Biology subjects

Murphy, A. M.

Publications and source records attributed to Murphy, A. M..

3 recordsLinked to original sources

bioRxiv2024

Investigating the interactions of the cucumber mosaic virus 2b protein with the viral 1a replicase component and the cellular RNA silencing factor Argonaute 1

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b proteins host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient it induces reinforcement of antiviral silencing by AGO2, and triggers increased resistance against aphids, CMVs insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein by sequestering sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated and red and green fluorescent protein fusions used to investigate subcellular colocalization with AGO1 and the 1a protein, and the effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b proteins interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b proteins ability to interact with AGO1, the 1a protein and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

plant biology↗

bioRxiv2023

Strain-specific differences in the interactions of the cucumber mosaic virus 2b protein with the viral 1a and host Argonaute 1 proteins

Abstract/SummaryThe cucumber mosaic virus (CMV) 2b protein is a potent counter-defense protein and symptom determinant that inhibits antiviral silencing by titration of short double-stranded RNAs. Expression of the 2b protein from the CMV Subgroup IA strain Fny-CMV in transgenic Arabidopsis thaliana plants disrupts microRNA-mediated cleavage of host mRNAs by binding ARGONAUTE 1 (AGO1), leading to symptom-like phenotypes. This also triggers AGO2-mediated resistance against CMV and strong resistance to CMVs aphid vectors, which would be deleterious to viral fitness. However, in authentic viral infections the Fny-CMV 1a protein modulates 2b-AGO1 interactions, which inhibits induction of AGO2-mediated virus resistance and resistance to aphid vectors. Contrastingly, the 2b proteins encoded by the Subgroup II LS-CMV strain or the recently discovered Subgroup IA strain Ho-CMV induce no apparent symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation and co-immunoprecipitation showed that the Fny-CMV and Ho-CMV 2b proteins interact with the Fny-CMV and LS-CMV 1a proteins whilst the CMV-LS 2b protein does not. However, the Fny-CMV, Ho-CMV and LS-CMV 2b proteins all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the host cell nucleus and cytoplasm, the corresponding AGO1-2b complexes for LS-CMV and Ho-CMV accumulated almost exclusively in nuclei. AGO2 transcript accumulation was used to assess the inhibition of AGO1-mediated miRNA-regulated mRNA cleavage. While Fny-CMV 2b induced a five-fold increase in AGO2 accumulation, the LS-CMV and Ho-CMV 2b proteins induced only two-fold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization sites of 2b-AGO1 complexes influences the degree to which a 2b protein can inhibit microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover.

plant biology↗

bioRxiv2023

The HCM-Linked Mutation Arg92Leu in TNNT2 Allosterically Alters the cTnC-cTnI Interface and Disrupts the PKA-mediated Regulation of Myofilament Relaxation

BackgroundImpaired left ventricular relaxation, high filling pressures, and dysregulation of Ca2+ homeostasis are common findings contributing to diastolic dysfunction in hypertrophic cardiomyopathy (HCM). Studies have shown that impaired relaxation is an early observation in the sarcomere-gene-positive preclinical HCM cohort which suggests potential involvement of myofilament regulators of relaxation. Yet, a molecular level understanding of mechanism(s) at the level of the myofilament is lacking. We hypothesized that mutation-specific, allosterically mediated, changes to the cardiac troponin C-cardiac troponin I (cTnC-cTnI) interface can account for the development of early-onset diastolic dysfunction via decreased PKA accessibility to cTnI. MethodsHCM mutations R92L-cTnT (Arg92Leu) and {Delta}160E-cTnT (Glu160 deletion) were studied in vivo, in vitro, and in silico via 2D echocardiography, western blotting, ex vivo hemodynamics, stopped-flow kinetics, time resolved fluorescence resonance energy transfer (TR-FRET), and molecular dynamics simulations. ResultsThe HCM-causative mutations R92L-cTnT and {Delta}160E-cTnT result in different time-of-onset of diastolic dysfunction. R92L-cTnT demonstrated early-onset diastolic dysfunction accompanied by a localized decrease in phosphorylation of cTnI. Constitutive phosphorylation of cTnI (cTnI-D23D24) was sufficient to recover diastolic function to Non-Tg levels only for R92L-cTnT. Mutation-specific changes in Ca2+ dissociation rates associated with R92L-cTnT reconstituted with cTnI-D23D24 led us to investigate potential involvement of structural changes in the cTnC-cTnI interface as an explanation for these observations. We probed the interface via TR-FRET revealing a repositioning of the N-terminus of cTnI, closer to cTnC, and concomitant decreases in distance distributions at sites flanking the PKA consensus sequence. Implementing TR-FRET distances as constraints into our atomistic model identified additional electrostatic interactions at the consensus sequence. ConclusionThese data indicate that the early diastolic dysfunction observed in a subset of HCM is likely attributable to structural changes at the cTnC-cTnI interface that impair accessibility of PKA thereby blunting {beta}-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.

biophysics↗