Eeyarestatin I inhibits the p97 ATPase to induce tumor cell apoptosis

通过内质网相关降解（ERAD）从内质网（ER）中消除错误折叠的蛋白质涉及底物从ER腔逆转运到胞质溶胶中，由蛋白酶体降解。对于许多底物，逆易位需要泛素化酶和p97-Ufd 1-Np 14 ATP酶复合物的作用，所述泛素化酶使从ER腔出现的底物聚泛素化，所述p97-Ufd 1-Np 14 ATP酶复合物使聚泛素化的底物移位到胞质溶胶中。由p97提取的多肽最终被转移到蛋白酶体中进行破坏。Eeyarestatin I（Eer I）以前被确定为一种有效的逆转录酶抑制剂，但其作用机制尚不清楚。我们的工作表明，EerI与p97 ATP酶结合，阻断p97相关的去泛素化（PAD），这是降解错误折叠的ER蛋白所必需的。我们进一步确定ataxin-3（atx 3），一种p97相关的去泛素化酶，以前参与ERAD，作为EerI影响的目标之一。在没有PAD的情况下，携带未加工的多聚泛素链的底物到达蛋白酶体，但仍被蛋白酶体结合而不被降解。我们的分析建立了一个新的去泛素化过程中蛋白酶体依赖的蛋白质周转的作用。 鉴于泛素-蛋白酶体系统（UPS）最近已成为癌症治疗药物开发的主要目标，我们研究EerI是否具有抗癌活性。事实上，我们发现Eeyarestatin I（Eer I）具有与硼替佐米（bortezastatin）相似的抗肿瘤和生物活性，硼替佐米是一种蛋白酶体抑制剂，临床上用于治疗多种类型的肿瘤。与硼替佐米一样，Eer I诱导的细胞毒性需要仅促凋亡蛋白NOXA的BH 3上调。 我们进一步证明，EerI和硼替佐米激活NOXA通过一个意想不到的机制，需要两个过程之间的合作：第一，这些代理商引起一个集成的应激反应程序在ER激活CREB/ATF转录因子ATF 3和ATF 4。我们表明，ATF 3和ATF 4形成一个复合物能够结合到NOXA启动子，以促进其转录。其次，Eer I和硼替佐米还降低泛素化组蛋白H2 A的水平，以减轻其对NOXA转录的抑制。我们的研究结果确定了一类抗癌药物，整合ER应激反应与表观遗传机制，以诱导细胞死亡。 我们最近开发了体外结合和基于细胞的功能测定，以证明EerI中的含硝基呋喃（NFC）基团是负责细胞毒性的功能域。使用SPR和下拉分析，我们表明，EerI直接结合p97 ATP酶，ERAD机器的一个重要组成部分，通过NFC域。EerI中的芳香族结构域虽然不是p97相互作用所必需的，但可以将EerI定位于ER膜，这提高了其靶向特异性。用另一个保持膜定位的含苯结构域取代芳香族模块产生结构上不同的化合物，尽管如此，该化合物具有与Eer I相似的生物活性。 我们的研究结果揭示了一类双功能化学试剂，可以优先抑制膜结合的p97，破坏ER稳态，诱导肿瘤细胞死亡。这些结果还表明AAA ATP酶p97可能是癌症治疗的潜在药物靶点。

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD) involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which dislocates polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. Eeyarestatin I (EerI) was previously identified as a potent inhibitor of retrotranslocation, but the mechanism of its action is unclear. Our work demonstrates that EerI associates with the p97 ATPase to block p97-associated deubiquitination (PAD), which is essential for the degradation of misfolded ER proteins. We further identify ataxin-3 (atx3), a p97-associated deubiquitinating enzyme previously implicated in ERAD, as one of the targets affected by EerI. In the absence of PAD, substrates carrying unprocessed polyubiquitin chains reach the proteasome, but remain bound by the proteasome without being degraded. Our analyses establish a role for a novel deubiquitinating process in proteasome dependent protein turnover. Given that the ubiquitin-proteasome system (UPS) has recently emerged as a major target for drug development in cancer therapy, we study whether EerI has anti-cancer activity. Indeed, we found that Eeyarestatin I (EerI) has anti-tumor and biologic activities similar to bortezomib, a proteasome inhibitor used in clinic to treat several types of tumors. Like bortezomib, EerI-induced cytotoxicity requires the upregulation of the BH3 only pro-apoptotic protein NOXA. We further demonstrate that both EerI and bortezomib activate NOXA via an unanticipated mechanism that requires cooperation between two processes: First, these agents elicit an integrated stress response program at the ER to activate the CREB/ATF transcription factors ATF3 and ATF4. We show that ATF3 and ATF4 form a complex capable of binding to the NOXA promoter to facilitate its transcription. Second, EerI and bortezomib also decrease the level of ubiquitinated histone H2A to relieve its inhibition on NOXA transcription. Our results identify a class of anti-cancer agents that integrate ER stress response with an epigenetic mechanism to induce cell death. We recently developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI. Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapy.