|Ph.D Student||Ben-Saadon Ronen|
|Subject||Identification of Signals that Target Proteins for|
Degradation via the Ubiquitin Pathway: A Role for
the N-Terminal as a Conjugation Site
|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 the majority of the substrates of the ubiquitin system. For several substrates it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, internal lysines also play a role in modulating their stability. To better understand the physiological significance of this novel modification, it was important to identify proteins which their degradation is completely independent on internal lysines. Also, though experimental data strongly support the existence of N-terminal ubiquitination (NTU), a direct evidence for this modification had been missing. Here we demonstrate that naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor p16INK4a are targeted via N-terminal ubiquitination. Interestingly, p16INK4a is degraded in a cell density-dependent manner. Importantly, we provide the first direct evidence for N-terminal ubiquitination: analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. We also demonstrate that a short N-terminal segment of the myogenic transcription factor MyoD is necessary and sufficient for its degradation by NTU, suggesting that it may serve as a recognition motif for the ligase. During our efforts to identify the E3 that targets p16 we found that the polycomb repressive complex 1 (PRC1), RING finger-containing proteins, Bmi1 and Ring1B, that are known repressors of p16, do not promote its ubiquitination and degradation, at least not directly. Ring1B is an E3 that mediates its own polyubiquitination and monoubiquitination of histone H2A. By further studying the activity of Bmi1 and Ring1B, we found that Ring1B and Bmi1 are degraded by an exogenous E3, independent of their RING domain. Moreover, while the RING domain of Bmi1 is required for its biological activity, Bmi1 is not a self ubiquitinating E3. Consistent with the non-proteolytic self ligase activity of Ring1B, it generates atypical mixed ubiquitin chains that use lysines 6, 27, and 48 of ubiquitin, and are required for its ability to ubiquitinate H2A. Importantly, we demonstrate that Bmi1 regulates Ring1B in several levels. The RING domain of both proteins mediate their association, subsequent stabilization, and probably prevent their accumulation as free, unbound PRC1 components. Independent of stabilizing Ring1B, Bmi1 regulates the self ubiquitinating activity of the ligase and stimulates its H2A ligase activity.