Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH₄) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH₄ triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators.

中文翻译：

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Hsp70:CHIP 泛素化功能障碍但不是天然神经元 NO 合酶。

正在开发热休克蛋白 (Hsp) 70 调节剂，以增强各种蛋白质错误折叠疾病中有毒蛋白质的去除。在我们对 Hsp90 和 Hsp70 伴侣系统的客户神经元一氧化氮合酶 (nNOS) 的研究过程中，我们已经确定了血红素或四氢生物蝶呤 (BH ₄) 改变和丢失通过 Hsp70 相互作用蛋白 (CHIP) 的 Hsp70 相关 E3 连接酶 c 端触发泛素化和随后的细胞降解。尽管在细胞中 Hsp90 和 Hsp70 协同工作以维持蛋白质质量控​​制，但在本研究中，我们专门开发了一种检测方法来评估 Hsp70:CHIP 复合物对灭活 nNOS 的选择性。我们开发了一种高度灵敏的 ELISA 来测量 Hsp70:CHIP 依赖​​性 nNOS 泛素化，而不受 CHIP 直接泛素化的干扰，正如 Bcl-2 相关的 athanogene 1-M 完全消除泛素化所证明的那样。为了进一步验证我们证明的测定，JG-98，一种作用于 Hsp70 但不作用于其无活性结构类似物 JG-258 的红花青苷化合物，可将 nNOS 的泛素化增强 3 倍。利用该测定，我们已经证明 Hsp70：CHIP 复合物优先泛素化缺乏血红素的 nNOS（apo-nNOS），而不是含有血红素的 nNOS（holo-nNOS）。此外，nNOS 结合 BH 的消耗₄触发 Hsp70:CHIP 复合物对全息-nNOS 的泛素化。最重要的是，JG-98 被证明可以增强只有功能失调的 nNOS 的泛素化，同时不影响天然的功能性 nNOS。因此，增强 Hsp70:CHIP 介导的泛素化不影响天然蛋白质的发现具有重要的药理学意义。此外，开发一种简便的体外 Hsp70 方法：CHIP 介导的泛素化将有利于测试其他 Hsp70 调节剂。