Subdomain Location of Mutations in Cardiac Actin Correlate with Type of Functional Change

Determining the molecular mechanisms that lead to the development of heart failure will help us gain better insight into the most costly health problem in the Western world. To understand the roles that the actin protein plays in the development of heart failure, we have taken a systematic approach toward characterizing human cardiac actin mutants that have been associated with either hypertrophic or dilated cardiomyopathy. Seven known cardiac actin mutants were expressed in a baculovirus system, and their intrinsic properties were studied. In general, the changes to the properties of the actin proteins themselves were subtle. The R312H variant exhibited reduced stability, with a Tm of 53.6°C compared to 56.8°C for WT actin, accompanied with increased polymerization critical concentration and Pi release rate, and a marked increase in nucleotide release rates. Substitution of methionine for leucine at amino acid 305 showed no impact on the stability, nucleotide release rates, or DNase-I inhibition ability of the actin monomer; however, during polymerization, a 2-fold increase in Pi release was observed. Increases to both the Tm and DNase-I inhibition activity suggested interactions between E99K actin molecules under monomer-promoting conditions. Y166C actin had a higher critical concentration resulting in a lower Pi release rate due to reduced filament-forming potential. The locations of mutations on the ACTC protein correlated with the molecular effects; in general, mutations in subdomain 3 affected the stability of the ACTC protein or affect the polymerization of actin filaments, while mutations in subdomains 1 and 4 more likely affect protein-protein interactions

Summary of the intrinsic properties of ACTC mutant proteins related to the development of cardiomyopathies in humans.

†<p>labeled with tetramethylrhodamine.</p>*<p><i>p</i><0.05 by <i>t</i>–test.</p>**<p><i>p</i><0.15 by <i>t–test.</i></p><p>All values are the average of at least three replicates, showing the standard deviation of each.</p

Correlation of location and effects of ACTC mutations.

<p>The locations of ACTC mutations associated with cardiomyopathies are shown in black (HCM) and red (DCM) letters. The regions of molecular effects are circled and spacefilling models presented in red (protein stability changes), green (actin polymerization changed), and blue (no significant intrinsic property changes). Those residues shown in grey have not been characterized. Coordinate data for ATP-bound TMR-actin (1J6Z) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036821#pone.0036821-Graceffa1" target="_blank">[39]</a> were visualized using PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036821#pone.0036821-Schrodinger1" target="_blank">[40]</a>.</p

Nucleotide release from monomeric ACTC variant proteins.

<p>The release of ε-ATP from WT ACTC followed a 2^nd-order decay model (•). The R312H ACTC protein showed faster nucleotide release kinetics (Ο) that fitted the 2^nd-order decay model with a significantly different fast decay rate.</p

Cardiac actin mutations related to cardiomyopathies.

<p><b><i>A</i></b><i>.</i> The protein structure of actin (PDB 1J6Z) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036821#pone.0036821-Graceffa1" target="_blank">[39]</a> showing the locations of mutations related to HCM (in green spacefilling) or DCM (in red spacefilling) and bound ATP (sticks) visualized using PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036821#pone.0036821-Schrodinger1" target="_blank">[40]</a>. The D-loop of actin is highlighted with a dashed circle. ACTC substitution mutations related to HCM shown are: H88Y, F90del, R95C, E99K, P164A, Y166C, A230V, S271F, A295S, R312C, A331P, and M305L. The two ACTC mutants associated with DCM are R312H and E361G. The amino acid numbers listed are based on the sequence of the protein after posttranslational processing of the N-terminus, which removes the first two amino acids of the nascent polypeptide. Note that mutations at Arg-312 are associated with both HCM and DCM. <b><i>B</i></b><i>.</i> Purification of baculovirus expressed ACTC mutant protein. <i>Sf</i>9 cells were infected with recombinant baculovirus expressing the A230V ACTC mutant protein at an MOI of 1 for 72 hours. Shown is a 10% SDS-PAGE of samples taken throughout the purification: lane 1, crude lysate; lane 2, supernatant fraction of lysates; lane 3, lysate following filtration and applied to a DNase-I affinity column; lane 4, purified A230V ACTC protein eluted from the DNase-I affinity column. Arrows indicate the position of actin and the polyhedrin protein expressed by the baculovirus in <i>Sf</i>9 cells.</p

Intrinsic cardiac actin mutant monomer properties.

<p><b><i>A</i></b><i>.</i> The melting temperature of WT ACTC (•) (black circle) was similar to that of TMR-modified E99K ACTC (•) (grey circle) (56.8±1.3°C and 56.7±0.23°C, respectively). Unmodified E99K ACTC (Ο) had a melting temperature of 68.8±0.35°C. <b><i>B</i></b><i>.</i> Correlation of measured <i>T</i>_m and DNase-I IC₅₀ values. The values for five of the tested ACTC proteins fitted a single exponential decay equation (y =  y₀+A<i>e</i>^-bx, where y₀ was 53.4±0.15, A was 19.27±2.65, and b was 0.131±0.01 (S.E.)) with an R² of 0.998). Both ACTC variants with measured <i>T</i>_m values higher than WT displayed higher DNase-I IC₅₀ values (E99K ACTC is not shown), while the A230V ACTC displayed a lower Tm value with an IC₅₀ similar to WT ACTC. <b><i>C.</i></b> DNase-I inhibition curves with WT ACTC protein show little change over time (day 2, Ο; day 5, ?; day 7, ▿). Data points are averages of triplicate measurements, with error bars showing standard deviation. <b><i>D.</i></b> DNase-I inhibition by R312H ACTC protein shows a higher initial IC-50 value (day 2, ?). At day 7 (Ο), the IC₅₀ data could not be fitted because it had increased.</p