Acyl-CoA thioesterase-2 facilitates ß-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner.

Acyl-Coenzyme A (acyl-CoA) thioesters are compartmentalized intermediates that participate in in multiple metabolic reactions within the mitochondrial matrix. The limited availability of free CoA (CoASH) in the matrix raises the question of how the local acyl-CoA concentration is regulated to prevent trapping of CoASH from overload of any specific substrate. Acyl-CoA thioesterase-2 (ACOT2) hydrolyzes long-chain acyl-CoAs to their constituent fatty acids and CoASH, and is the only mitochondrial matrix ACOT refractory to inhibition by CoASH. Thus, we reasoned that ACOT2 may constitutively regulate matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up when lipid supply and energy demands were modest. When energy demand and pyruvate availability were elevated, lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2 , and overt inhibition of ß-oxidation was demonstrated in isolated mitochondria from Acot2 -depleted glycolytic SM. In mice fed a high fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. These observations suggest that ACOT2 supports CoASH availability to facilitate ß-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

Frataxin deficiency lowers lean mass and triggers the integrated stress response in skeletal muscle.

Friedreich's ataxia (FRDA) is an inherited disorder caused by reduced levels of frataxin (FXN), which is required for iron-sulfur cluster biogenesis. Neurological and cardiac comorbidities are prominent and have been a major focus of study. Skeletal muscle has received less attention despite indications that FXN loss affects it. Here, we show that lean mass is lower, whereas body mass index is unaltered, in separate cohorts of adults and children with FRDA. In adults, lower lean mass correlated with disease severity. To further investigate FXN loss in skeletal muscle, we used a transgenic mouse model of whole-body inducible and progressive FXN depletion. There was little impact of FXN loss when FXN was approximately 20% of control levels. When residual FXN was approximately 5% of control levels, muscle mass was lower along with absolute grip strength. When we examined mechanisms that can affect muscle mass, only global protein translation was lower, accompanied by integrated stress response (ISR) activation. Also in mice, aerobic exercise training, initiated prior to the muscle mass difference, improved running capacity, yet, muscle mass and the ISR remained as in untrained mice. Thus, FXN loss can lead to lower lean mass, with ISR activation, both of which are insensitive to exercise training.

Refinement of Highly Flexible Protein Structures using Simulation-Guided Spectroscopy.

Highly flexible proteins present a special challenge for structure determination because they are multi-structured yet not disordered, so their conformational ensembles are essential for understanding function. Because spectroscopic measurements of multiple conformational populations often provide sparse data, experiment selection is a limiting factor in conformational refinement. A molecular simulations- and information-theory based approach to select which experiments best refine conformational ensembles has been developed. This approach was tested on three flexible proteins. For proteins where a clear mechanistic hypothesis exists, experiments that test this hypothesis were systematically identified. When available data did not yield such mechanistic hypotheses, experiments that significantly outperform structure-guided approaches in conformational refinement were identified. This approach offers a particular advantage when refining challenging, underdetermined protein conformational ensembles.