Rushbrook At any point in time, a cellular protein is in a state of flux which represents the balance between synthesis and degradation. While much has been learned about the two main degradation systems, the calpains and the ATP-dependent proteasome, there is no overall consensus on their relative importance or how they might interact. A myosin heavy chain (HC) protein transition in the developing avian pectoralis major muscle provides a particularly attractive model system. HC IV is expressed between 19-days in ovo and 3-days posthatch. Between 3- and 5-days posthatch, it is dramatically replaced by HC V. The half-life of HC IV is in markedly less that that of adult myosin which is approximately 30 days. The adult HC is methylated at four positions, monomethyl Lys-36, trimethyl Lys-131 and -552 and 3-methyl His-756. The functions of these modifications are obscure. HC IV at 2-days posthatch and the isoforms which precede it in the embryo completely lack modification of Lys-36. The entering HC V at 5-days posthatch, however, is clearly methylated at this position. These observations suggest a role for methylation of Lys-36 in protection of the HC against degradation. Lys-131 is not methylated in HC V at 5-days posthatch, ruling out such a role. The methylation states of Lys-552 and His-756 during the transition are unknown. The project incorporates the following hypothesis. Gradual increase in cytosolic [Ca2+] in the days after hatching results in a calpain-mediated degradation of unmethylated HC IV at a site which activates a PEST sequence. This leads to ubiquitination and degradation via the proteasome system. Methylation at Lys-36 and possibly other residues, with the appearance of isoform V at 5-days posthatch, leads to conformational changes which stabilize against calpain clevage. The goals of the project are: 1. To complete information regarding methylation changes associated with the HC IV -> transition. 2. To determine whether HCs IV and V in the posthatch PM are differentially susceptible to clevage by calpain. 3. To determine whether the ATP-dependent proteasome system is involved in HC IV degradation. 4. To carry out preliminary characterization of the methylase(s) which appear during the transition from HC IV to HC V.
In muscle cells, specialized proteins form long filaments which overlap slightly. When the muscle is stimulated to contract, the overlap of the filaments increases, shortening the muscle. In a healthy person, these proteins are constanly being synthesized and degraded. In times of disease and famine, their breakdown rate increases to provide small molecules that the body uses for energy. While two major systems exist for degradation of cellular proteins, the calpains and the proteasome, there is no agreement as to their relative importance. In this project, the cellular mechanism for degradation of a muscle protein which appears for a very short time during development will be determined. In addition, usual differences in structure between it an its more mature counterparts will be investigated as the possible origin of the strikingly greater degradation rate for the developmental form.
