CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS: FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
Open Access
- Author:
- Tuckow, Alexander Paul
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 29, 2010
- Committee Members:
- Leonard Shelton Jefferson Jr., Dissertation Advisor/Co-Advisor
Leonard Shelton Jefferson Jr., Committee Chair/Co-Chair
Scot R Kimball, Committee Member
Charles H Lang, Committee Member
Lisa M Shantz, Committee Member
Willard M Freeman, Committee Member - Keywords:
- translation initiation
protein synthesis
skeletal muscle - Abstract:
- Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric complex that functions as a guanine nucleotide exchange factor (GEF) toward its substrate, eIF2. The activity of eIF2B is tightly regulated and is rate controlling with regard to global protein synthesis by impacting the rate of the initiation phase of mRNA translation. Alterations in skeletal muscle eIF2B activity and expression of its catalytic epsilon subunit (eIF2Bε) have been observed in a number of physiological conditions where protein synthesis is altered. One objective of the dissertation was to directly examine the effects of exogenously expressed eIF2Bε on GEF activity and protein synthetic rates in rat skeletal muscle in vivo. Using electro gene transfer, a plasmid encoding FLAG-eIF2Bε was transfected into the tibialis anterior (TA) of one hindlimb while the contralateral TA received a control plasmid. Overexpression of the eIF2Bε subunit resulted in increased GEF activity in TA homogenates of healthy rats demonstrating that the expressed protein can enhance catalytic activity. In an effort to restore a deficit in eIF2B activity, an established model of chronic sepsis was utilized in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of FLAG-eIF2Bε in the TA rescued the sepsis-induced deficit in eIF2B GEF activity and muscle protein synthesis. The results demonstrate that modulation of the expression of eIF2Bε may be sufficient to correct deficits in skeletal muscle protein synthesis associated with sepsis and possibly other muscle wasting conditions. Unexpectedly, attempts at longer-term experiments revealed difficulty in maintaining elevated expression of eIF2Bε in rat skeletal muscle beyond a few days despite the ability to express other genes in muscle for several weeks. Thus, a second objective of the research was to identify the mechanism(s) accounting for the rapid loss of exogenously expressed eIF2Bε protein. Evidence obtained in this regard points to a covalent modification of the eIF2Bε protein (ubiquitination) that targets it for proteasome-mediated degradation. Following co-immunoprecipitation of eIF2Bε with ubiquitin from lysates of C2C12 myoblasts treated with proteasome inhibitor, tandem mass spectrometry analysis was performed to locate posttranslational modifications on the eIF2Bε protein. Although the site(s) of ubiquitin modification have not yet been identified, the mass spectrometry analysis resulted in the identification of two novel phosphorylation sites in the rat eIF2Bε protein. The newly identified mechanism for regulating eIF2Bε expression via the ubiquitin-proteasome system may be particularly important with regard to its potential role in muscle wasting conditions where protein synthesis is impaired. Statins are a widely prescribed and effective class of lipid lowering drugs with many beneficial effects; however, one of the less desirable side effects of statin use is the incidence of muscle related complaints. To explore the mechanism(s) contributing to the statin-induced alterations in protein metabolism, C2C12 myoblasts were treated with simvastatin or vehicle for 24 h. Cells exposed to simvastatin exhibited reduced rates of protein synthesis as evidenced by [35S]methionine and [35S]cysteine incorporation into protein. The reduction in protein synthesis occurred with concomitant repression of the GEF activity of eIF2B. Simvastatin treatment also resulted in reduced expression of several subunits of the eIF2B heteropentameric complex at the protein level. These results suggest that repression of eIF2B expression and activity may contribute, at least in part, to the statin-induced reduction in protein synthesis. Finally, experiments were also undertaken to elucidate the role of the 5'- and 3'- untranslated regions (UTRs) of the human eIF2Bε mRNA in the translational regulation of the gene. Luciferase reporter constructs were generated containing the 5'- and 3'-UTRs of eIF2Bε, alone or in combination, in the appropriate location relative to a firefly luciferase coding sequence. Several cell lines were transfected with the reporter plasmids and subjected to various cell culture conditions followed by luciferase activity assays. Similar experiments were performed in an attempt to validate microRNAs predicted to target the 3'-UTR of eIF2Bε. Overall, the work described in this dissertation contributes several novel findings for an important point of regulation in the control of skeletal muscle protein synthesis. Specifically, the present findings emphasize the functional significance of enhanced expression of eIF2Bε in muscle and provide further insight into the regulation of its expression. Advancing our understanding of the role of eIF2B and the regulation of expression of its subunits has important implications for future interventions aimed at the prevention of muscle wasting.