Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Abstract

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.actin | myosin II | smooth muscle | thoracic aortic aneurysms | vascular disease T horacic aortic aneurysms and dissections (TAAD) are the 18th most common cause of death in individuals in the United States (1). The high degree of mortality is partly due to the fact that aneurysms tend to be asymptomatic until a lifethreatening acute aortic dissection occurs. Familial TAAD is an autosomal dominant disorder with variable penetrance, which is characterized by enlargement or dissection of the thoracic aorta (reviewed in 2). The most prevalent genetic cause of familial TAAD, responsible for ∼15% of all cases, are mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2. More than 40 mutations in ACTA2 have been identified to date (3-5). Intriguingly, ACTA2 mutations also differentially predispose individuals to occlusive vascular diseases, such as premature coronary artery disease and strokes (6). ACTA2 mutations thus can lead to either dilation of large elastic arteries like the aorta or occlusion of smaller muscular arteries.SM α-actin is the most abundant protein in vascular smooth muscle cells, constituting ∼40% of the total protein and ∼70% of the total actin, with the rest composed of β-and γ-cytoplasmic actin. Actin is critical for contraction and force production by smooth muscle cells, as well as for their proliferation and migration. Dissected aortas show s...

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

“…The R258 residue, which corresponds to amino acid R256 in the actin protein due to posttranslational processing that removes the N-terminal Met and Cys residues (18), lies at the interface between the two strands of filamentous actin. Mutation of R258 to C or H is understood to disrupt allosteric communication to binding sites on the surface of actin (17,19). Intermediate effects on SM α-actin functions were observed in 1:1 mixtures of WT and R258C proteins, consistent with anticipated disruption of actin-dependent properties in heterozygous smooth muscle cells of patients harboring this mutation.…”

“…Effects of the mutation on fibroblast motility and contraction are remarkably consistent with predictions from biophysical studies on expressed vascular actins, but not unsurprisingly so. In vitro studies with expressed actins showed that the R258C mutation leads to defects in multiple facets of SM α-actin properties, including reduced actin filament stability and less productive interactions with smooth muscle myosin (17). However, in 50:50 mixtures of WT and R258C, polymerization rates were intermediate and skewed more toward the rate observed for WT SM α-actin.…”

Mutant vascular actin is a TAAD misbehaving

“…Mutations in α-smooth muscle isoactin (αSMA) cause vascular defects leading primarily to thoracic aneurysm and dissection (TAAD). In PNAS, Lu et al (3) addresses two of these mutations, primarily R258C and secondarily R258H, to try to gain insight into how changes induced by these mutations lead to TAAD by affecting actin function. Vascular material from an affected individual carrying the R258C mutation shows that the muscle layer of the vessel wall is abnormal, with disorganized structure and a dearth of contractile filaments compared with normal smooth muscle, suggesting abnormal filament assembly, protein instability, or failure of the assembled cytoskeleton to withstand the forces imposed on it by myosin during contraction of the muscle (4,5).…”

“…Lu et al (3) show that the R258C mutation in smooth muscle actin resulted in a decreased rate of polymerization compared with that of WT actin, a less stable filament, and increased susceptibility to severing by the filament-severing protein cofilin (10). Earlier yeast work showed that polymerization rates and filament stability were similarly affected by the corresponding R258H mutation.…”

“…Although in the yeast paper, cofilin-severing was not studied, earlier studies with two other TAAD mutations at positions 115 and 116 in a helix leading to the interface involving R258 produced filament weakening and increased cofilin susceptibility. Lu et al (3) emphasize that the mutation produces effects far distant from the actual site of the mutation, implicating a high degree of protein allostery. Earlier, Malloy et al (7) and later Rubenstein and Wen (2) proposed an allosteric model in which a helix containing residues 115 and 116 acted as an allosteric conduit connecting the filament surface with the triad of residues containing R258, E197, and 115 on the other strand, which act as a major determinant of filament stability.…”