טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFajerman Ifat
SubjectMechanisms of Recognition and Degradation of the Id2 Protein
by the Ubiquitin Proteolytic Pathway
DepartmentDepartment of Medicine
Supervisor ? 18? Aaron Ciechanover


Abstract

Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin, and degradation of the tagged substrate by the 26S proteasome. The Id2 protein belongs to Id family of proteins. Id proteins play key roles in negative regulation of cell differentiation and they are essential for cell cycle progression. They are all short-lived proteins with a highly complex pattern of temporal and tissue-specific expression.  We have shown that degradation of the Id2 protein is mediated by the ubiquitin proteasome system. Recent studies from our laboratory have shown that degradation of MyoD, EBV-LMP1, and HPV-16 E7 requiers a novel pathway of ubiquitination. The first ubiquitin moiety is attached linearly to the free N-terminal residue, and not, as it was the accepted rule for many years, to an internal Lys residue of the substrate. We have shown that conjugation of Id2 protein occurs via this novel ubiquitination pathway.  The proof that a protein is ubiquitinated at the N-terminus has been, so far, indirect. We present here direct evidence that corroborates the existence of N-terminal ubiquitination. We used the HPV-58 E7, which is naturally occurring lysine-less protein. Studies from our laboratory have shown that this protein is substrate of the ubiquitin proteasome system. TEV protease recognition site was inserted into E7 after the first Met or after the first 21 amino acids. Conjugation followed by TEV cleavage generated a ubiquitin molecule with additional residues according to the cleavage site. Here, for the first time, we show that attachment of the first ubiquitin moiety to the free NH2 terminal group of the initiator amino acid does indeed exist.