Mol083873 104..113

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.