Allele-specific Effects of Thoracic Aortic Aneurysm and Dissection α-Smooth Muscle Actin Mutations on Actin Function

Abstract

Twenty-two missense mutations in ACTA2, which encodes ␣-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human ␣-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actinbinding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection.

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

“…Interestingly, Lys-113 is part of a helix, Lys-113-Thr-125, that contains two other TAAD mutations we have previously characterized, N115T and R116Q, in addition to Lys-118, the site of two deafness-causing mutations (14,45). If our hypothesis is correct, the mutations become part of a pathogenic module that, as a unit, can affect F-actin conformation.…”

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

“…The effect of vascular-disease causing mutations in actin on regulation by cofilin was recently studied by our group 27 . Based on our initial findings, Aip1p, an actin binding protein that facilitates cofilin-dependent actin severing, was investigated.…”

Insights into the effects of disease‐causing mutations in human actins

“…Consistent with this observation, based on electron microscopic example of negativestained samples, only short fragments could be obtained following substantial dialysis against G-buffer which immediately polymerized to actin filaments upon re-addition of polymerization-inducing salts. The R116Q mutation displays an elevated critical concentration, retarded polymerization kinetics, a pronounced polymerization lag phase, increased rates of depolymerization, and a hypersensitivity to cofilin severing [Bergeron et al, 2011]. The mutations at 116, 117, and 370 also produced many common effects in vivo.…”

“…Nineteen of these were originally described, and these account for about 15% of the genetic causes of this disease [Guo et al, 2007]. Initial studies focused on N115T, R116Q, and R256H [Bergeron et al, 2011;Malloy et al, 2012]. Based on these findings, they hypothesized that a helical element beginning on the surface of the filament at K118 extended inward terminating at residue K113 at the strand-strand interface.…”

“…Subsequent work with these alleles did play a major role in defining some of these interactions, such as fimbrin [Holtzman et al, 1994] and myosin [Miller et al, 1996] binding sites. Some of these mutated clusters also occurred at the sites of pathogenic mutations when they were ultimately identified, such as residues 116 to 118 in [Zhu et al, 2003;Bergeron et al, 2011;Johnston et al, 2013] and Residues E99 and E100 [Bookwalter and Trybus, 2006;Debold et al, 2010]. In one respect, it is difficult to use the Wertman strains to study disease phenotypes, since, with them, one has the potential contributions of other mutated residues in a cluster affecting the phenotype.…”