|Ph.D Student||Shabek Nitzan|
|Subject||Ubiquitin Meets its Fate: Mechanisms of Ubiquitin|
|Department||Department of Medicine||Supervisor||? 18? Aaron Ciechanover|
|Full Thesis text|
Ubiquitin (Ub), as a principal component of the ubiquitin-proteasome system, must be maintained at adequate levels to support cellular homeostasis under basal and stressed conditions. While it is clear that Ub is degraded, the underlying mechanisms of recognition of Ub as a substrate, the enzymatic components involved, and the mode of regulation of this degradation pathway are still unknown. Here we demonstrate that conjugation of monomeric Ub requires both its internal lysines and N-terminal residue, but the degradation is rather slow. Rapid degradation of the monomeric species requires a C-terminal tail of a minimal length (>20 residues) fused to it. The tail, which may represent the substrate or part of it, or a naturally occurring extended form of Ub, probably allows efficient entry of the protein into the 20S catalytic chamber, using Ub as a binding anchor to the 19S regulatory particle. Importantly, we demonstrate in a direct manner that Ub is degraded along with its target substrate in a “piggyback” mechanism, and that stress-induced accelerated degradation of cellular substrates results in depletion of Ub. Furthermore, we show that addition of substrates enhances proteasome-dependent degradation of Ub which is preceded by specific conjugation. Thus we propose that Ub can be degraded by the proteasome via three routes - along with its conjugated substrate, when extended with a C-terminal tail, and as a free monomer. Moreover, we demonstrate that the degradation of tailed Ub does not require further ubiquitination, highlighting the ability of the proteasome to recognize and degrade monoubiquitinated substrates. Thus we suggest that the minimal requirements for degradation of a substrate by the 26S proteasome are binding Ub moiety and a long enough extension that will allow it to be ratcheted into the proteolytic chamber. We further show that peptides/substrates shorter than 120 residues can be degraded following monoubiquitination, but longer peptides/substrates have to undergo polyubiquitination to support their efficient binding to the proteasome and proteolysis. Moreover, we show that short-tailed Ubs, such as UBB, bind to the proteasome, but since they cannot be efficiently degraded, they inhibit the degradation of other proteasomal substrates. The inhibition is dependent on their ability to be ubiquitinated which probably tightens their binding to the proteasome. Interestingly, the inhibition affects only substrates that must undergo ubiquitination for their subsequent degradation. The degradation of ODC that is targeted by the proteasome in a ubiquitin-independent manner, is not affected by the short-tailed ubiquitinated Ub.