Rapamycin Allosterically Inhibits the Proteasome

Abstract

Rapamycin is a canonical allosteric inhibitor of the mammalian tarpet of rapamycin (mTOR) kinase with immunosuppressive and proapoptotic activities. We found that in vitro rapamycin also regulates the proteasome, which is an essential intracellular protease of the ubiquitin-proteasome pathway. Rapamycin inhibits proteinase and selected peptidase activities of the catalytic core proteasome at low micromolar concentrations. Moreover, the drug interferes with binding of the 19S cap essential for processing of polyubiquitinylated substrates and with the PA200 proteasome activator to the 20S catalytic core proteasome. These protein complexes are known to bind to specific grooves on the a face region of the 20S core. Treatment with rapamycin affects the conformational dynamics of the proteasomal gate, which is centrally positioned within the a face and allosterically regulated element responsible for the intake of substrates. We showed that rapamycin shares all the proteasome targeting properties not only with other two-domain, closed-ring analogs (rapalogs) but also with its single domain mimics and seco-rapamycin, which is the first in vivo open-ring metabolite of rapamycin that does not affect mTOR. We hypothesize that rapamycin and related compounds bind to the a face and allosterically impact proteasome function. This article discusses the implications of our findings for the mechanism of in vivo actions of rapamycin and for the design of novel allosteric drugs targeting the proteasome.

Harnessing Proteasome Dynamics and Allostery in Drug Design

“…AFM imaging of the human 20S proteasomes revealed that rapamycin shifts the conformational equilibrium toward a higher abundance of the open particles (Fig. 4E) (102). We speculate that on docking to the a face, rapamycin exploits the allosteric route used by gate-opening regulatory modules.…”

“…At high nanomolar concentrations of rapamycin, post-acidic cleavages are inhibited by a noncompetitive mechanism. At low micromolar concentrations, the ChT-L peptidase is strongly inhibited, while the T-L peptidase activity is increased twofold (102). Interestingly, rapamycin abolishes activation of the 20S core by 19S complex or by peptides containing the HbYX motif.…”

“…Interestingly, rapamycin abolishes activation of the 20S core by 19S complex or by peptides containing the HbYX motif. The most straightforward explanation of the finding would be competition of rapamycin and the peptides for binding to the a face (102). Indeed, molecular modeling strongly suggests that rapamycin may fit very well into selected grooves on the a face (Bohmann, Gaczynska, and Osmulski, Unpublished observations).…”

“…In vivo treatment with rapamycin induces numerous pleiotropic effects, including immunosuppression, proapoptotic and anti-cancer actions, as well as prolonging longevity (55,58,133). In vitro, at low micromolar concentrations rapamycin inhibits the degradation of casein, a poorly structured protein, by the latent human 20S core (102). The various peptidase activities of the 20S core respond to varying concentrations of rapamycin differently.…”

“…When the rapamycin-treated 20S proteasomes are subsequently treated with a substrate, the partition of open and closed forms stays at 60% and 40%, respectively, and does not shift to 75% and 25%, even if the active sites of the core are not directly blocked and, thus, ought to be capable of engaging substrates (Fig. 4E) (102). The nature of the link between apparent rapamycin-induced perturbation in conformational dynamics and the effects on proteasome catalytic activity is purely speculative at this point.…”