Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunction

Sirtuin expression in HEK293 does not affect (A) the rate of cellular growth; (B) mitochondrial mass as judged by western blotting of electron transport chain components; (C)steady-state ATP under basal conditions or after the addition of the metabolic inhibitors etomoxir (Eto, 100 µM), oligomycin (oligo, 1 µM), 2-deoxyglucose (2DG, 100 mM), or combinations thereof; or (D) intramitochondrial NAD⁺.

<p>Growth was measured in quadruplicate wells in two separate experiments which were averaged. ATP was measured in triplicate wells containing equal numbers of cells in two separate experiments which were averaged. NAD+ was measured in three separate preparations of mitochondria and the results averaged. All data are means and standard deviations.</p

Effect of mitochondrial sirtuin expression on glycolysis.

<p>Seahorse extracellular acidification rates (A) were measured in quadruplicate wells containing equal numbers of cells. The experiment was repeated with similar results. Data collected over the first 30 minutes were averaged to yield the basal glycolytic rate (B). Oligomycin-stimulated glycolysis (C) was calculated by subtracting the basal values from the maximum values obtained immediately after oligomycin injection. The oxygen consumption/extracellular acidification ratio (D) was calculated by dividing the values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g001" target="_blank">Figure 1D</a> by those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g003" target="_blank">Figure 3B</a>. All graphs depict means and standard deviations, and *P<0.05. mpH = milli pH units.</p

Seahorse XF24 extracellular flux analysis of sirtuin-expressing HEK293 cells under 5 mm glucose conditions.

<p>(A) Oxygen consumption and (B) extracellular acidification rates were measured under the same protocol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g003" target="_blank">3</a>. All graphs depict means and standard deviations. mpH = milli pH units.</p

SIRT3 and SIRT5 deacylate VLCAD at overlapping sites.

<p>A) Recombinant, unmodified VLCAD (Ctrl) was subjected to chemical succinylation (top) or acetylation (bottom) which was verified by western blotting with anti-succinyllysine (SuK) or anti-acetyllysine (AcK) antibodies. B) Chemically succinylated (Suc) and acetylated (Ac) VLCAD proteins were reacted with SIRT5 and SIRT3, respectively. Changes in succinylation or acetylation were then evaluated by western blotting, with anti-His blotting as loading control. C) Only SIRT3 reacts with chemically acetylated VLCAD as determined by incubating increasing amounts of acetylated VLCAD with SIRT3, SIRT4, or SIRT5 in the presence of radiolabeled NAD+. Shown are the means of duplicate assays. D) Acetylated VLCAD was treated with SIRT3 or inactive mutant SIRT3 (Control). Quantitative mass spectrometry was used to determine the relative abundance of acetylated peptides. Shown are acetylation sites with >2-fold change. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122297#pone.0122297.s001" target="_blank">S1 Dataset</a> for details. E) Succinylated VLCAD was treated with SIRT5 or inactive mutant SIRT5 (Control) and succinylated peptides were quantified by mass spectrometry. Shown are succinylation sites with >2-fold change. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122297#pone.0122297.s002" target="_blank">S2 Dataset</a> for details. D and E both depict the means and standard deviations of quadruplicate assays.</p

VLCAD from SIRT3 and SIRT5 knockout mice shows reduced affinity for cardiolipin.

<p>A) The fat blot method was used to evaluate endogenous VLCAD binding to cardiolipin in fasted (20 hr) mouse liver lysates. B) Densitometry was used to quantify binding from panel A. C) Lysate from VLCAD-/- liver was tested as a negative control and shows no detectable signal. D) Western blot was used to confirm that total VLCAD expression is not significantly different between wild-type, SIRT3 KO, and SIRT5 KO mice.</p

The SIRT3/SIRT5 target site K299 is critical for FAD binding and VLCAD activity.

<p>A) Chemical acetylation and succinylation both reduce enzymatic activity of recombinant VLCAD. B) Incubation of acetylated VLCAD with SIRT3 rescues activity, while incubation of succinylated VLCAD with SIRT5 does not (not shown). C) Mutant K298R retains sensitivity to acylation-induced loss of activity,suggesting that K298 does not play a mechanistic role in the reduced activity. D) Likewise, mutant K507R retains sensitivity to acylation-induced loss of activity, suggesting that K507 also does not play a mechanistic role in the reduced activity. E) K299 is highly sensitive to conservative substitution with arginine. K299R lost the yellow color characteristic of FAD and consequently became inactive. All bar graphs depict means and standard deviations of triplicate assays. *P<0.01 versus wild-type or control.</p

SIRT3 and SIRT5 deacylate lysines that localize to the active site and putative membrane binding domain of VLCAD.

<p>K299 (red) hydrogen bonds with neighboring S304 (green), and both are within interacting distance of the essential FAD cofactor (yellow) which is non-covalently bound in the VLCAD active site. B) Amino acid alignment of the region surrounding K299, showing conservation of this residue across diverse species. C) The portion of VLCAD spanning residues 486–518, which includes sirtuin target sites K492 and K507, is disordered in the crystal structure. PsiPred was used to generate a model of the disordered segment which was overlaid upon the structure of a VLCAD monomer. Hydrophobic residues are rendered red, positively charged residues blue, and negatively charged residues green. The active site is indicated as FAD in yellow and acyl-CoA substrate in red. D) Amino acid alignment of the putative membrane-binding amphipathic helix.</p