Cooperativity in Proteasome Core Particle Autocatalytic Processing

Proteasomes are complex proteases that can be found in bacteria, archaea, and eukaryotes. The formation of core particle (CP) is not trivial as it involves 28 polypeptides forming a cylindrical structure that undergoes maturation by processing of propeptides present on the β subunits. The propeptides play an important role during assembly and different roles have been described. Here, using bioinformatics approaches we identified three distinct regions in bacterial β propeptides. Molecular Dynamics simulations indicated the presence of a flexible loop‐like region (region III) that extended out of the half CP assembly intermediate for periods. We therefore hypothesized a role for region III in the dimerization of two half CPs. To test this, we used in vitro reconstitution assays with Rhodococcus erythropolis α and β subunits. Mutations in region III caused a severely reduced rate of dimerization. Region II has previously been shown to be structured and function in the association of β with two α's. When we mutated the N‐terminal region, region I, we saw an increase in rate of dimerization but a much slower rate of maturation. Since maturation requires autocatalytic removal of β‐propeptides, this suggests that the region I plays a role in efficient cleavage of the propeptide from β. The reduced maturation rate of Region I mutants allowed us to capture both pre‐holoproteasome (i.e. assembled CP with propeptides present) and holoproteasome complexes simultaneously in a reconstitution. The absence of any other intermediate forms, where a subset of the propeptides were processed, indicates that while the propeptides are processed slowly in general, the switch to holoproteasome happens within a very short time frame. This suggests the existence of cooperativity phenomenon in CP maturation. Probing for mechanisms that might explain this cooperativity, we tested for the ability of a processed β subunit to trim the propeptide of a neighboring β subunit, which we could envision as a trigger for faster processing or a conformational change. Indeed, we were able to detect cross processing of propeptides in this Rhodococcus erythropolis reconstitution assay by neighboring active β subunits both from within the same β ring as well as across the dimer interface. In conclusion, we identified novel roles of the bacterial propeptide in regulating the dimerization through region III, as well as its role in the regulating the maturation of pre‐holoproteasome through region I. Presence of two distinct populations of active and inactive CPs shows that dimerization and maturation are distinct processes. Support or Funding Information NIGMS R15 GM112142, R01 GM118660, K‐INBRE P20 GM103418 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .