|M.Sc Student||Kaufman Tahel|
|Subject||Identification of the E3, Ubiquitin Ligase, which|
Facilitates the Formation of N-Terminal Conjugated
|Department||Department of Medicine||Supervisor||? 18? Aaron Ciechanover|
|Full Thesis text|
Many cellular proteins are targeted for degradation by the ubiquitin proteasome system (UPS). Following tagging with ubiquitin via three sequential enzymatic reactions, the ubiquitinated substrate is degraded by the 26S proteasome complex with release of free and reusable ubiquitin. One of the substrates of the ubiquitin proteasome system is the myogenic transcription factor MyoD that is degraded along the myogenic differentiation program in both phosphorylation-dependent and independent manners. It was assumed that the first event in the degradation of MyoD is the attachment of ubiquitin to a Lys residue within the protein. However, mutagenesis of all Lys to Arg residues in MyoD demonstrated that the lysine-less MyoD (LL MyoD) is still a UPS substrate, though less effective than the WT protein, and that blocking of the α-NH2 group in the N-terminus of LL MyoD inhibited its degradation significantly. This result suggested that an additional important ubiquitin docking site resides on the α-NH2 group of the protein. These findings also suggested that more than one E3 regulates the degradation of MyoD. Thus to better understand the mechanism by which MyoD is degraded, it was necessary to identify the E3(s) that mediate the ubiquitination of MyoD.
In this study we used two strategies in order to identify these E3s. The first was protein chromatography. We identified two proteins, HERC4 and KPC1, as candidate E3 ubiquitin ligases that mediate the attachment of ubiquitin to internal Lys and/or to the N-terminus of MyoD.
The second approach was genetic screening in Saccharomyces cerevisiae. Recent findings in our laboratory demonstrated that WT MyoD and LL MyoD are degraded in yeast with t1/2 of ~10 min. Using the EuroSCARF systematic deletion library, we screened for a yeast strain in which WT and LL MyoD are stabilized. We identified TOM1, a HECT domain E3 ubiquitin ligase known to mediate ubiquitination of many proteins. We demonstrated that following deletion of TOM1, LL MyoD becomes stable. Moreover, we showed that expression of TOM1 in ∆tom1 yeast strain restored the degradation of LL MyoD to a rate similar to the WT strain. Although, MyoD is not a native substrate of the yeast ubiquitin-proteasome system, these results demonstrate for the fist time, that a specific E3 is involved in the degradation of a protein that lacks internal Lys residues, probably via attachment of ubiquitin to the α-NH2 group of the protein.