Biology subjects

O'Reilly, N.

Publications and source records attributed to O'Reilly, N..

8 recordsLinked to original sources

bioRxiv2024

Evolution and inhibition of the FIKK effector kinase family in P. falciparum

Among the [~]200 Plasmodium species that infect vertebrates, six infect humans. Of these, P. falciparum causes >95% of all [~]500,000 annual fatalities. Phylogenetically, P. falciparum belongs to the Laverania subgenus, a group of Plasmodium species that infect great apes. Common to Laverania species is the family of FIKK kinases. One million years ago, a single FIKK kinase conserved in all Plasmodium species gained an export element in the Laverania subgenus and expanded into the family of [~]20 atypical FIKK kinases, most of which are exported into the host cell. The fikk genes are conserved in syntenic loci across the Laverania, arguing for a rapid expansion controlling important functions in host cell remodelling and pathogenesis. We provide evidence that the FIKK paralogues evolved specific and mutually exclusive phosphorylation motif preferences, conserved across their Laverania orthologues, in a short evolutionary timeframe. Surprisingly, we find that FIKK13 has evolved exclusive tyrosine-phosphorylation preference, which was thought to be absent in Plasmodium species. Combining a crystal structure with AlphaFold2 predictions, we identify residues that determine kinase-specificity within the FIKK family in a fast-evolving flexible loop. Finally, we show that all expressed members of the FIKK kinase family can be chemically inhibited in vitro using a single compound. Such a pan-specific inhibitor of this kinase family important for virulence could reduce the ability of the parasite to gain escape-mutations and resistance.

microbiology↗

bioRxiv2023

Dichotomy of neutralizing antibody, B cell and T cell responses to SARS-CoV-2 vaccination and protection in healthy adults

Heterogeneity in SARS-CoV-2 vaccine responses is not understood. Here, we identify four patterns of live-virus neutralizing antibody responses: individuals with hybrid immunity (with confirmed prior infection); rare individuals with low responses (paucity of S1-binding antibodies); and surprisingly, two further groups with distinct serological repertoires. One group - broad responders - neutralize a range of SARS-CoV-2 variants, whereas the other - narrow responders - neutralize fewer, less divergent variants. This heterogeneity does not correlate with Ancestral S1-binding antibody, rather the quality of the serological response. Furthermore, IgDlowCD27-CD137+ B cells and CCR6+ CD4+ T cells are enriched in broad responders before dose 3. Notably, broad responders have significantly longer infection-free time after their third dose. Understanding the control and persistence of these serological profiles could allow personalized approaches to enhance serological breadth after vaccination.

immunology↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

biochemistry↗

bioRxiv2020

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

Homologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, while RFS-1/RIP-1 acts as a chaperone to promote 3 to 5 filament growth via highly dynamic engagement with 5 filament ends. Inhibiting ATPase or mutation in RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

biophysics↗

bioRxiv2020

Identification of Chlorophyll a-b Binding Protein AB96 as a novel TGFbeta1 binding agent.

The discovery of compounds and proteins from plants has greatly contributed to modern medicine, especially in malaria where both quinine and artemisinin have been the cornerstone of therapeutics. Here we describe the first known plant-derived cytokine binding agent. Chlorophyll a-b binding protein AB96, present to varying levels in the leaves of all chlorophyll-containing plants, binds to active TGF{beta}1. Active TGF{beta}1 contributes to the pathology of many infectious and neoplastic diseases and therefore, by inhibiting these processes, chlorophyll a-b binding protein opens up new approaches with therapeutic potential.

immunology↗

bioRxiv2020

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

ABSTRACTPPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1’s hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified numerous fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

molecular biology↗