Background

Misfolded proteins are formed in the endoplasmic reticulum (ER) due to diverse stimuli including oxidant production, calcium disturbance, and inflammatory factors. Accumulation of these non-native proteins in the ER evokes cellular stress involving the activation of unfolded protein response (UPR) and the execution of ER-associated degradation (ERAD). Naturally-occurring plant compounds are known to interfere with UPR due to their antioxidant and anti-inflammatory activities, leading to inhibition of ER stress. However, there are few studies dealing with the protective effects of natural compounds on the functionality of ERAD.

Purpose

The current study examined whether asaronic acid enhanced ubiquitin-proteasomal degradation in J774A.1 murine macrophages exposed to 7β-hydroxycholesterol, a risk factor for atherosclerosis. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. Little is known regarding the effects of asaronic acid on the ERAD process and the ubiquitin-proteasomal degradation.

Methods and results

Murine macrophages were incubated with 28 μM 7β-hydroxycholesterol in absence and presence of 1–20 μΜ asaronic acid for up to 24 h. Nontoxic asaronic acid in macrophage diminished the activation of the ER stress sensors of ATF6, IRE1 and PERK stimulated by 7β-hydroxycholesterol. This methoxybenzoic acid down-regulated the oxysterol-induced expression of EDEM1, OS9, Sel1L-Hrd1 and p97/VCP1, all required for the recognition, recruitment and dislocation of misfolded proteins. On the other hand, asaronic acid enhanced the ubiquitin-proteasomal degradation of non-native proteins dislocated to the cytosol by 7β-hydroxycholesterol, which entailed the induction of the chaperones of Hsp70 and CHIP and the increased colocalization of ubiquitin and proteasomes. Taken together, asaronic acid attenuated the induction of the UPR-associated sensors and the dislocation-linked transmembrane components in the ER. Conversely, this compound enhanced the proteasomal degradation of dislocated non-native proteins in concert with the chaperones of Hsp70 and CHIP through ubiquitination.

Conclusion

These observations demonstrate that asaronic acid may be a potent atheroprotective agent as a natural chaperone targeting ER stress-associated macrophage injury.

中文翻译：

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细辛酸抑制内质网应激传感器并增强负载 7β-羟基胆固醇的巨噬细胞中泛素-蛋白酶体降解的功能

背景

在内质网 (ER) 中由于多种刺激（包括氧化剂产生、钙紊乱和炎症因子）形成错误折叠的蛋白质。这些非天然蛋白质在 ER 中的积累引起细胞应激，包括未折叠蛋白反应 (UPR) 的激活和 ER 相关降解 (ERAD) 的执行。已知天然存在的植物化合物因其抗氧化和抗炎活性而干扰 UPR，从而抑制内质网应激。然而，很少有研究涉及天然化合物对 ERAD 功能的保护作用。

目的

目前的研究检查了细辛酸是否增强了 J774A.1 小鼠巨噬细胞中的泛素-蛋白酶体降解，该细胞暴露于 7β-羟基胆固醇，这是动脉粥样硬化的危险因素。细辛酸（2,4,5-三甲氧基苯甲酸）被确定为紫苏成分之一，具有抗糖尿病和抗炎作用。关于细辛酸对 ERAD 过程和泛素-蛋白酶体降解的影响知之甚少。

方法和结果

在不存在和存在 1–20 μM 细辛酸的情况下，将鼠巨噬细胞与 28 μM 7β-羟基胆固醇一起孵育长达 24 小时。巨噬细胞中的无毒细辛酸减少了由 7β-羟基胆固醇刺激的 ATF6、IRE1 和 PERK 的内质网应激传感器的激活。这种甲氧基苯甲酸下调氧甾醇诱导的 EDEM1、OS9、Sel1L-Hrd1 和 p97/VCP1 的表达，这些都是错误折叠蛋白的识别、募集和错位所必需的。另一方面，细辛酸增强了被 7β-羟基胆固醇脱位到细胞质中的非天然蛋白质的泛素-蛋白酶体降解，这需要诱导 Hsp70 和 CHIP 的伴侣蛋白以及泛素和蛋白酶体的共定位增加。综合起来，细辛酸减弱了内质网中与 UPR 相关的传感器和与位错相关的跨膜成分的诱导。相反，这种化合物通过泛素化与 Hsp70 和 CHIP 的分子伴侣协同增强了脱位的非天然蛋白质的蛋白酶体降解。

结论

这些观察结果表明，作为靶向内质网应激相关巨噬细胞损伤的天然伴侣，细辛酸可能是一种有效的动脉粥样硬化保护剂。