Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.
The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases.DOI: http://dx.doi.org/10.7554/eLife.01603.001
Myosin-10 is an actin-activated ATPase that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration and mitotic spindle maintenance. The myosin-10 duty-cycle ratioi.e. the fraction of time the motor remains tightly bound to actin during its total ATPase cycle -from previous biochemical studies are inconclusive, thus whether this myosin displays intermediate or high duty ratio is still under debate. To study this motor protein's mechano-chemical properties we have used a recombinant, truncated form of myosin-10 consisting of the first 940 amino acids, followed by a GCN4 leucine zipper motif to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules (~87%) are dimeric with a head-to-head contour distance of~50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of 150 -400 nm s À1 . Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (~13 s À1 ) is similar to the maximum ATPase activity (~12 -14 s À1 ) and, therefore, contributes to rate-limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (~0.5 pN) myosin-10 interacts intermittently with actin and produces a working stroke of~17 nm, composed of an initial 15 nm and subsequent 2 nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six,~35 nm, steps per binding interaction. Here we describe the kinetics and mechanics of myosin-10, interrogating bulk and single molecule biophysical/biochemical properties to further our understanding of its ATP-driven, motor mechanism and how this relates to its cellular functions.
The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases.
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