Biology subjects

Smerdon, S. J.

Publications and source records attributed to Smerdon, S. J..

7 recordsLinked to original sources

bioRxiv2025

Inactivation of a protein hydroxylase complex impairs replication fork restart in cancer and neurodevelopmental disorders

Protein hydroxylation is a post-translational modification that is commonly catalysed by enzymes of the oxygen- and 2-oxoglutarate (2OG)-dependent oxygenase family. The Jumonji-C (JmjC)-only sub-family of 2OG-oxygenases catalyse the hydroxylation of protein and tRNA substrates involved in fundamental cellular processes. Jumonji-domain 5 (JMJD5) is a highly conserved and essential 2OG-oxygenase that, thus far, is the only arginyl hydroxylase assigned in eukaryotes. We recently reported that JMJD5 hydroxylase activity is required for DNA replication fidelity, and that pathogenic variants cause replication stress (RS) and genome instability (GIN) in a novel neurodevelopmental disorder. Because of the prevalence of RS and GIN in cancer, and reported roles of JMJD5 in tumorigenesis, we here investigate the impact of JMJD5 cancer mutations on its role in replication fidelity. We describe the structural impact of cancer missense mutations on the hydroxylase activity of JMJD5 and its interaction with RCCD1, an abundant binding partner encoded by a gene associated with susceptibility to a variety of tumour types. We show that the JMJD5:RCCD1 interaction is disrupted by cancer mutations and that the complex is essential for suppressing RS and GIN in tumour cells. Finally, we describe a novel interaction of the complex with RAD51 paralogues and demonstrate the importance of the JMJD5:RCCD1 interaction for normal replication fork restart. Our findings further highlight the importance of protein hydroxylases in fundamental cellular processes and the consequences of JMJD5 and RCCD1 deregulation in human disease.

cancer biology↗

bioRxiv2025

Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. To gain further insight into how the ubiquitin system regulates the DDR, we performed a CRISPR-Cas9 knockout screen focused on E3 ligases and related proteins with the DNA topoisomerase I inhibitor, camptothecin. This uncovered the CTLH ubiquitin E3 ligase complex -- and particularly one of its core subunits, MAEA -- as a critical regulator of the cellular response to single-ended DNA double-strand breaks (seDSBs) and replication stress. In tandem, we identified patients with variants in MAEA who present with neurodevelopmental deficits including global developmental delay, dysmorphic facial features, brain abnormalities, intellectual disability, and abnormal movement. Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart as a likely cause of disease. We propose that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which compromises genome integrity and cell division during development.

genetics↗

bioRxiv2024

MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks

Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.

cell biology↗

bioRxiv2023

Rewiring protein binding specificity in paralogous DRG/DFRP complexes

The Developmentally Regulated GTP-binding (DRG) proteins are an ancient subfamily of GTPases implicated in the regulation of translation and cell growth. In eukaryotes, there are two paralogs: DRG1 and DRG2, both of which have a conserved binding partner called DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively. These binding partners are required for the function of DRGs, including their stabilisation at the protein level. Moreover, DFRPs interact with their respective DRG via a conserved region called the DFRP domain. Despite being highly similar, DRG1 and DRG2 have strict binding specificity for their respective DFRP. Using AlphaFold generated structure models of the human DRG/DFRP complexes, we have biochemically characterised their interactions and identified interface residues involved in determining specificity. This analysis revealed that as few as five mutations in DRG1 are able to switch its binding from DFRP1 to DFRP2. We show how two DRG1 residues in the core of the interface are most important for specifying the interaction with DFRP1 over DFRP2. We also demonstrate that whilst DFRP1 can stimulate the GTPase activity of DRG1, DFRP2 binding cannot. Overall, this work provides new insight into the structural determinants responsible for the binding specificities of the DRG:DFRP translation factor complexes, which are known to be essential for normal development in mice and humans.

biochemistry↗

bioRxiv2023

A protein hydroxylase couples epithelial membrane biology to nucleolar ribosome biogenesis

Jumonji-C (JmjC) ribosomal protein hydroxylases are an ancient class of oxygen- and Fe(II)-dependent oxygenases that spawned the wider JmjC family and Histone Lysine Demethylases (KDMs) in eukaryotes. Myc-induced Antigen (MINA) has been implicated in ribosome biogenesis and was assigned as a nucleolar-localized JmjC histidyl hydroxylase of the large ribosomal subunit protein RPL27A, consistent with reports that it supports cell growth and viability in a variety of tumor cell types. Reported roles in diverse aspects of disease biology may be consistent with additional MINA functions, although the molecular mechanisms involved remain unclear. Here, we describe an extra-nucleolar interaction of MINA with the Hinge domain of the membrane-associated guanylate kinase, MPP6. We show that MINA promotes the expression and membrane localization of MPP6 and that the MINA-MPP6 pathway is required for epithelial tight junction integrity and barrier function. The function of MINA in this novel pathway is suppressed by ribosomal RNA transcription and the nucleolar MINA interactome. In this way, MINA couples epithelial membrane biology to nucleolar ribosome biogenesis. Our work sheds light on how quiescent cells lose adhesion as they switch to proliferative states associated with increased ribosome biogenesis.

cell biology↗

bioRxiv2019

Lysine acetylation promotes PALB2 chromatin association to maintain genome stability

The tumour suppressor PALB2 stimulates error-free repair of DNA breaks, whilst its steady-state chromatin association protects active genes from genotoxic stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/B), commonly known to promote transcriptional activation, acetylate the PALB2 chromatin association motif (ChAM), providing a dynamic regulatory mechanism for PALB2. ChAM acetylation within a cluster of seven lysine residues (7K), detected in the chromatin-enriched fraction in undamaged cells, enhanced its association with nucleosomes while decreasing its non-specific binding to naked DNA. DNA damage triggered a rapid deacetylation of ChAM and a concomitant increase in PALB2 mobility. Significantly, a 7K-null mutation, which hindered ChAM binding to both nucleosomes and DNA, conferred deficiency in DNA repair and hypersensitivity to the anti-cancer drug olaparib. Thus, our study reveals a unique mechanism mediated by KAT2A/B-dependent acetylation of a non-histone protein, which fine-tunes the DNA damage response and hence promotes genome stability.

cell biology↗

bioRxiv2019

Btbd6-dependent Plzf recruitment to Cul3 E3 ligase complexes through BTB domain heterodimerization

The Cul3 adaptor Btbd6 plays crucial roles in neural development by driving the ubiquitin-dependent degradation of promyelocytic zinc finger transcription factor (Plzf). Btbd6 has conserved motifs, BTB-BACK-PHR, and by analogy with other BTB-BACK adaptors, might be expected to bind to Cul3 through the BTB-BACK domain, and to substrate through the PHR domain. However, we now present a mode of adaptor-substrate interaction through heterodimerisation between the normally homodimeric BTB domains of Btbd6 and Plzf. This heterodimerization appears to occur through monomer exchange that is detected only at or near physiological concentrations. The Btbd6-Plzf heterodimer thus formed assembles into a ternary complex with Cul3. In addition we show that the BTB and PHR domains of Btbd6 promote localisation in the nucleus and that the BACK domain contains a nuclear export signal. Our findings support a model whereby Btbd6 moves into and out of the nucleus, iteratively sweeping Plzf into the cytoplasm and enabling complex formation with Cul3 that presents Plzf for ubiquitination.\n\nHighlightsO_LIA general mechanism for recruitment of BTB domain-containing substrates by BTBdomain adaptors for the Cul3 E3 ligase complex\nC_LIO_LINuclear export of the Plzf/Btbd6 complex mediated by a NES within the Btbd6 BACK domain\nC_LIO_LICul3-dependent Plzf ubiquitylation through heterodimerisation of BTB domains on adaptor and substrate by monomer exchang\nC_LI

biochemistry↗