Bindung von Poly-Ubiquitinierten Substraten durch das 26S Proteasom
Zusammenfassung der Projektergebnisse
The 26S Proteasome is the main proteolytic particle in eukaryotic cells and degrades proteins, which have been marked for degradation by the attachment of a poly-Ubiquitin chain. After the binding of the substrates to the 26S particle, the protein is unfolded, deubiquitinated and translocated into the 20S proteolytic particle, where the protein is cleaved into smaller peptides. The aim of this proposal was to study the biochemical requirements for the binding of these ubiquitinated substrates to the Proteasome. To do so, we developed a new and rapid assay, which allowed us to study the initial binding as an isolated event without degradation of the substrate. We discovered that the binding of Ubiquitin conjugates can occur at 4̊C and is initially reversible. We further characterized the known binding sites Rpn10 and Rpn13 as high affinity binding sites for ubiquitinated proteins and discovered the existence of a still unknown lower affinity binding site in the Proteasome. Binding of Ubiquitin conjugates to both Rpn10 and Rpn13 is stimulated in the presence of ATP and its non-hydrolysable analogue ATPγS, but not by ADP. After the initial reversible binding the substrate assumes a tighter, nonreversible binding to the Proteasome. The conversion to this step requires elevated temperatures (37̊C) and ATP-hydrolysis. Furthermore the tighter binding requires an unfolded or easily unfoldable domain in the substrate and does not occur in tightly folded proteins. In addition the tighter binding can be inhibited by the addition of unfolded proteins, which can be degraded by the 26S without ubiquitination. Therefore this tighter binding is a newly discovered step in the binding of Ubiquitin conjugates to the 26S that correlates with the degradation of the substrate. The tighter binding precedes the removal of the Ubiquitin chain, however we discovered that the subsequent deubiquitination of the substrate by the proteasome associated deubiquitinating enzyme Usp14 increases peptide hydrolysis 2-7 fold by the 26S. Interaction of the Ubiquitin chain with the active site of Usp14 stimulates gate opening in the 20S particle thereby facilitating substrate entry. Interestingly the 26S Proteasome can also be stimulated in an Usp14 active site mutant, which indicates that Usp14 functions non-catalytically to open the gate. Furthermore the stimulation of gate opening by Ubiquitin conjugates by Usp14 requires nucleotide binding to the 19S ATPase subunits. Our findings support a new two-step model for the binding of Ubiquitin conjugates by the 26S, which consists of a reversible initial binding to Rpn10 and Rpn13 that is subsequently converted to a tighter binding depended on an unfolded domain in the substrate. The interaction of the protein with the 26S occurs most likely at the 19S ATPase subunits, which bind unfolded proteins and allows the deubiquitination of the substrate. The removal of the Ubiquitin chain by Usp14 in turn promotes the degradation of the protein by stimulating gate opening, which indicates that the activities of 26S (deubiquitination, unfolding and translocation into the 20S) are linked to ensure efficient degradation of the substrate.
Projektbezogene Publikationen (Auswahl)
- (2008) Binding of Ubiquitin conjugates to the 26S proteasome enhances its proteoltic capacity by enhancing gate opening in the 20S particle (Faseb Summer Research Conference 2008)
Peth, A., Uchiki, T., and Goldberg A.L.
- (2009). Getting to first base in proteasome assembly. Cell 138, 25-28
Besche, H.C., Peth, A., and Goldberg, A.L.
- (2009). Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Molecular Cell 36, 794-804
Peth, A., Besche, H.C., and Goldberg, A.L.
- (2010). ATP-dependent steps in the binding of Ubiquitin conjugates to the 26S proteasome that commit to degradation. Molecular Cell 40, 671-681
Peth, A., Uchiki, T., and Goldberg, A.L.
