Background: Puromycin and serum starvation have significant effects on the stability and half-life of proteins in skeletal muscle cells. Pyruvate dehydrogenase kinase 1 (PDK1) plays a fundamental role in the regulation of cellular metabolism by controlling the activity of the pyruvate dehydrogenase complex. The stability and half-life of PDK1 are crucial for the long-term regulation of energy balance because PDK1 controls the conversion of pyruvate to acetyl-coenzyme A. The half-lives of PDK1, the transcription factor STAT3, and the heavy chain of the slow myosin isoform (MyHC-β/slow) differ due to their specific roles in cellular processes: STAT3, as a transcription factor, has a short half-life; PDK1, important for metabolic regulation, has a longer half-life; while MyHC-β/slow, as a structural protein, exhibits the longest half-life due to the slow turnover of muscle fibers. In addition, puromycin and serum starvation may affect the mTOR (mechanistic target of rapamycin) signaling pathway, which is fundamental for regulating protein synthesis and adapting the cellular response to stress.
Aims: (1) To determine how puromycin and serum affect the expression and half-life of PDK1, STAT3 and MyHC-β/slow. (2) To examine the effect of serum and puromycin on the mTOR signaling pathway.
Hypotheses: 1) Under the influence of puromycin, the level of PDK1 decreases more slowly than the level of STAT3 and faster than the level of myosin heavy chain (MyHC-β/slow); 2) Puromycin and serum starvation decrease mTOR signaling pathway activity; 3) Serum starvation nonspecifically decreases the level of all proteins in skeletal muscle cells.
Methods: We used rat L6 skeletal muscle cells as the experimental model and determined protein expression by Western blotting and gene expression by RT-qPCR.
Results: Puromycin decreased the expression of total proteins, PDK1 and MyHC-β/slow, and also has increased phosphorylation of ribosomal protein S6 (S6-RP) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the rat L6 cell line. Serum starvation significantly decreased the expression of total proteins in rat skeletal muscle cells, but was shown to increase mRNA transcription for MyHC-β/slow.
Conclusions: Puromycin and serum withdrawal reduce PDK1 and MyHC-β/slow levels faster than STAT3, which remains more stable. Puromycin increases phosphorylation and activation of mTOR signalling pathway proteins (4E-BP, P70 ribosomal protein S6 kinase, S6-RP), while serum withdrawal reduces this effect. Serum withdrawal increases MyHC-β/slow expression despite an overall decrease in protein expression.
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