Neuronal nitric oxide synthase (nNOS) splice variant function: Insights into nitric oxide signaling from skeletal muscle.

Defects in neuronal nitric oxide synthase (nNOS) splice variant localization and signaling in skeletal muscle are a firmly established pathogenic characteristic of many neuromuscular diseases, including Duchenne and Becker muscular dystrophy (DMD and BMD, respectively). Therefore, substantial efforts have been made to understand and therapeutically target skeletal muscle nNOS isoform signaling. The purpose of this review is to summarize recent salient advances in understanding of the regulation, targeting, and function of nNOSµ and nNOSß splice variants in normal and dystrophic skeletal muscle, primarily using findings from mouse models. The first focus of this review is how the differential targeting of nNOS splice variants creates spatially and functionally distinct nitric oxide (NO) signaling compartments at the sarcolemma, Golgi complex, and cytoplasm. Particular attention is given to the functions of sarcolemmal nNOSµ and limitations of current nNOS knockout models. The second major focus is to review current understanding of cGMP-mediated nNOS signaling in skeletal muscle and its emergence as a therapeutic target in DMD and BMD. Accordingly, we address the preclinical and clinical successes and setbacks with the testing of phosphodiesterase 5 inhibitors to redress nNOS signaling defects in DMD and BMD. In summary, this review of nNOS function in normal and dystrophic muscle aims to advance understanding how the messenger NO is harnessed for cellular signaling from a skeletal muscle perspective.

Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner.

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery.

Multivalent binding and facilitated diffusion account for the formation of the Grb2-Sos1 signaling complex in a cooperative manner.

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion, and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms.

AIM: Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSµ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. RESULTS: GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSµ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1-/- muscle. Functional analyses of GC1-/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1-/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. INNOVATION: GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. CONCLUSIONS: These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

Exercise modulates redox-sensitive small GTPase activity in the brain microvasculature in a model of brain metastasis formation.

Tumor cell extravasation into the brain requires passage through the blood-brain barrier (BBB). There is evidence that exercise can alter the oxidation status of the brain microvasculature and protect against tumor cell invasion into the brain, although the mechanisms are not well understood. In the current study, we focused on the role of microenvironment generated by exercise and metastasizing tumor cells at the levels of brain microvessels, influencing oxidative stress-mediated responses and activation of redox-sensitive small GTPases. Mature male mice were exercised for four weeks using a running wheel with the average voluntary running distance 9.0 ± 0.3 km/day. Mice were then infused with 1.0 × 10(6) D122 (murine Lewis lung carcinoma) cells into the brain microvasculature, and euthanized either 48 hours (in short-term studies) or 2-3 weeks (in long-term studies) post tumor cell administration. A significant increase in the level of reactive oxygen species was observed following 48 hours or 3 weeks of tumor cells growth, which was accompanied by a reduction in MnSOD expression in the exercised mice. Activation of the small GTPase Rho was negatively correlated with running distance in the tumor cell infused mice. Together, these data suggest that exercise may play a significant role during aggressive metastatic invasion, especially at higher intensities in pre-trained individuals.