Arrington David D, Schnellmann Rick G
Department of Pharmaceutical Sciences, Medical University of South Carolina, 280 Calhoun St., Charleston, SC 29425, USA.
Am J Physiol Cell Physiol. 2008 Feb;294(2):C641-50. doi: 10.1152/ajpcell.00400.2007. Epub 2007 Dec 19.
Oxygen-regulated protein 150 (ORP150) is an inducible endoplasmic reticulum (ER) chaperone molecule that is upregulated after numerous cellular insults and has a cytoprotective role in renal, neural, and cardiac models of ischemia-reperfusion injury. ORP150 also has been shown to play a role in cellular Ca(2+) homeostasis, and in turn, regulating calpain activity. In this study, we identified ORP150 in whole rat renal cortical mitochondria and matrix fractions, demonstrated the targeting of an ORP150-GFP construct to the mitochondria of NIH-3T3 cells, and showed that the NH(2)-terminal 13 amino acids of ORP150 are sufficient for this translocation. ORP150 expression was found to be regulated by the anti-C/enhancer-binding protein homologous protein (CHOP)/GADD153 transcription factor and ORP150 levels increased in the mitochondria and ER of COS-7 cells after diverse stresses, including hypoxia, serum starvation, prolyl hydroxylase inhibition with dimethyloxaloylglycine, and exposure to tunicamycin, ethidium, bromide, and 2-deoxyglucose. Induction of the mitochondrial specific stress response in COS-7 cells through expression of an ornithine transcarbamylase mutant (Delta OTC) increased mitochondrial ORP150 levels and mitochondrial calpain activity. To determine whether mitochondrial ORP150 and mitochondrial calpain 10 interact, rat cortical mitochondria exposed to Ca(2+) resulted in ORP150 cleavage in a calpain inhibitor-dependent manner, revealing that ORP150 is a substrate and may be regulated by calpain 10. These data reveal a novel cellular localization for ORP150 and that mitochondrial ORP150 is upregulated by CHOP/GADD153 in response to mitochondrial and ER stress. Our data also reveal that ORP150 is a substrate for mitochondrial calpain 10.
氧调节蛋白150（ORP150）是一种可诱导的内质网（ER）伴侣分子，在多种细胞损伤后上调，在肾、神经和心脏缺血再灌注损伤模型中具有细胞保护作用。ORP150也已被证明在细胞钙（Ca2+）稳态中起作用，进而调节钙蛋白酶活性。在本研究中，我们在整个大鼠肾皮质线粒体和基质组分中鉴定出ORP150，证明了ORP150-GFP构建体靶向NIH-3T3细胞的线粒体，并表明ORP150的NH2末端13个氨基酸足以实现这种转运。发现ORP150的表达受抗C/增强子结合蛋白同源蛋白（CHOP）/GADD153转录因子调节，在包括缺氧、血清饥饿、用二甲基草酰甘氨酸抑制脯氨酰羟化酶以及暴露于衣霉素、溴化乙锭和2-脱氧葡萄糖等多种应激后，COS-7细胞的线粒体和内质网中ORP150水平升高。通过表达鸟氨酸转氨甲酰酶突变体（Delta OTC）在COS-7细胞中诱导线粒体特异性应激反应，增加了线粒体ORP150水平和线粒体钙蛋白酶活性。为了确定线粒体ORP150和线粒体钙蛋白酶10是否相互作用，暴露于Ca2+的大鼠皮质线粒体导致ORP150以钙蛋白酶抑制剂依赖的方式裂解，表明ORP150是一种底物，可能受钙蛋白酶10调节。这些数据揭示了ORP150的一种新的细胞定位，并且线粒体ORP150在响应线粒体和内质网应激时被CHOP/GADD153上调。我们的数据还揭示了ORP150是线粒体钙蛋白酶10的底物。
