Myosin is reversibly inhibited by S-nitrosylation

Abstract

Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO* donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO* or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions.

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+ Sensitivity in Intact Cardiomyocytes

“…In a previous study, we demonstrated an increase in nitrite and protein-SNO in vertebrate skeletal muscle following exercise and characterized the effects of GSNO-dependent S-nitrosylation on myosin ATPase in vitro (41). Maximum inhibition was 20-30%, attributable to a small population of SNO-cysteines, and it was readily reversible.…”

Diverse post-translational modifications of the pannexin family of channel-forming proteins

“…Targeted modification of cysteine thiols by the bioactive gas nitric oxide (NO) promotes reversible S-nitrosylation of a number of membrane proteins, having distinct effects on protein function. [20][21][22][23][24][25] S-nitrosylation of Panx1 can occur at cysteines 40 and 346, with modification of both residues imparting inhibition of Panx1 channel currents and the ability of the channels to release ATP. 25 To date, however, the ability of Panx2 and Panx3 to be modified by S-nitrosylation remains to be determined.…”

Uterine Smooth MuscleS-Nitrosylproteome in Pregnancy

“…Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain (Evangelista et al, 2010). Inhibition of the Mg 2ϩ -ATPase activity of myosin and actomyosin by GSNO provides a plausible explanation for the functional effects of SNO donors in muscle fibers (Nogueira et al, 2009). We show for the first time that myosin-11 and myosin regulatory light peptide 9 are able to become S-nitrosylated during pregnancy but not in nonpregnant USM.…”

“…Myosin and actin have both been shown to be S-nitrosylated in skeletal muscle with myosin being reversibly inhibited by S-nitrosylation with GSNO (Nogueira et al, 2009). Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain (Evangelista et al, 2010).…”