Luo Moulun, Ma Wuqiong, Zapata-Bustos Rocio, Willis Wayne T, Mandarino Lawrence J
Department of Medicine, Division of Endocrinology, USA.
Center for Disparities in Diabetes, Obesity, and Metabolism, The University of Arizona, Tucson, AZ, 85724, USA.
Biochem Biophys Rep. 2021 Feb 15;26:100928. doi: 10.1016/j.bbrep.2021.100928. eCollection 2021 Jul.
VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.
VWA8（含血管性血友病因子A结构域蛋白8）是一种定位于线粒体基质的AAA+ATP酶，在高能量组织中广泛表达。最初发现，在喂食高脂肪饮食的小鼠肝脏中，VWA8的丰度更高，在分化的小鼠AML12肝细胞中删除VWA8基因意外地产生了一种表型，即线粒体和非线粒体氧化代谢增强、主要由NADPH氧化酶产生的活性氧（ROS）增加以及肝细胞核因子4α（HNF4a）表达增加。本研究的目的，一是确定VWA8基因缺失的肝细胞中线粒体氧化能力增强是否是电子传递链和氧化磷酸化各方面能力增强的结果，二是测量线粒体嵴的密度和线粒体含量，以确定线粒体氧化能力增强是否伴随着嵴面积和线粒体丰度增加。使用CRISPR/Cas9删除VWA8基因的肝细胞中，电子传递链复合物I、II、III和IV的活性均更高。先前使用无偏差蛋白质组学方法在VWA8基因缺失的肝细胞中测定的电子传递链复合物I、III和ATP合酶中蛋白质丰度的比较结果与活性测定结果一致。使用电子显微镜和体视学方法对电子传递链复合物所在部位的线粒体嵴进行了定量分析。VWA8基因缺失的细胞中，每线粒体面积的嵴密度几乎高出两倍（P<0.01），线粒体面积高出两倍（P<0.05）。本研究结果使我们能够得出结论，尽管VWA8基因缺失的细胞中ROS持续产生增加，但仍维持了整体线粒体代偿反应，导致线粒体氧化能力总体增强。
