Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes

von
Willebrand A domain-containing protein 8 (VWA8) is a poorly characterized,
mitochondrial matrix-targeted protein with an AAA ATPase domain
and ATPase activity that increases in livers of mice fed a high-fat
diet. This study was undertaken to use CRISPR/Cas9 to delete VWA8
in cultured mouse hepatocytes and gain insight into its function.
Unbiased omics techniques and bioinformatics were used to guide subsequent
assays, including the assessment of oxidative stress and the determination
of bioenergetic capacity. Metabolomics analysis showed VWA8 null cells
had higher levels of oxidative stress and protein degradation; assays
of hydrogen peroxide production revealed higher levels of production
of reactive oxygen species (ROS). Proteomics and transcriptomics analyses
showed VWA8 null cells had higher levels of expression of mitochondrial
proteins (electron transport-chain Complex I, ATP synthase), peroxisomal
proteins, and lipid transport proteins. The pattern of higher protein
abundance in the VWA8 null cells could be explained by a higher level
of hepatocyte nuclear factor 4 α (HNF4α) expression. Bioenergetic
assays showed higher rates of carbohydrate oxidation and mitochondrial
and nonmitochondrial lipid oxidation in intact and permeabilized cells.
Inhibitor assays
localized sites of ROS production to peroxisomes and NOX1/4. The rescue
of VWA8 protein restored the wild-type phenotype, and treatment with
antioxidants decreased the level of HNF4α expression. Thus,
loss of VWA8 produces a mitochondrial defect that may be sensed by
NOX4, leading to an increase in the level of ROS that results in a
higher level of HNF4α. The compensatory HNF4α response
results in a higher oxidative capacity and an even higher level of
ROS production. We hypothesize that VWA8 is an AAA ATPase protein
that plays a role in mitochondrial protein quality.

冯·维尔莱布兰特结构域蛋白8（VWA8）是一种描述不充分的、靶向线粒体基质的蛋白，其具备AAA ATPase结构域及ATPase活性，在喂食高脂饮食的小鼠肝脏中该活性有所上升。本研究旨在利用CRISPR/Cas9技术敲除培养小鼠肝细胞中的VWA8，以深入探究其功能。通过无偏见的组学技术和生物信息学方法指导后续实验，包括氧化应激的评估和生物能量能力的测定。代谢组学分析显示，VWA8基因敲除的细胞具有更高的氧化应激和蛋白质降解水平；氢过氧化物的产生实验揭示了活性氧（ROS）产量的增加。蛋白质组学和转录组学分析显示，VWA8基因敲除的细胞中，线粒体蛋白（电子传递链复合物I、ATP合酶）、过氧化物酶体蛋白和脂质转运蛋白的表达水平升高。VWA8基因敲除细胞中蛋白质丰度升高的模式可以归因于肝细胞核因子4α（HNF4α）表达水平的增加。生物能量学实验显示，在完整细胞和通透化细胞中，碳水化合物氧化和线粒体及非线粒体脂质氧化速率均有所升高。抑制剂实验将ROS的产生定位到过氧化物酶体和NOX1/4。VWA8蛋白的恢复恢复了野生型表型，而抗氧化剂处理则降低了HNF4α的表达水平。因此，VWA8的缺失导致线粒体缺陷，这种缺陷可能被NOX4感知，从而导致ROS水平升高，进而导致HNF4α水平升高。补偿性HNF4α反应导致氧化能力增强以及ROS产生水平更高。我们假设VWA8是一种AAA ATPase蛋白，它在线粒体蛋白质量调控中发挥着重要作用。