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·威勒布兰特结构域蛋白8（VWA8）是一种特征描述欠佳的线粒体基质靶向蛋白，其具有AAA ATPase结构域和ATPase活性，该活性在食用高脂肪饮食的小鼠肝脏中增加。本研究旨在利用CRISPR/Cas9技术敲除培养小鼠肝细胞中的VWA8，以深入了解其功能。本研究采用非偏倚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学能力的测定。代谢组学分析显示，VWA8敲除细胞具有更高的氧化应激和蛋白质降解水平；过氧化氢产生水平的检测揭示了活性氧（ROS）产生水平的增加。蛋白质组学和转录组学分析显示，VWA8敲除细胞中，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更高。VWA8敲除细胞中蛋白丰度升高的模式可归因于肝细胞核因子4α（HNF4α）表达水平的升高。生物能量学实验显示，完整和渗透细胞中碳水化合物氧化以及线粒体和非线粒体脂质氧化的速率更高。抑制剂实验将ROS的产生定位到过氧化物酶体和NOX1/4。VWA8蛋白的挽救作用恢复了野生型表型，而抗氧化剂处理降低了HNF4α的表达水平。因此，VWA8的丢失导致线粒体缺陷，该缺陷可能被NOX4感知，从而导致ROS水平升高，进而导致HNF4α水平升高。HNF4α的补偿性反应导致氧化能力增强，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作用。