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Three dimentional architectures of dynamic interactions of 26S proteasome

Final Report Summary - PROTEAMICS (Three dimentional architectures of dynamic interactions of 26S proteasome)

In eukaryotic cells, the ubiquitin-proteasome system is responsible for the elimination of proteins: misfolded and damaged are selected by ‘proteins quality control’ system and degraded by the 26S proteasome. This system regulates many fundamental cellular processes such as DNA repair, protein quality control and signaling pathway and its malfunction leads to several disorders, including neurodegenerative disease, cancer and cardiovascular disease. The 26S proteasome is a huge protein machinery that selectively degrades intercellular proteins in an ATP dependent manner. It is composed of 33 canonical subunits, forming two sub-complexes: 20S Core Particle (CP) and 19S Regulatory Particles (RP). The CP is built of four coaxially stacked rings of α- and β-subunits in the order of αββα, keeping its catalytic sites within the chamber. Substrate access to the proteolytic chamber is restricted by the α subunit N-terminal extensions, assembling a gate at the central channel. To open the gate, the 19S RP associates to the CP via the C-terminal hydrophobic-tyrosine-X (HbYX) motifs. The RP is composed of 19 subunits and the core of the RP is formed by a heterohexameric ATPase associated with various cellular activities (AAA+) ATPase, which is the driver of large-scale conformational dynamics of the RP. The AAA+ ATPase prepares substrates for degradation in coordination with at least three ubiquitin receptors (Rpn1, Rpn10 and Rpn13) and a deubiquitylating subunit Rpn11. We have use cryo-electron microscopy to reveal the architecture of the 26S proteasome, showing how its subunits change the conformation upon ATP hydrolysis. Most of structural information of the 26S proteasome including the subunit localization has been obtained by single particle EM analysis (Wehmer and Sakata, 2016). Classification of large electron microscopy dataset led us to identify three major conformations: A ground state (s1) where the proteasome is ready to accept substrates, an intermediate state (s2) where the substrate becomes positioned above the ‘opening’ of the ATPase module and a commitment state (s3) where substrate is translocated into the core complex and the degradation process becomes irreversible. Intriguingly, the central channel of the AAA+ ring and the CP ring are not axially aligned in the 26S proteasome structure in s1 state, whereas 26S proteasome in s3 state exhibits dynamic conformational changes of the 19S RP, which is characterized by an axial-alignment of the central channels from the ATPase to the CP rings. The conformational change from s1 to s3 allows substrates to access the catalytic chamber. Conformational changes in the AAA+ ring play a key role in substrate unfolding and translocation. The ATPase motor is suggested to translocate the polypeptide by a paddling movement of the pore-1 loop that protrudes into the central channel and interacts with hydrophobic patches of the extended polypeptide chains of substrate. My project focused on the regulatory mechanism of the 26S proteasome and our studies that capture several functional states revealed a detailed view of the mechanisms by which the 26S proteasome processes polypeptides for degradation.
First, we reported the structural basis for regulation of the 26S proteasome by its cofactor Ubp6. 26S proteasome is transiently associated with several cofactors, including deubiquitylating enzymes (DUB), ubiquitin ligase and ubiquitin receptors. One of those cofactors, DUB Ubp6 was reported that its binding to the 19S RP enhances the proteolytic activity of the 20S CP. We have solved proteasome structures together with Ubp6 by cryo-EM and observed conformational changes of the proteasome induced by Ubp6 binding. The catalytic domain of Ubp6 binds to the cleft between the OB domain and the ATPase domain of Rpt1, while the N-terminal Ubl domain binds to Rpn1. Ubp6 modulates the conformational landscape of the proteasome, favouring an intermediate state (s2) between the resting and the substrate processing conformations. This is the first study showing the structural basis of the regulatory role of a PIP (Aufderheide et al., 2015). Thus, the PIPs regulate the proteasome function by not only providing an additional function but also by altering the conformation of the proteasome.
Using direct electron detector, we reported a high resolution structure of human proteasome at a resolution of 3.9 Å (Schweitzer et al., 2016). The structure allowed us to make a precise atomic model and provided mechanical insights into the proteasome function. Furthermore, using nucleotide analogs, we explored conformational landscape of the proteasome (Wehmer et al., 2017). One of those structures captured in the presence of nucleotide analogs, we found an activated structure where the CP gate is open and an access to the CP catalytic chamber is allowed. This structure revealed that the proteasome changes shape when it recognizes a target protein, allowing that protein to enter. In the open-gate structure, arrangement of the AAA+ ATPases is different from that of previously reported structures. Interestingly, all three HbYX motifs show clear densities inside the α pockets in the s1, s2 and s3 states, although the CP gate is closed. In addition to three HbYX motifs, the C-terminal tail of Rpt6 was detected at the interface between α3 and α4 subunits in the open-gate state, suggesting that HbYX engagement is not sufficient to open the CP gate and that insertion of the Rpt6 C-termini triggers to open the CP gate. Our study showed that the gate-opening of the 20S is coupled with the conformational change of the AAA+ ATPases. We further analyzed the gate-opening conformations and elucidated that the CP gate is regulated by the movement of the AAA+ ATPases. The C-terminal tails of the AAA+ ATPases change the position upon ATP binding/hydrolysis and stimulate the gate-opening by disrupting the interaction network of the N-terminus of α subunits (Eisele et al, in preparation). Our study elucidates the structural mechanism how the C-terminal tails of the ATPase induce the conformational change of the CP gate. Detailed structural information of our study provide not only structural basis for gate-opening mechanism upon ATP hydrolysis but also a useful platforms for drug discovery. For example, cancer cell where protein synthetic rate is high and cell cycle is rapid, highly depend on the quality control system. Indeed, the CP inhibitor bortezomib has marked efficacy against multiple myeloma and several lymphoma subtypes. In contrast to the CP inhibitors which several of them are already used for cancer therapy, development of the RP inhibitors have been much less successful, partly due to lack of structural information. Our study which gained better understanding of the structure and mechanistic function of the 19S RP will help drug discovery efforts.