Marín-Buera Lorena, García-Bartolomé Alberto, Morán María, López-Bernardo Elia, Cadenas Susana, Hidalgo Beatriz, Sánchez Ricardo, Seneca Sara, Arenas Joaquín, Martín Miguel A, Ugalde Cristina
Instituto de Investigación, Hospital Universitario 12 de Octubre, Madrid 28041, Spain.
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid, Spain.
J Proteomics. 2015 Jan 15;113:38-56. doi: 10.1016/j.jprot.2014.09.007. Epub 2014 Sep 18.
We have analyzed the cellular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in BCS1L, a major genetic cause of mitochondrial complex III enzyme deficiency. Mutant fibroblasts exhibited low oxygen consumption rates and intracellular ATP levels, indicating that the main altered molecular event probably is a limited respiration-coupled ATP production through the OXPHOS system. Two-dimensional DIGE and MALDI-TOF/TOF mass spectrometry analyses unambiguously identified 39 proteins whose expression was significantly altered in complex III-deficient fibroblasts. Extensive statistical and cluster analyses revealed a protein profile characteristic for the BCS1L mutant fibroblasts that included alterations in energy metabolism, cell signaling and gene expression regulation, cytoskeleton formation and maintenance, and intracellular stress responses. The physiological validation of the predicted functional adaptations of human cultured fibroblasts to complex III deficiency confirmed the up-regulation of glycolytic enzyme activities and the accumulation of branched-chain among other amino acids, suggesting the activation of anaerobic glycolysis and cellular catabolic states, in particular protein catabolism, together with autophagy as adaptive responses to mitochondrial respiratory chain dysfunction and ATP deficiency. Our data point to an overall metabolic and genetic reprogramming that could contribute to explain the clinical manifestations of complex III deficiency in patients.
Despite considerable knowledge about their genetic origins, the pathophysiological mechanisms that contribute to the clinical manifestations of mitochondrial disorders remain poorly understood. We have investigated the molecular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in the BCS1L gene, a primary cause of mitochondrial complex III enzyme deficiency. Two-dimensional DIGE together with MALDI-TOF/TOF mass spectrometry and physiological validation analyses revealed a significant metabolic and genetic reprogramming as an adaptive response to mitochondrial respiratory chain dysfunction. Our data provide information about specific protein targets that regulate the transmitochondrial functional responses to complex III deficiency, thereby opening new doors for future research.
我们分析了原发性皮肤成纤维细胞中发生的细胞途径和代谢适应性变化，这些成纤维细胞来自患有线粒体复合物III酶缺乏症的主要遗传病因——BCS1L基因突变的患者。突变的成纤维细胞表现出低氧消耗率和细胞内ATP水平，表明主要的分子改变事件可能是通过氧化磷酸化系统进行的有限的呼吸偶联ATP产生。二维差异凝胶电泳（DIGE）和基质辅助激光解吸/电离飞行时间/飞行时间（MALDI-TOF/TOF）质谱分析明确鉴定出39种蛋白质，其在复合物III缺陷的成纤维细胞中的表达有显著改变。广泛的统计和聚类分析揭示了BCS1L突变成纤维细胞特有的蛋白质谱，包括能量代谢、细胞信号传导和基因表达调控、细胞骨架形成和维持以及细胞内应激反应的改变。对人类培养的成纤维细胞对复合物III缺乏的预测功能适应性的生理学验证证实了糖酵解酶活性的上调以及支链氨基酸等其他氨基酸的积累，表明厌氧糖酵解和细胞分解代谢状态的激活，特别是蛋白质分解代谢，以及自噬作为对线粒体呼吸链功能障碍和ATP缺乏的适应性反应。我们的数据表明存在整体代谢和基因重编程，这可能有助于解释患者线粒体复合物III缺乏的临床表现。
尽管对线粒体疾病的遗传起源有相当多的了解，但导致线粒体疾病临床表现的病理生理机制仍知之甚少。我们研究了来自BCS1L基因突变患者的原发性皮肤成纤维细胞中发生的分子途径和代谢适应性变化，BCS1L基因突变是线粒体复合物III酶缺乏的主要原因。二维DIGE结合MALDI-TOF/TOF质谱分析和生理学验证分析揭示了显著的代谢和基因重编程，作为对线粒体呼吸链功能障碍的适应性反应。我们的数据提供了有关调节对复合物III缺乏的跨线粒体功能反应的特定蛋白质靶点的信息，从而为未来的研究打开了新的大门。
