Disease mechanisms of spectrin superfamily proteins

Abstract

Spectrin superfamily proteins are a group of evolutionarily linked proteins that share in actin crosslinking functions. Characterized by an N-terminal actin binding domain (ABD), comprised of tandem calponin homology (CH) domains, a variable number of spectrin repeat domains, and in some cases an EF hand motif, these cytoskeletal proteins are an integral feature of the submembranous cellular structure. Mutations within the ACTN2 and SPTBN2 genes that encode α-actinin-2 and β-III-spectrin, respectively are known to cause disease. Predominately expressed in cardiomyocytes of the heart, α-actinin-2 mutations localizing to the ABD are linked to cardiomyopathy, leading to heart failure and sudden cardiac death. ABD localized β-III-spectrin mutations cause the rare neurodegenerative disease, spinocerebellar ataxia type 5 (SCA5). Previous studies of a L253P mutation within the β-III-spectrin ABD revealed an increased actin-binding affinity. Positioning of the L253P mutation to the interface of CH1 and CH2 was demonstrated to promote a conformational opening, leading to high affinity actin interactions. These studies investigate the molecular consequences of nine additional β-III-spectrin ABD-localized, SCA5 missense mutations (V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R) and three α-actinin-2 ABD-localized, cardiomyopathy missense mutations (A119T, M228T, and T247M). Importantly, all β-III-spectrin and α-actinin-2 mutations localizing the CH1-CH2 interface increased actin-binding affinity, while the actin-binding surface localized α-actinin-2 mutation, A119T, decreased actin-binding affinity. Using biochemical and biophysical approaches, we demonstrate that the mutant β-III-spectrin and α-actinin-2 ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all ABD mutations are destabilizing, suggesting structural disruptions to the ABDs. Additional in vivo studies of the β-III-spectrin mutations indicate an accumulation of protein within neurons, likely a result of increased actin binding-affinity. Altogether, the data indicate aberrant actin-binding affinity is a shared molecular consequence of ABD localized mutations of spectrin superfamily proteins.