Biochemical basis of human disease-causing actin mutations

Actin isoform specific mutations have been identified as causes for various human diseases. These include twelve missense mutations in γ-nonmuscle actin leading to early onset autosomal dominate nonsyndromic hearing loss and twenty two missense mutations in α-smooth muscle actin leading to thoracic aortic aneurysms and dissections (TAAD). The molecular mechanisms leading to these human pathologies are mainly unknown, principally due to the inability to isolate pure mutant γ-nonmuscle actin and αsmooth muscle actin in quantities required for biochemical analysis. To begin to address these problems, I have individually expressed the human nonmuscle actin isoforms, βand γnonmuscle actin, in a baculovirus expression system and characterized their biochemical properties. Surprisingly, despite a conserved amino acid difference of only 4 residues at or near the N-terminus, Ca-γ-actin exhibits slower monomeric and filamentous biochemical properties than β-actin. In the Mg-form, the difference between the two is smaller. Mixing experiments with Ca-actins reveal the two will readily copolymerize. Calcium bound in the high affinity binding site of γ-actin may cause a selective energy barrier relative to β-actin that retards the equilibration between Gand Fmonomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the γ-actin-rich cochlear hair cell stereocilium where local mM calcium concentrations may exist. In hair cells γ-nonmuscle actin seems to play a central role in stereocilia maintenance. To determine how the deafness causing D51N-γ-mutant actin mutation leads to deafness, I expressed and characterized it in the γ-actin background. The D51N mutation, lethal when cloned into yeast, displayed decreased filament stability and polymerization kinetics of an actin more dynamic than γ-actin. This result suggests that the hearing effects of the γ-actin mutations on the hearing apparatus are not simply caused by an inability to polymerize. The observed increased polymerization rates and