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1.
Cells ; 12(16)2023 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-37626910

RESUMO

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein ß-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the ß-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 an early age of symptom onset. Altogether, the data indicate that increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.


Assuntos
Actinas , Ataxias Espinocerebelares , Humanos , Actinas/genética , Espectrina/genética , Mutação/genética , Mutação de Sentido Incorreto , Ataxias Espinocerebelares/genética
2.
Biochem Biophys Res Commun ; 670: 12-18, 2023 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-37271035

RESUMO

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 α-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 α-actinin-2 actin binding domain (ABD). We investigated the molecular consequences of three ABD localized, HCM-associated variants: A119T, M228T and T247 M. 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 α-actinin-2.


Assuntos
Cardiomiopatias , Cardiomiopatia Dilatada , Cardiomiopatia Hipertrófica , Humanos , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Hipertrófica/genética , Mutação
3.
bioRxiv ; 2023 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-36865188

RESUMO

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein ß-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the ß-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.

4.
J Biol Chem ; 299(3): 102956, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36731793

RESUMO

ß-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 L253P mutation in the cytoskeletal protein ß-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 ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-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 cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.


Assuntos
Actinas , Espectrina , Ataxias Espinocerebelares , Humanos , Actinas/genética , Actinas/metabolismo , Descoberta de Drogas , Neurônios/metabolismo , Espectrina/metabolismo , Ataxias Espinocerebelares/tratamento farmacológico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
5.
Sci Rep ; 12(1): 1726, 2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-35110634

RESUMO

Recent structural studies of ß-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the ß-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated ß-spectrin fails to rescue lethality resulting from a ß-spectrin loss-of-function allele, indicating that the N-terminus is essential to ß-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 ß-spectrin and spectrin-related disease proteins.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neurônios/metabolismo , Espectrina/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Masculino , Mutação , Plasticidade Neuronal , Neurônios/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espectrina/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
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