to disrupt this complex have failed to generate therapeutic modalities and suggest that current strategies should be reevaluated. HPV’s E6 protein promotes cancer by degrading p53. This study reveals the cryoEM structure of HPV16 E6 in complex with E6AP and p53, highlighting their picomolar affinity and large protein-protein interaction interface.

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5′−3′ exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy. Broken chromosomes are lethal to cells with breaks ends often containing complex DNA damage. Here, the authors reveal a key role for the SNM1A enzyme in cleaning up break ends. Key anticancer drugs, and therapeutic radiation produce complex DNA breaks, making SNM1A an appealing therapeutic target.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN–CtIP-dependent end resection, which generates the 3′ single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3′–5′ exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair. DNA resection is the first step of double-strand break repair by homologous recombination. Broderick et al. find that EXD2 plays a key role in this process by acting as an essential cofactor for the MRN complex.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN. Hepatitis C virus (HCV) infection blunts induction of hepcidin expression by bone morphogenetic protein 6 (BMP6), probably via TNF-mediated downregulation of the BMP co-receptor HJV, while BMP6 regulates a gene repertoire reminiscent of type I IFN signalling. BMP6 and related activin proteins potently block replication of HCV, hepatitis B virus and Zika virus independently of IFN.

The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19. Competing Interest Statement The authors have declared no competing interest.

Understanding the control of viral infections is of broad importance. Chronic HCV infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic BMP/SMAD signaling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, likely via TNF-mediated downregulation of the BMP co-receptor HJV. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted outcome of infection, suggesting BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of Type I IFN signaling, including upregulating IRFs and downregulating an inhibitor of IFN signaling, USP18. Moreover, in BMP stimulated cells, SMAD1 occupied loci across the genome similar to those bound by IRF1 in IFN stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related Activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and Activin A suppressed growth of HBV in cell culture, and Activin A inhibited ZIKV replication alone and in combination with IFN. The data establish an unappreciated important role for the BMPs and Activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Covalent peptides have found widespread applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and highly tunable affinity selection method to enrich covalent reactive peptides from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform enabled by tandem high resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfides to build reversible peptide-protein conjugates that can enrich crosslinked peptides that after reduction can be sequenced with MS/MS. Using this platform, we achieved maturation of covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). The resulting peptides selectively covalently crosslink Cys58 of E6 with 96% yield in 4 hours at 37 °C and Cys113 of Pin1 with >99% yield in 1 hour at room temperature, respectively. This approach enables the identification of highly selective covalent peptide inhibitors for diverse molecular targets, introducing an applicable method to assist pre-clinical therapeutic development pipelines.

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these ‘dirty ends’, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5ʹ-3ʹ exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

ABSTRACT Artemis (DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in DNA double-strand break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Consistently, Artemis deficiency leads to radiosensitive congenital severe immune deficiency (RS-SCID). Artemis belongs to a structural superfamily of nucleases that contain conserved metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. Here, we present crystal structures of the catalytic domain of wild type and variant forms of Artemis that cause RS-SCID Omenn syndrome. The truncated catalytic domain of the Artemis is a constitutively active enzyme that with similar activity to a phosphorylated full-length protein. Our structures help explain the basis of the predominantly endonucleolytic activity of Artemis, which contrast with the predominantly exonuclease activity of the closely related SNM1A and SNM1B nucleases. The structures also reveal a second metal binding site in its β-CASP domain that is unique to Artemis. By combining our structural data that from a recently reported structure we were able model the interaction of Artemis with DNA substrates. Moreover, co-crystal structures with inhibitors indicate the potential for structure-guided development of inhibitors. Competing Interest Statement The authors have declared no competing interest.