Mechanism of autocatalytic activation during proteasome assembly.

Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the ß5 and ß2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.

Rational design of proteasome inhibitors based on the structure of the endogenous inhibitor PI31/Fub1.

Proteasome inhibitors are widely used anticancer drugs. The three clinically approved agents are modified small peptides that preferentially target one of the proteasome's three active sites (ß5) at physiologic concentrations. In addition to these drugs, there is also an endogenous proteasome inhibitor, PI31/Fub1, that enters the proteasome's interior to simultaneously yet specifically inhibit all three active sites. Here, we have used PI31's evolutionarily optimized inhibitory mechanisms to develop a suite of potent and specific ß2 inhibitors. The lead compound strongly inhibited growth of multiple myeloma cells as a standalone agent, indicating the compound's cell permeability and establishing ß2 as a potential therapeutic target in multiple myeloma. The lead compound also showed strong synergy with the existing ß5 inhibitor bortezomib; such combination therapies might help with existing challenges of resistance and severe side effects. These results represent an effective method for rational structure-guided development of proteasome inhibitors.

Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis.

Assembly of the proteasome's core particle (CP), a barrel-shaped chamber of four stacked rings, requires five chaperones and five subunit propeptides. Fusion of two half-CP precursors yields a complete structure but remains immature until active site maturation. Here, using Saccharomyces cerevisiae, we report a high-resolution cryogenic electron microscopy structure of preholoproteasome, a post-fusion assembly intermediate. Our data reveal how CP midline-spanning interactions induce local changes in structure, facilitating maturation. Unexpectedly, we find that cleavage may not be sufficient for propeptide release, as residual interactions with chaperones such as Ump1 hold them in place. We evaluated previous models proposing that dynamic conformational changes in chaperones drive CP fusion and autocatalytic activation by comparing preholoproteasome to pre-fusion intermediates. Instead, the data suggest a scaffolding role for the chaperones Ump1 and Pba1/Pba2. Our data clarify key aspects of CP assembly, suggest that undiscovered mechanisms exist to explain CP fusion/activation, and have relevance for diseases of defective CP biogenesis.

Yeast PI31 inhibits the proteasome by a direct multisite mechanism.

Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome's barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.