The long term objectives are to elucidate the molecular mechanisms involved in muscle protein catabolism during excess/deficit of hormones and cytokines. Chronic excess of tumor necrosis factor-alpha (TNF-alpha) inhibits global protein synthesis in rat fast twitch muscles such as extensor digitorum tongus (EDL). TNF-alpha induced inhibition of muscle protein synthesis is caused by an impairment of peptide-chain initiation which is reversed by the nutritional therapy of rats with branched-chain amino acids. The initiation of mRNA translation is a complicated multi-step process involving over a dozen eukaryotic initiation factors (eIFs). However, two steps have been identified to be subject to physiological regulation: i) binding of initiator met-tRNAi to the 40S ribosomal subunit to form 43S pre-initiation complex, mediated by eIF-2 and ii) the binding of the 43S to the 5' end of mRNA, mediated by eIF-4. One of the eIF 4 family members, eIF-4E, binds to the m7GTP cap at the 5' end of mRNA. The initiation of the peptide-chain can be inhibited by the sequestering of this cap-binding protein by another translational regulator, 4E-DPI as 4e-BPI.eIF-4E complex. Phosphorylation of 4E-BPI reduces its affinity for eIF-4E and phosphorylation of eIF-4E increases its affinity for m7GTP cap of mRNA. The proposed study will test the hypothesis that TNF-alpha inhibits peptide-chain initiation in rat muscles by decreasing the availability and/or activity of eIF-4E to facilitate the binding of 43S pre- initiation complex to mRNA. The proposed study, using rats as a model system, will focus on the following specific aims: i) Evaluation of the effects of chronic TNF-alpha excess on the concentration and phosphorylation state of eIF-4E and 4E-BP1 in EDL, ii) evaluation of the effects of chronic TNF-alpha excess on the concentration of 4E-BP1.eIF- 4E & eIF-4G.eIF-4E complexes in EDL, and iii) evaluation of the effects of 1-leucine on TNF-alpha induced regulation of eIF-4E, eIF-4g, NAD 4e-BP1. Quantitation of eIF-4E and 4E-BP1 will be done by densitometric analysis of protein immunoblots after affinity chromatography of eIF-4e and immunoprecipitation of 4E-BP1 in muscle extracts. The association of 4E-BP1 and eIF-4G with eIF-4ED will be quantitated by protein immunoblot analysis after immunoprecipitation of the 4E-BPI.eIF-4E and eIF-4G.eIF-4E complexes with a monocolonal antibody against eIF-4E. Phosphorylated and unphosphorylated forms of eIF-4E will be separated by isoelectric focusing on a slab gel. Phosphorylation of 4E-BP1 will be determined on the basis of its electrophoretic mobility on SDS- polyacrylamide gel; slow migrating forms represent more highly phosphorylated 4E-BPI. There is an increased secretion of TNF-alpha during fever, sepsis, severe injury, trauma, burns or cancer; therefore, elucidation of molecular mechanisms involved in the TNF-induced impairment of peptide-chain initiation has appreciable biomedical significance.
| Cheema, Ijaz R; Jones, Donald L (2003) TNF-alpha and cortisone impair peptide chain initiation by altering the availability of initiation factor EIF-4E. Front Biosci 8:s1051-5 |
