Figure 4. hACOX1^N237S promotes dimer formation and elevated ACOX1 protein levels.

(A) The current model for hACOX1 dimerization (left) and the 3D protein structure of a hACOX1 dimer bound to 2 flavine adenine dinucleotides (FAD) (right). (B) Enlarged view of a portion of the FAD contact region of hACOX1. The black arrow points to asparagine 237, and N237 is part of the FAD binding pocket of hACOX1. (C) hACOX1^N237S protein accumulates in vivo when compared to hACOX1^WT. Scale bar: 10μm. (D) hACOX1^N237S accumulates in the form of dimers. hACOX1 protein levels are elevated in larval extracts in which N237S is expressed. Quantification of relative dimer/monomer ACOX1 level from the blot of (D). n = 4 independent blots, Mean ± s.e.m. ***p< 0.001, Statistical analyses were determined by 2-sided Student’s t-test. (E) Purified hACOX1^N237S protein show higher activity than hACOX1^WT when normalized for protein level. (n = 3 for each), Mean ± s.e.m. **p< 0.01, Statistical analyses were determined by 2-sided Student’s t-test. (F) Gas chromatography mass spectrometry for total VLCFA, and the relative level of VLCFA (C28:0/C22:0). n = 7 (da>dACOX1^WT, 10 adult flies extracts). n = 9 (da>dACOX1^N250S), Statistical analyses were determined by 2-sided Student’s t-test. n.s., not significant. (G) Quantification of relative 4HNE (STAR Methods) level normalized by actin, n = 4 independent blots (50 larvae per each blot), Statistical analyses are one-way ANOVA followed by a Tukey post-hoc test. Mean ± s.e.m. ***p < 0.001, n.s., not significant.