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.

含冯·威勒布兰德A结构域的蛋白8（VWA8）是一类功能尚不明晰的靶向线粒体基质的蛋白，携带AAA ATP酶结构域并具有ATP酶活性，在高脂饮食喂养小鼠的肝脏中表达水平上调。本研究通过CRISPR/Cas9技术在体外培养的小鼠肝细胞中敲除VWA8，以期解析其生物学功能。研究采用无偏倚组学技术与生物信息学方法指导后续实验，包括氧化应激检测与生物能学能力测定。代谢组学分析显示，VWA8敲除细胞的氧化应激与蛋白质降解水平更高；过氧化氢生成实验证实其活性氧（reactive oxygen species，ROS）生成量显著升高。蛋白质组学与转录组学分析表明，VWA8敲除细胞中线粒体蛋白（电子传递链复合物I、ATP合酶）、过氧化物酶体蛋白以及脂质转运蛋白的表达水平均显著上调。VWA8敲除细胞中蛋白丰度升高的现象，可通过肝细胞核因子4α（hepatocyte nuclear factor 4 α，HNF4α）表达水平上调得到解释。生物能学实验显示，完整细胞与透化细胞的碳水化合物氧化、线粒体及非线粒体脂质氧化速率均有所提升。抑制剂实验将ROS生成位点定位至过氧化物酶体与NOX1/4。回补VWA8蛋白可恢复野生型表型，而抗氧化剂处理能够降低HNF4α的表达水平。综上，VWA8缺失会引发线粒体功能缺陷，该缺陷可被NOX4感知，进而导致ROS水平升高，最终促使HNF4α表达上调。这种代偿性HNF4α应答会提升细胞的氧化能力，进而进一步增加ROS生成量。我们推测，VWA8作为一类AAA ATP酶蛋白，在线粒体蛋白质质量调控中发挥重要作用。