|M.Sc Student||Torres Liron|
|Subject||The Role of N Terminal Acetylation in Protecting Proteins|
from Ubiquitination and Degradation by Preventing
|Department||Department of Medicine||Supervisor||? 18? Aaron Ciechanover|
|Full Thesis text|
Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of many substrates of the ubiquitin system. For several substrates, such as the myogenic transcription factor MyoD, it has been shown that the ubiquitin moiety is conjugated to the N-terminal residue. Another modification that occurs on the N-terminal residue of proteins is N-terminal acetylation. N-terminal acetylation is a co-translational modification occurring when the peptide is 30 to 50 amino acids long and is considered irreversible. N-terminal acetylation is catalyzed by a family of N-acetyl transferases (NATs). In yeast there are 3 different NATs (NatA, B and C). Both N-terminal acetylation and degradation act on the same region in the protein, but the effect of N-terminal acetylation on the degradation state of proteins has not been examined yet, whether it will remain stable or degraded by the 26S proteasome. We chose MyoD as a model protein to examine this balance. First, we demonstrated in yeast that degradation of MyoD is completely dependent on its N-terminal ubiquitination which enabled us to study the process in a simple model system that can also be manipulated genetically. After examining the N-terminal domain sequence of MyoD, we found that it can be a candidate for NatB N-terminal acetylation (Met-Glu-); however, since proline is known to inhibit N-terminal acetylation, the two proline residues at position 6 and 7 may block acetylation. We demonstrated that mutating the proline residues to alanine (MyoD P6, 7A) stabilized the protein in yeast strains with active NatB, but did not affect its ability to be degraded in the ΔNatB yeast strain. Mass spectrometry analysis confirmed that indeed stabilization was due to N-terminal acetylation. We concluded that N-terminal acetylation stabilizes MyoD and prevents its degradation. We observed similar stabilization of mutant MyoD in COS7 cells. In vitro results also show stabilization of MyoD P6,7A. However, Mass Spectrometry analysis of MyoD isolated from cells revealed that in cells, the acetylation status is more complex: we isolated a mixed population of acetylated and non-acetylated MyoD of both identified WT and mutant MyoD. In addition to our efforts to find the affect of N-terminal acetylation on the degradation of MyoD, we searched for a yeast protein, that is naturally N-terminally acetylated and that its acetylation affects its degradation, in order to find a biological significance for this modification. To date we did not find a yeast protein in which his degradation is dependent on its acetylation state.