Mutant vascular actin is a TAAD misbehaving.

Actin plays a number of important roles in mammalian cells. Mammals express six different isoforms of actin in a tissueand temporal-specific manner (1). Mutations in each isoform have been linked to specific diseases in humans, and in most instances a mutation in one of the two copies of the gene is sufficient to cause the disease (2). 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 R258Cmutation 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). However, the advanced disease stage at the time of tissue resection prevents early stages of the disease from being addressed. This same site has been mutated in other actin isoforms, in each case leading to a disease. An R256 mutation to either H or L in α-skeletal muscle isoactin causes nemaline myopathy and an R256W mutation in β-nonmuscle isoactin causes Baraitser–Winter Syndrome (2). Normal blood vessel wall function requires normal kinetics of actin polymerization. The filaments must have normal enough intermonomer contacts to allow formation of structures with normal dynamics. The structure of the filament must allow proper control by the numerous regulatory proteins with which it interacts. Finally, the interaction of smooth muscle myosin with the actin filament must produce enough contractile force for normal regulation of the circulation. An actin mutation can potentially affect any of these parameters. Gaining insight into the effects of these mutations at a biochemical level requires sufficient pure mutant actin isoform for biochemical experimentation. The usual approach incorporates a model expression system in which one can express a mutated protein and isolate it in a chemically and isoform pure state. For studying vascular smooth muscle function, it would be best to use smooth muscle proteins, including αSMA. These proteins have evolved together either within a specific cell type or within an organism to maximize functional efficiency of that particular system. Disease could result from quantitative differences in the output of these systems, so using model hybrid systems based on protein homology could produce misleading data in terms of gaining insight into a disease process. Because of technological problems in obtaining mutated vascular smooth muscle actin, initial studies into the effect of TAADcausing actin mutations first used the budding yeast Saccharomyces cerevisiae. Actin from budding yeast is 86% identical to αSMA in terms of amino acid structure (6), and all of the sites at which disease-causing mutations occur in the smooth muscle actin are conserved in the yeast protein. One of the mutations studied with yeast was the R258H mutation (R256H in the yeast nomenclature). Malloy et al. (7) demonstrated that this mutation produced adverse cytoskeletal behavior in vivo and that the mutated actin formed less stable filaments and was subject to abnormal regulation by yeast formin Bni1p. Further observation suggested that R258 was part of an interstrand ionic system within the filament interior, also containing K115 and E197, which could be allosterically regulated by the binding of proteins, such as Arp2/3 and formin, on the filament surface (8). Lu et al. (3) make a significant achievement in establishing an in vitro system for studying the effects of TAAD-causing mutations in αSMA per se (Fig. 1). Previous attempts to express this isoactin in baculovirus-infected cells had been unsuccessful. This was particularly surprising because α-cardiac, α-skeletal, and βand γ-nonmuscle isoactins had been expressed using this system. However, by using a system for expressing toxic actins in Dictyostelium (9), Lu et al. produced enough WT and mutant αSMA to carry out their experiments. This achievement not only allowed the investigators to establish the effects of the mutation on the actin actually present in the diseased cell, but it also provided an opportunity to compare the results obtained with yeast vs. smooth muscle actin in cases where similar experiments were performed. 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 Fig. 1. Location of the TAAD mutations at R258 in actin. In A and B, the site of the mutation is shown in red. (A) Monomeric or G-actin. The four subdomains of the protein are denoted. The area of the monomer to which profilin binds is denoted. (B) A trimer of actin monomers in the context of an actin filament. Each monomer is denoted by an m number. The binding site for myosin bridges the gap between two longitudinally apposed monomers.