Figure 6. Fatty acid binding protein orthologues in C. elegans confirm the link between these proteins, fatty acid levels, mitochondrial function and lifespan. (A) Sequence alignment of FABP3 and C. elegans orthologues LBP-4, -5 and -6. A long chain fatty acid transporter protein in H. sapiens, M. musculus and C. elegans. Multiple sequence alignment of three C. elegans lipid binding protein family members, LBP-4, -5 and -6, with fatty acid binding protein 3 from H. sapiens. Amino acid residues are coloured shades of blue signifying their sequence homology; lightest is 30%, darkest is 100%. Sequence homology is 23.74%, 45.52% and 48.15% for LBP-4, -5 and -6 respectively. Sequence alignment was carried out using Jalview. (B) A phylogenetic tree was produced using PAM 250 scoring to relate human, mouse and C. elegans FABP3 orthologues. See Supplementary Table 1 for scoring. (C) Oxygen consumption measurements were made on LBP adult knock down adult worms at 4, 8 and 12 days and compared with controls. Reduction of LBP levels caused changes in oxygen consumption in each group compared with controls. The most marked difference in oxygen consumption was recorded in the LBP6 knockdown at days 4 and 8. (D) LBP-4, -5 and -6 knockdowns significantly reduce lifespan. C. elegans treated with LBP-4, -5 or -6 RNAi show significant reduction in lifespan p=0.0022 (Log rank test). Median lifespans were 9.5, 10 and 8 days for LBP-4, -5 and -6 respectively. 10 worms were followed for each treatment. (E) Measurement of free fatty acid quantities in the LBP knock downs confirmed a reduction in free fatty acid abundance in each of the orthologue knock-downs, reaching significance in LBP-4 (p value = 0.027). (F) LBP-4, -5 and -6 knockdown affect mitochondrial form and function. Worms carry the transgene ccIs4251 with GFP fusion proteins localised to the body muscle mitochondria and nuclei. Oxygen consumption in worms at different age points was measured using a Loligo systemsTM electrode array (E). CB5600 worm mitochondria subjected to knock-down of LBP-4, -5 and -6. WT worms grown on OP50 (A). Mitochondria become condensed and disordered when subjected to RNAi for LBP-4 (B), -5 (C) and -6 (F).