El Hindy Maya, Hezwani Mohammed, Corry David, Hull Jonathon, El Amraoui Farah, Harris Matthew, Lee Christopher, Forshaw Thomas, Wilson Andrew, Mansbridge Abbe, Hassler Martin, Patel Vinood B, Kehoe Patrick Gavin, Love Seth, Conway Myra Elizabeth
1 Faculty of Health and Life Sciences, University of the West of England , Coldharbor Lane, Bristol, United Kingdom .
Antioxid Redox Signal. 2014 Jun 1;20(16):2497-513. doi: 10.1089/ars.2012.4869. Epub 2013 Dec 21.
The human branched-chain aminotransferase proteins (hBCATm and hBCATc) are regulated through oxidation and S-nitrosation. However, it remains unknown whether they share common redox characteristics to enzymes such as protein disulfide isomerase (PDI) in terms of regulating cellular repair and protein misfolding.
Here, similar to PDI, the hBCAT proteins showed dithiol-disulfide isomerase activity that was mediated through an S-glutathionylated mechanism. Site-directed mutagenesis of the active thiols of the CXXC motif demonstrates that they are fundamental to optimal protein folding. Far Western analysis indicated that both hBCAT proteins can associate with PDI. Co-immunoprecipitation studies demonstrated that hBCATm directly binds to PDI in IMR-32 cells and the human brain. Electron and confocal microscopy validated the expression of PDI in mitochondria (using Mia40 as a mitochondrial control), where both PDI and Mia40 were found to be co-localized with hBCATm. Under conditions of oxidative stress, this interaction is decreased, suggesting that the proposed chaperone role for hBCATm may be perturbed. Moreover, immunohistochemistry studies show that PDI and hBCAT are expressed in the same neuronal and endothelial cells of the vasculature of the human brain, supporting a physiological role for this binding.
This study identifies a novel redox role for hBCAT and confirms that hBCATm differentially binds to PDI under cellular stress.
These studies indicate that hBCAT may play a role in the stress response of the cell as a novel redox chaperone, which, if compromised, may result in protein misfolding, creating aggregates as a key feature in neurodegenerative conditions such as Alzheimer's disease.
人类支链氨基转移酶蛋白（hBCATm和hBCATc）通过氧化和S-亚硝基化进行调节。然而，就调节细胞修复和蛋白质错误折叠而言，它们是否与蛋白质二硫键异构酶（PDI）等酶具有共同的氧化还原特性仍不清楚。
在此，与PDI类似，hBCAT蛋白表现出通过S-谷胱甘肽化机制介导的二硫醇-二硫化物异构酶活性。CXXC基序活性硫醇的定点诱变表明，它们对于最佳蛋白质折叠至关重要。Far Western分析表明，两种hBCAT蛋白都可以与PDI结合。免疫共沉淀研究表明，hBCATm在IMR-32细胞和人脑中直接与PDI结合。电子显微镜和共聚焦显微镜验证了PDI在线粒体中的表达（使用Mia40作为线粒体对照），发现PDI和Mia40都与hBCATm共定位。在氧化应激条件下，这种相互作用减弱，表明hBCATm所提出的伴侣作用可能受到干扰。此外，免疫组织化学研究表明，PDI和hBCAT在人脑脉管系统的相同神经元和内皮细胞中表达，支持这种结合的生理作用。
本研究确定了hBCAT的一种新的氧化还原作用，并证实hBCATm在细胞应激下与PDI的结合存在差异。
这些研究表明，hBCAT可能作为一种新的氧化还原伴侣在细胞应激反应中发挥作用，如果这种作用受损，可能导致蛋白质错误折叠，形成聚集体，这是阿尔茨海默病等神经退行性疾病的关键特征。
