No abstract

Highlights d High-resolution structures of RyR1 resolved in liposomes d The cryo-EM structure of RyR1 in liposomes closely matches detergent structures d The channel remains dynamic in liposomes

Mechanistic target of rapamycin (mTOR) signaling is necessary to generate a mechanically induced increase in skeletal muscle mass, but the mechanism(s) through which mechanical stimuli regulate mTOR signaling remain poorly defined. Recent studies have suggested that Ras homologue enriched in brain (Rheb), a direct activator of mTOR, and its inhibitor, the GTPase-activating protein tuberin (TSC2), may play a role in this pathway. To address this possibility, we generated inducible and skeletal muscle-specific knock-out mice for Rheb (iRhebKO) and TSC2 (iTSC2KO) and mechanically stimulated muscles from these mice with eccentric contractions (EC). As expected, the knock-out of TSC2 led to an elevation in the basal level of mTOR signaling. Moreover, we found that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both inducible and skeletal muscle-specific knock-out mice for Rheb and iTSC2KO mice. Using mass spectrometry, we identified six sites on TSC2 whose phosphorylation was significantly altered by the EC treatment. Employing a transient transfection-based approach to rescue TSC2 function in muscles of the iTSC2KO mice, we demonstrated that these phosphorylation sites are required for the role that TSC2 plays in the EC-induced activation of mTOR signaling. Importantly, however, these phosphorylation sites were not required for an insulin-induced activation of mTOR signaling. As such, our results not only establish a critical role for Rheb and TSC2 in the mechanical activation of mTOR signaling, but they also expose the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway.As the largest organ in the body, skeletal muscles comprise ϳ45% of our total body mass and play essential roles in voluntary movement, metabolic health, and maintaining quality of life (1-4). Indeed, both sedentary and active adults will lose 35-40% of their skeletal muscle mass by the age of 80, and this loss in muscle mass is associated with disability, loss of independence, an increased risk of morbidity and mortality, as well as an estimated $18.5 billion in annual healthcare costs in the United States alone (2, 5-7). Thus, the development of therapies that can maintain, restore, or even enhance muscle mass is a clinically and fiscally significant goal (8). However, to succeed in developing such therapies, we must first understand the molecular mechanisms that regulate skeletal muscle mass.Skeletal muscle is a highly plastic tissue, and it can change its mass in response to a number of environmental factors. At the most basic level, changes in muscle mass are driven by an alteration in the balance between the rate of protein synthesis and the rate of protein degradation, with a net positive balance leading to muscle growth (i.e. hypertrophy) and a net negative balance leading to muscle loss (i.e. atrophy) (9, 10). Over the last two decades, it has become apparent that a protein kinase called the mammalian/mechanistic target of rapamycin (mTO...

Thyroglobulin is a homodimeric glycoprotein that is essential for the generation of thyroid hormones in vertebrates. Upon secretion into the lumen of follicles in the thyroid gland, tyrosine residues within the protein become iodinated to produce monoiodotyrosine (MIT) and diiodotyrosine (DIT). A subset of evolutionarily conserved pairs of DIT (and MIT) residues can then engage in oxidative coupling reactions that yield either thyroxine (T4; produced from coupling of a DIT `acceptor' with a DIT `donor') or triiodothyronine (T3; produced from coupling of a DIT acceptor with an MIT donor). Although multiple iodotyrosine residues have been identified as potential donors and acceptors, the specificity and structural context of the pairings (i.e. which donor is paired with which acceptor) have remained unclear. Here, single-particle cryogenic electron microscopy (cryoEM) was used to generate a high-resolution reconstruction of bovine thyroglobulin (2.3 Å resolution in the core region and 2.6 Å overall), allowing the structural characterization of two post-reaction acceptor–donor pairs as well as tyrosine residues modified as MIT and DIT. A substantial spatial separation between donor Tyr149 and acceptor Tyr24 was observed, suggesting that for thyroxine synthesis significant peptide motion is required for coupling at the evolutionarily conserved thyroglobulin amino-terminus.