Biology subjects

Plevin, M. J.

Publications and source records attributed to Plevin, M. J..

7 recordsLinked to original sources

bioRxiv2025

Coenzyme A homeostasis regulates hypoxic signalling via UBFD1in triple-negative breast cancer

Hypoxia-inducible factors (HIFs) orchestrate cellular responses to oxygen deprivation and are frequently activated in triple-negative breast cancer (TNBC), driving metastasis and poor prognosis1,2. However, the full mechanisms of how HIFs are activated are not completely understood. It has been long noted that certain tumours have decreased levels of coenzyme A compared to surrounding tissues3,4, but the functional consequences of this remain unknown. Here, we identify Coenzyme A Synthase (CoAsy) as a novel regulator of HIF signalling. We demonstrate that CoAsy loss stabilises HIF-1 and HIF-2 independently of the canonical oxygen sensing pathway. Proximity-labelling proteomics revealed that CoAsy deficiency disrupts the association between HIF-1 and the proteasome through UBFD1, a novel CoA-binding protein that scaffolds HIF for degradation. UBFD1s CoA-dependent interaction with HIF- is mediated by its PH domain, which undergoes CoAlation, an understudied post-translational modification. COASY loss of heterozygosity occurs in [~]1/3 of breast cancer patients and correlates with increased HIF activity, lack of hormone receptor expression and poor patient outcome. In vivo studies confirm that restoring CoAsy expression in tumours suppresses metastasis to the lung. These findings uncover a critical metabolic checkpoint regulating hypoxic signalling and identify CoAsy as a potential biomarker and therapeutic target in aggressive breast cancers.

molecular biology↗

bioRxiv2025

An open-sourced 3D printable microscope with a large CNC stage

3D printed laboratory tools are a fast-developing field, and microscopes in particular have seen several high-quality projects reported in recent years. However, currently available projects for 3D printed microscopes have not been optimised for molecular biology applications. We present an open-source, 3D-printable optical microscope that has been specifically designed for affordability, ease of use, and versatility in molecular biology applications, particularly for fluorescence detection, time lapse imaging, and higher throughput scanning of microtitre plates. The microscope integrates a large computer numerically controlled (CNC) stage, allowing precise scanning of wells in standard microtiter plates, and is equipped with a Raspberry Pi HQ camera module and customizable optical components, including standard microscope objectives. The modular design allows for a range of illumination setups, such as a 3 W blue LED for basic epifluorescence, and supports additional modifications, including dark field or RGB matrix lighting. The design opens new possibilities for cheap home-built microscopes for bioscience applications that are modular and easily adaptable for specific user needs.

biophysics↗

bioRxiv2024

A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage

CO2 fixation is commonly limited by inefficiency of the CO2-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO2 in phase-separated condensates called pyrenoids, which complete up to one-third of global CO2 fixation. Condensation of Rubisco in pyrenoids is dependent on interaction with disordered linker proteins that show little conservation between species. We developed a sequence-independent bioinformatic pipeline to identify linker proteins in green algae. We report the linker from Chlorella and demonstrate that it binds a conserved site on the Rubisco large subunit. We show the Chlorella linker phase separates Chlamydomonas Rubisco and that despite their separation by [~]800 million years of evolution, the Chlorella linker can support the formation of a functional pyrenoid in Chlamydomonas. This cross-species reactivity extends to plants, with the Chlorella linker able to drive condensation of some native plant Rubiscos in vitro and in planta. Our results represent an exciting frontier for pyrenoid engineering in plants, which is modelled to increase crop yields.

plant biology↗

bioRxiv2023

A C-terminal HaloTag Impairs AGO2 Function

A full understanding of RNA silencing requires appropriate molecular biology tools to explore the roles of Argonaute 2 (AGO2) and the RNA-induced silencing complex (RISC). Approaches relying on affinity tagging and antibodies have important limitations that can lead to artificial results. Both the N- and C-terminal domains of AGO2 have been shown to be important for correct activity and yet the consequences of appending tags to either terminus have not been fully investigated. N-terminal tags are frequently used to study AGO2 biology. Recently, an N-terminal HaloTag-Ago2 fusion was reported and examined in mice. While the versatile HaloTag provided new opportunities to study RISC biology, the tagged construct showed certain activity changes compared to unmodified AGO2. CRISPaint, a new CRISPR-Cas9 technique, permits the creation of endogenous C-terminal tag fusions. We used CRISPaint to generate the first reported recombinant AGO2 construct with a C-terminal tag: an endogenous C-terminal HaloTag fusion to AGO2 (AGO2HALO) in human (A549) cells. We found that the AGO2HALO fusion protein has a reduced capacity to interac with the key protein binding partner TNRC6A and that the C-terminal HaloTag does not affect cell viability. However, the AGO2HALO fusion significantly impairs RNA cleavage and RNA silencing activity compared to control cells and reduces nuclear localisation of the fusion protein. Using plasmid constructs and transient transfection, we compared AGO2 tagged with EGFP at the N- or C-terminus in siRNA and miRNA reporter gene assays, and cellular localisation. N-terminally tagged AGO2 functioned and localised similarly to WT untagged AGO2, whereas C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing and exhibited poor nuclear and P-body localisation. We conclude that the fusion of a C-terminal HaloTag to AGO2 is not appropriate for studying AGO2 and RISC. Our results assert the importance of comprehensively validating recombinant tagging strategies to ensure that any experimental results generated do not arise from, or are not obscured by, critical functional defects.

molecular biology↗

bioRxiv2023

Fundamental steps in Minichromosome Maintenance complex assembly are conserved from archaea to eukaryotes

Minichromosome maintenance (MCM) proteins are the replicative helicase in both archaea and eukaryotes. Recent cryo-EM studies have defined key steps in the assembly pathway of the eukaryotic MCM2-7 complex and determined roles for each subunit. While archaeal MCMs are simpler in composition, we know little about how these homohexameric MCMs assemble. Investigation of archaeal MCMs has largely focussed on proteins from thermophilic organisms, which typically form robust oligomers under ambient experimental conditions, preventing dissection of their assembly pathway. Here, we describe an uncharacterised MCM from the mesophilic archaeon Mancarchaeum acidophilum (MacMCM). A 3D structure of MacMCM reveals subunit-subunit interfaces that are more similar to yeast MCM2-7 than previously studied MCMs from thermophilic archaea. We show that assembly of a MacMCM homohexamer on DNA proceeds via comparable steps to MCM2-7. These results reveal an ancient mechanism underlies assembly of the MCM complex, which is conserved from archaea to eukaryotes.

biochemistry↗

bioRxiv2020

Periscope Proteins are variable length regulators of bacterial cell surface interactions

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly-available long-read genome sequencing data, we propose this new class could contain over 50 distinct proteins, including those implicated in host colonisation and biofilm formation by human pathogens. In large multi-domain proteins, sequence divergence between adjacent domains appears to reduce inter-domain misfolding. Periscope Proteins break this "rule", suggesting their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria and the immune system.

microbiology↗

bioRxiv2019

Post-translational epigenetics: PRMT7 regulates RNA-binding capacity and protein stability to control Leishmania parasite virulence

RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine MethylTransferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and stability of{delta} -amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation upon RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

genetics↗