Sprangers, Remco; Li, Xiaoming; Mao, Xinliang; Rubinstein, John L.; Schimmer, Aaron D.; Kay, Lewis E.

Biochemistry (2008), 47(26), 6727-6734 CODEN: BICHAW; ISSN: 0006-2960. English.

The proteasome plays a central role in maintaining cellular homeostasis, in controlling the cell cycle, in removing misfolded proteins that can be toxic, and in regulating the immune system. It is also an important target for novel anticancer drugs, such as bortezomib, a potent inhibitor that has been used successfully in the treatment of multiple myeloma. Here, we show that the antimalaria drug chloroquine inhibits proteasome function in eukaryotic cell exts. and in prepns. of purified 20S archaeal proteasome from Thermoplasma acidophilum. Methyl-TROSY-based NMR spectroscopy expts. conducted with the 670-kDa 20S proteasome localize chloroquine binding to regions between the .alpha. and .beta. subunits of the .alpha.-.beta.-.beta.-.alpha. barrel-like structure, ~20 .ANG. from the proteolytic active sites in this 7-fold sym. mol. Complementary amide TROSY expts. that provide further probes of proteasome-inhibitor interactions were performed on a novel 180 kDa single-ring construct containing only .alpha. subunits, the proper assembly of which was confirmed by electron microscopy. In contrast to the chloroquine-proteasome interaction described here, all previously reported inhibitors of the proteasome, including MG132, bind the catalytic region directly. Consistent with the NMR chem. shift perturbation data reported here that place chloroquine binding distal from sites of proteolysis, we show that MG132 and chloroquine can bind the proteasome simultaneously, further establishing that they exploit two completely sep. binding pockets. Our data thus establish a novel class of proteasome inhibitor that functions via a mechanism distinct from binding to active sites.