Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular Severo Ochoa CSIC-UAM, Departamento de Biología Molecular. Universidad Autónoma de Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), IdiPAZ, Madrid, Spain.
J Cell Biochem. 2013 Jan;114(1):183-91. doi: 10.1002/jcb.24316.
Oxidative stress has been described as a putative disease mechanism in pathologies associated with an elevation of homocysteine. An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders of cobalamin metabolism, particularly with methylmalonic aciduria (MMA) combined with homocystinuria cblC type. In this work, we have evaluated several parameters related to oxidative stress and apoptosis in fibroblasts from patients with homocystinuria due to defects in the MTR, MTRR, and MTHFR genes involved in the remethylation pathway of homocysteine. We have also evaluated these processes by knocking down the MTRR gene in cellular models, and complementation by transducing the wild-type gene in cblE mutant fibroblasts. All cell lines showed a significant increase in ROS content and in MnSOD expression level, and also a higher rate of apoptosis with similar levels to the ones in cblC fibroblasts. The amount of the active phosphorylated forms of p38 and JNK stress-kinases was also increased. ROS content and apoptosis rate increased in control fibroblasts and in a glioblastoma cell line by shRNA-mediated silencing of MTRR gene expression. In contrast, wild-type MTRR gene corrected mutant cell lines showed a decrease in ROS and apoptosis levels. To the best of our knowledge, this study provides the first evidence that an impaired remethylation capacity due to low MTRR and MTR activity might be partially responsible for stress response.
氧化应激被描述为与同型半胱氨酸升高相关的病理学中的一种潜在疾病机制。活性氧 (ROS) 产生增加和细胞凋亡率增加与钴胺素代谢的几种紊乱有关，尤其是与甲基丙二酸尿症 (MMA) 伴同型胱氨酸尿症 cblC 型有关。在这项工作中，我们评估了几种与氧化应激和细胞凋亡相关的参数，这些参数涉及到同型半胱氨酸再甲基化途径中涉及的 MTR、MTRR 和 MTHFR 基因缺陷导致的同型胱氨酸尿症患者的成纤维细胞。我们还通过敲低细胞模型中的 MTRR 基因，并通过转导 cblE 突变型成纤维细胞中的野生型基因来评估这些过程。所有细胞系均显示 ROS 含量和 MnSOD 表达水平显著增加，细胞凋亡率也较高，与 cblC 成纤维细胞相似。应激激酶 p38 和 JNK 的磷酸化形式的活性也增加。通过 shRNA 介导的 MTRR 基因表达沉默，对照成纤维细胞和神经胶质瘤细胞系中的 ROS 含量和细胞凋亡率增加。相比之下，野生型 MTRR 基因纠正了突变细胞系中的 ROS 和细胞凋亡水平降低。据我们所知，这项研究首次提供了证据，表明由于低 MTRR 和 MTR 活性导致的再甲基化能力受损可能是应激反应的部分原因。
