|Ph.D Student||Isakov Elada|
|Subject||Proteasome Dynamics as Exemplified by P97 and S5a|
|Department||Department of Medicine||Supervisor||DR. Ariel Stanhill|
|Full Thesis text|
The 26S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation. In this thesis we exemplify proteasome modularity in two scenarios. The first entails a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26S proteasome via the 19S particle thereby forming an additional hexameric ATPase ring with unfolding ability, in order to relieve repression. P97 bound proteasomes showed selective binding towards the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97 bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new check point in UPS impairment. The second example deals with substrate targeting with polyubiquitin chains and substrate recognition by the proteasome as the first step towards protein degradation. The recognition of the polyubiquitinated species by the proteasome is achieved by ubiquitin receptors. The later part of my thesis deals with the ubiquitin receptor S5a/Rpn10. S5a contains two UIM domains that interact with ubiquitinated substrates and a single VWA domain that associates with the 26S proteasome. Here we show that S5a is important for normal cellular degradation as S5a knock-down cells show higher basal poly-ubiquitin levels and endogenous S5a undergo up regulation in this cells three weeks after produced. Additionally, we have found that S5a undergoes multi-ubiquitination in its VWA domain. To test the functional importance of these post translational events on S5a function and to examine the importance of each of the S5a domains to protein degradation, we have used an in vivo system that monitors ATF4 degradation. Our results reveal the significance of multy-ubiquitination on S5a for substrate degradation, and the importance of the C-terminal domain of S5a but not the UIM domains in normal proteasome degradation mainly in steps after substrate recognition. The present study demonstrates the proteasome dynamics as shown by P97 and S5a. P97 interacts with the proteasome under protein misfolding conditions and induces proteasome accessibility, while S5a undergoes post translational modifications that affect proteasome structure and activity. The ability to modulate proteasome activity and properly respond to stress conditions is of great importance in cellular regulation.