TROSY-based NMR evidence for a novel class of 20S proteasome inhibitors
TROSY-based NMR evidence for a novel class of 20S proteasome inhibitors
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DOI：
10.1021/bi8005913
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发表时间：
2008-07-01
期刊：
影响因子：
2.9
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Sprangers, Remco;Li, Xiaoming;Kay, Lewis E.
蛋白酶体在维持细胞内稳态、控制细胞周期、清除可能有毒的错误折叠的蛋白质以及调节免疫系统方面发挥着核心作用。它也是新型抗癌药物的重要靶点，如Bortezomib，一种已成功用于治疗多发性骨髓瘤的有效抑制剂。在这里，我们展示了抗疟疾药物氯喹抑制真核细胞提取物和从嗜酸热浆中纯化的20S古生菌蛋白酶体的蛋白酶体功能。用670 kDa的20S蛋白酶体进行的基于甲基TROSY的核磁共振波谱实验定位了氯喹与α-β-β-α桶状结构的α和β亚基之间的区域，距离这个7倍对称分子中的蛋白分解活性部位20埃。在一个仅含有α亚基的180 kDa单环结构上进行了互补酰胺TROSY实验，为蛋白酶体与抑制剂的相互作用提供了进一步的探针，电子显微镜证实了该结构的正确组装。与这里描述的氯喹-蛋白酶体相互作用不同，所有先前报道的蛋白酶体抑制剂，包括MG132，都直接与催化区结合。与报道的核磁共振化学位移扰动数据一致，我们证明了MG132和氯喹可以同时与蛋白酶体结合，进一步证明了它们利用了两个完全分离的结合口袋。因此，我们的数据建立了一类新的蛋白酶体抑制剂，其功能通过与活性位点结合的不同机制发挥作用。
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 extracts and in preparations of purified 20S archaeal proteasome from Thermoplasma acidophilium. Methyl-TROSY-based NMR spectroscopy experiments 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 angstrom from the proteolytic active sites in this 7-fold symmetric molecule. Complementary amide TROSY experiments 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 chemical 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 separate binding pockets. Our data thus establish a novel class of proteasome inhibitor that functions via a mechanism distinct from binding to active sites.