The effects of catabolic and anabolic steroids on amino acid incorporation by skeletal-muscle ribosomes.
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ABSTRACT:
SUBMITTER: Bullock G
PROVIDER: S-EPMC1198827 | biostudies-other | 1968 Jul
REPOSITORIES: biostudies-other
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Project description:Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo β-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.
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Project description:Chronic metabolic acidosis plays a role in cachexia by enhancing total proteolysis in skeletal muscle. Glucocorticoid also triggers proteolysis and plays a permissive role in the effect of acidosis. The System A amino acid transporter SNAT2/SLC38A2 is ubiquitously expressed in mammalian cells including muscle, performing Na+-dependent active import of neutral amino acids, and is strongly inhibited by low pH. Exposure of rat skeletal muscle cell line L6-G8C5 to low pH rapidly inhibits SNAT2 transport activity and enhances total proteolysis rate. Pharmacological inhibition or silencing of SNAT2 also enhances proteolysis. This study tests the hypothesis that the glucocorticoid dexamethasone (DEX), like low pH, inhibits SNAT2 activity in L6-G8C5 myotubes, thus contributing to total proteolysis. Incubation with 500 nM DEX for 4 h reduced the System A amino acid transport rate to half the rate in control cultures. This inhibition depended on glucocorticoid receptor-mediated gene transcription, but SNAT2 mRNA levels were unaffected by DEX. In contrast, the SNAT2 protein assessed by immunoblotting was significantly depleted. The co-inhibitory effects of DEX and low pH on System A transport activity were additive in stimulating total proteolysis. In keeping with this mechanism, DEX's inhibitory effect on SNAT2 transport activity was significantly blunted by the proteasome inhibitor MG132. Proof of principle was achieved in similar experiments using recombinant expression of a GFP-tagged SNAT2 fusion protein in HEK293A cells. It is concluded that DEX acutely depletes the SNAT2 transporter protein, at least partly through proteasome-dependent degradation of this functionally important transporter.
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Project description:Bisphosphonates (BPs) are the first-line treatment of bone loss resulting from various pathological conditions. Due to their high affinity to bone they have been used to develop conjugates with pro-anabolic or anti-catabolic drugs. We recently demontrated that hydrogen sulfide (H2S), promotes osteogenesis and inhibits osteoclast differentiation. Here we developed an innovative molecule, named DM-22, obtained from the combination of alendronate (AL) and the H2S-releasing moiety aryl-isothiocyanate. DM-22 and AL were assayed in vitro in the concentration range 1-33??M for effects on viability and function of human osteoclasts (h-OCs) and mesenchymal stromal cells (h-MSCs) undergoing osteogenic differentiation. Amperometric measures revealed that DM-22 releases H2S at a slow rate with a thiol-dependent mechanism. DM-22 significantly inhibited h-OCs differentiation and function, maintaining a residual h-OCs viability even at the high dose of 33??M. Contrary to AL, in h-MSCs DM-22 did not induce cytotoxicity as revealed by LDH assay, significantly stimulated mineralization as measured by Alizarin Red staining and increased mRNA expression of Collagen I as compared to control cultures. In conclusion, DM-22 is a new BP which inhibits h-OCs function and stimulate osteogenic differentiation of h-MSCs, without cytotoxicity. DM-22 is an ideal candidate for a novel family of osteoanabolic drugs.
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