Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin
Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin
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DOI：
10.1091/mbc.e03-07-0506
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发表时间：
2004-04-01
影响因子：
3.3
通讯作者：
Tortorella, D
中科院分区：
文献类型：
--
作者：
Fiebiger, E;Hirsch, C;Tortorella, D
哺乳动物内质网(ER)到胞浆的降解途径用于处理错误折叠的蛋白质，是以蛋白质降解受损为特征的疾病治疗干预的一个有吸引力的靶点。这样做的能力受到可用的少数特定抑制剂的阻碍，以及我们对这一途径中涉及的个别步骤的有限了解。表达I类主要组织相容性复合体(MHC)重链增强型绿色荧光蛋白(EGFP)融合蛋白和催化I类MHC EGFP报告基因错位的人巨细胞病毒蛋白US11的细胞只显示很少的荧光。蛋白酶体抑制剂通过稳定EGFP标记的MHC I类分子来增加它们的荧光。我们利用信号强度的变化作为读数来筛选16,320个化合物的化学库，并鉴定了两个结构相关的化合物(eeyarestatin I和II)，它们干扰了EGFP-重链及其内源性未经修饰的I类MHC重链的降解。Eeyarestatin I还抑制了第二个错误折叠的I型膜蛋白T细胞受体a的降解。这两个化合物都稳定了内质网膜中的这些错位底物，但没有阻止胞浆底物的蛋白酶体翻转。因此，新的抑制剂必须干扰蛋白酶体降解之前的步骤。因此，eeyarestatin I的使用允许定义一种新的位错中间体。
The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor a. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.