|Ph.D Student||Yu Zanlin|
|Subject||Proteasome Lid Subunit Rpn11: Allocation of Functions to its|
C- and N-Terminal Regions
|Department||Department of Biology||Supervisors||Professor Michael Glickman|
|Dr. Elah Pick|
|Full Thesis text|
In eukaryocytes, protein turnover is strictly and precisely regulated by ubiquitin proteasome pathway. Functional proteins that have finished their role and therefore should be removed, as well as many misfolded or mistranslated proteins are covalently labelled by ubiquitin via a cascade of three ubiquitinating enzymes: ⑴ ubiquitin activating enzyme, ⑵ ubiquitin conjugating enzyme, ⑶ a substrate-specific ubiquitin ligase. After the ubiquitin modified substrate is recognized and bound by the proteasome, the substrate is unfolded and translocated into the proteasome chamber for degradation by protease active sites. During this multi-step process, the polyubiquitin chain is released from the substrate under the function of proteasome-deubiquitinating enzymes (DUBs). This research thesis will focus on the contribution of one such DUB, Rpn11, a component of proteasome lid subcomplex. Exactly at which stage Rpn11 intervenes into the proteasome sequence of events is still unclear, as is its contribution to proteolysis. Therefore, understanding its position in the proteasome, its close-partner associations, and its active site properties will have profound mechanistic insights.
In this study, I present the studies of both the C-terminus and N-terminus domains of Rpn11. I demonstrate that the C-terminus of Rpn11 serves as a bridge connecting two modules of proteasome lid. Truncation of C-terminus of Rpn11 in an rpn11-m1 mutant strain generates a partially assembled proteasome lacking one of the lid modules, yet sustains viability. In addition, we studied self-assembly of proteasome module 1 and found that it may occur in a stepwise manner initiated by dimerization of Rpn11-Rpn8 heterodimer. We thus conclude that Base-CP may serve as a proteasome assembly platform and propose a possible model pathway of proteasome assembly.
To observe the Rpn11-Rpn8 interaction surface and the detailed deubiquitinating activity site of Rpn11 and counterpart site of Rpn8, we crystalized the Rpn11-Rpn8 protein and solved crystal structure. The Zinc ion binding pocket in the MPN domain of Rpn11 is composed of an aspartate side chain, two histidines and a water molecule ligand. A remote glutamate residue is required to stabilize the water liganded to Zn2. The backbones of Rpn11 and Rpn8 share a high similarity, despite the activity differences. The insight provided form structural comparison predicts that multiple mutations in Rpn8 might activate its potential deubiquitinating activity by reengineering its capacity to bind Zn2. The tandem roles of Rpn11 and Rpn8 in context of their close proximity within the lid are discussed.