Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes.

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.

Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane.

VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.

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.

Characterization of the novel protein KIAA0564 (Von Willebrand Domain-containing Protein 8).

The VWA8 gene was first identified by the Kazusa cDNA project and named KIAA0564. Based on the observation, by similarity, that the protein encoded by KIAA0564 contains a Von Willebrand Factor 8 domain, KIAA0564 was named Von Willebrand Domain-containing Protein 8 (VWA8). The function of VWA8 protein is almost unknown. The purpose of this study was to characterize the tissue distribution, cellular location, and function of VWA8. In mice VWA8 protein was mostly distributed in liver, kidney, heart, pancreas and skeletal muscle, and is present as a long isoform and a shorter splice variant (VWA8a and VWA8b). VWA8 protein and mRNA were elevated in mouse liver in response to high fat feeding. Sequence analysis suggests that VWA8 has a mitochondrial targeting sequence and domains responsible for ATPase activity. VWA8 protein was targeted exclusively to mitochondria in mouse AML12 liver cells, and this was prevented by deletion of the targeting sequence. Moreover, the VWA8 short isoform overexpressed in insect cells using a baculovirus construct had in vitro ATPase activity. Deletion of the Walker A motif or Walker B motif in VWA8 mostly blocked ATPase activity, suggesting Walker A motif or Walker B motif are essential to the ATPase activity of VWA8. Finally, homology modeling suggested that VWA8 may have a structure most confidently similar to dynein motor proteins.

Regulation of NOX1 expression by GATA, HNF-1alpha, and Cdx transcription factors.

NOX1, a member of the NOX family of NADPH oxidases, is expressed primarily in colon epithelium, where it may function in host defense and growth regulation. We investigated factors responsible for its transcriptional regulation in vitro and its expression in vivo. Analysis of promoter constructs in the CaCo2 cell line identified a complex element between -422 and -291 critical for promoter activity. This element contained four sites that bound GATA-4, -5, and -6 in vitro with varied affinities. One site also bound the caudal-related homeodomain proteins Cdx1 and Cdx2, whereas another also bound hepatocyte nuclear factor-1alpha (HNF-1alpha). GATA-6, HNF-1alpha, and Cdx2 also bound to this region in the intact chromatin of CaCo2 cells. These factors demonstrated cooperativity when transactivating the NOX1 promoter. NOX1 mRNA was detected in human colon epithelial cells along the crypt-villus axis. A gradient of NOX1 mRNA expression was seen in the colons of normal as well as germ-free mice, with significantly higher levels in distal compared with proximal segments. The expression gradients of NOX1 mRNA in the colon paralleled those of GATA-6, HNF-1alpha, and Cdx1. These data indicate that developmental, tissue-restricted transcription factors play a key role in NOX1 regulation in vivo.

Development of a synthetic promoter for macrophage gene therapy.

Macrophages have the potential to deliver therapeutic genes to many target tissues. Macrophage-specific synthetic promoters (SPs) generated by random ligation of myeloid/macrophage cis elements had activity up to 100-fold that of a native macrophage promoter in macrophage cell lines, but were minimally active in nonmyeloid cells. Mouse bone marrow cells (BMCs) transduced ex vivo with lentivectors expressing green fluorescent protein (GFP) driven either by an SP (SP-GFP) or a cytomegalovirus (CMV) promoter (CMV-GFP) were used for syngeneic transplantation of lethally irradiated mice. Blood leukocytes showed stable GFP expression for up to 15 months after transplantation. SP-GFP expression was selective for CD11b+ macrophages, whereas CMV-GFP expression was observed in erythrocytes, as well as in both CD11b+ and CD11b- leukocytes. Furthermore, SP-GFP expression was much stronger than CMV-GFP expression in CD11b+ macrophages. apoE-/- BMCs transduced with the lentiviral vector encoding human apoE were used to transplant apoE-/- mice. Macrophage expression of apoE from 10 to 26 weeks of age significantly reduced atherosclerotic lesions in recipient apoE-/- mice. Thus, the novel SPs, especially when combined with lentivectors, are useful for macrophage-specific delivery of therapeutic genes.