Biology subjects

Avery, A. W.

Publications and source records attributed to Avery, A. W..

4 recordsLinked to original sources

bioRxiv2023

Cardiomyopathy-Associated Variants Alter the Structure and Function of the α-Actinin-2 Actin-Binding Domain

Hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM) are characterized by thickening, thinning, or stiffening, respectively, of the ventricular myocardium, resulting in diastolic or systolic dysfunction that can lead to heart failure and sudden cardiac death. Recently, variants in the ACTN2 gene, encoding the protein -actinin-2, have been reported in HCM, DCM, and RCM patients. However, functional data supporting the pathogenicity of these variants is limited, and potential mechanisms by which these variants cause disease are largely unexplored. Currently, NIH ClinVar lists 34 ACTN2 missense variants, identified in cardiomyopathy patients, which we predict are likely to disrupt actin binding, based on their localization to specific substructures in the -actinin-2 actin binding domain (ABD). We investigated the molecular consequences of three ABD localized, HCM-associated variants: A119T, M228T and T247M. Using circular dichroism, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all three mutations are destabilizing, suggesting a structural disruption. Importantly, A119T decreased actin binding, and M228T and T247M cause increased actin binding. We suggest that altered actin binding underlies pathogenesis for cardiomyopathy mutations localizing to the ABD of -actinin-2.

biochemistry↗

bioRxiv2023

Increased actin binding is a shared molecular consequence of numerous spinocerebellar ataxia mutations in β-III-spectrin

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein {beta}-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the {beta}-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with early age of symptom onset. Altogether, the data indicate increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

biochemistry↗

bioRxiv2022

Identification of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia

{beta}-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells, and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the cytoskeletal protein {beta}-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small molecule actin-binding modulators of the L253P mutant {beta}-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P {beta}-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay, we screened a 2,684-compound library of FDA-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous co-sedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a SCA5-specific therapeutic. Furthermore, our validated FRET-based in vitro HTS platform is poised for screening large compound libraries for {beta}-III-spectrin ABD modulators.

biophysics↗

bioRxiv2021

Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction

Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dyneins function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the clusters size of ionotropic glutamate receptor (iGluR), and an increase in the amplitude and frequency of mEJPs. However, PI(4,5)P2 levels do not affect iGluR clustering and dynein does not affect the protein levels of iGluR subunits at the NMJ, suggesting a separate, transport independent function for dynein in iGluR cluster organization. As dynein punctae closely associate with iGluR clusters, we propose that dynein physically tethers iGluR clusters at the postsynaptic membrane to ensure proper synaptic transmission.

cell biology↗