Allele-specific effects of thoracic aortic aneurysm and dissection alpha-smooth muscle actin mutations on actin...

Twenty-two missense mutations in ACTA2, which encodes α-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 α-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 actin-binding 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.