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Functions of Acyl-CoA Binding Proteins in C. elegans Introduction Results Conclusions Future Perspectives Ida Coordt Elle 1, Karina Trankjær Simonsen 2,

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Presentation on theme: "Functions of Acyl-CoA Binding Proteins in C. elegans Introduction Results Conclusions Future Perspectives Ida Coordt Elle 1, Karina Trankjær Simonsen 2,"— Presentation transcript:

1 Functions of Acyl-CoA Binding Proteins in C. elegans Introduction Results Conclusions Future Perspectives Ida Coordt Elle 1, Karina Trankjær Simonsen 2, Pernille Kirstine Birck 1, Thuc Timothy Le 3, Nils Joakim Færgeman 1 Results 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark 2 Wine Research Center, University of British Columbia, Vancouver, British Columbia, Canada 3 Nevada Cancer Institute, Engelstad Research Building R1068, Las Vegas, Nevada,USA Paralogue#AADomain Structure ACBP-186 ACBP-2/ ECH ACBP-3116 ACBP-4146 ACBP-5274 ACBP-6115 MAA-1266 ACBPECHANKYRIN TMD COILED-COIL Acyl-CoA esters, the metabolically active form of fatty acids, are important intermediates in both anabolic and catabolic processes, but have also been identified as regulators of ion channels, enzymes, membrane fusion, and gene expression. Acyl-CoA binding protein (ACBP) is a small, primarily cytosolic protein, which binds acyl-CoA esters with high specificity and affinity. Several studies have indicated that ACBP binds acyl-CoAs and delivers them to various enzymes for membrane assembly, vesicle transport, β- oxidation, triglyceride storage, and complex lipid synthesis. All eukaryotic species express one or more ACBP paralogues of which some are basal forms and others are multi-domain proteins. C. elegans contains seven (possibly eight) ACBPs, and all of these have mammalian orthologues of similar domain structure. ACBP-1 is the shortest form in C. elegans and is considered the most basal form. ACBP-2 is a domain protein containing a C- terminal enoyl-CoA hydratase/isomerase domain similar to mammalian peroxisomal enoyl-CoA isomerase, but is likely expressed in the mitochondria, not in the peroxisomes. ACBP-3, -4, and -6 are short forms, while ACBP-5 contains ankyrin repeats, which probably mediate protein-protein interactions. MAA-1 is a membrane-associated ACBP involved in endosomal vesicle transport. The recently identified ACBP-7 is not included in this study. To investigate the localization of each of the ACBPs, we generated translational ACBP::GFP fusions and injected them into C. elegans to obtain stable lines expressing the fusion proteins. A: ACBP-1 is expressed in the intestine; the main fat storage tissue in C. elegans, indicating a direct function in triglyceride metabolism. B: ACBP-2 is expressed in the intestinal cells and hypodermis; the other fat storage tissue in the worm. C: ACBP-3 localizes to muscles and hypodermis. D + E: ACBP-4 is specifically associated with granular structures in the intestinal cells; likely lipid droplets. F: ACBP-5 is expressed in the intestine but at very low levels. G + H: ACBP-6 localizes to two specific neurons (possibly CAN and AVG), suggesting a sensory function related to acyl-CoA metabolism. The distinct expression patterns of C. elegans ACBPs demonstrate that these proteins probably perform specific functions, although we also have data indicating that lack of one form is compensated by up-regulation of expression of other forms. We then investigated lipid storage and catabolism in ACBP mutants. Total triglyceride contents were determined by an enzymatic method, which measures glycerol levels and therefore is not affected by possible differences in fatty acid composition. Lack of either ACBP-1, -2, and -3 results in reduced triglyceride levels. The acbp-1 mutant displays the most significant decrease, indicating a role for ACBP-1 in triglyceride storage in C. elegans. Surprisingly, the quadruple mutant displays normal total triglyceride levels. Asterisks indicate statistical significance; *: p<0.05; **: p<0.01; ***: p< We have performed CARS (Coherent Anti-Stokes Raman Scattering) microscopy on each of the acbp deletion mutants in order to examine differences in lipid droplet number and/or morphology. CARS microscopy relies on contrast derived from molecular vibrations in C-H bonds. This analysis revealed that acbp-1 contains a reduced number of lipid droplets that are severely enlarged compared to wild-type droplets, signifying a possible role for ACBP-1 in lipid droplet formation and/or fusion. The quadruple mutant displays a diffuse CARS signal in the intestine indicating compromised lipid droplet formation. A: N2 B: acbp-1 C: acbp-2 D: acbp-3 E: acbp-4 F: acbp-5 G: acbp-6 H: acbp-1;6;4;3 We have determined β-oxidation rates by feeding C. elegans tritiated fatty acids and measuring the formation of tritiated water. The acbp-1 and -3 deletion mutants display increased β-oxidation of oleic acid revealing a plausible explanation for their decreased triglyceride storage levels. The acbp-2 mutant displays decreased β- oxidation of oleic acid (unsaturated). Conversely, this mutant has increased β-oxidation of palmitic acid (saturated). The quadruple mutant displays dramatically increased β-oxidation indicating that lack of all basal ACBPs elicits a starvation-like phenotype. Median lifespan N214 acbp-19 acbp-212 acbp-314 acbp-415 acbp-514 acbp-614 Lifespan assays have been performed in three independent setups. The graph shown is representative of all data. The acbp-1 mutant has a significantly decreased lifespan compared to N2 (p<0.0001). The lifespan of the acbp-1;6;4;3 quadruple mutant has been examined in two separate experiments; it is not different from N2 lifespan. The results of the current study demonstrate that the ACBP paralogues in C. elegans are expressed in distinct tissues and have different effects on lipid metabolism. This indicates that each of the ACBPs performs a specific function, although we also see compensatory mechanisms in several deletion mutants. Much more work is required to reveal the molecular mechanisms of ACBP functions. Current efforts include analysis of acyl-CoA compositions by HPLC and lipidomic analysis by mass spectrometry. We plan to examine the role of ACBPs during different types of stress. Investigation of the skin barrier function along with lipidomic analysis have revealed that acbp-3 mutants are compromised under hypo-osmotic conditions likely due to reduced levels of sphingomyelin containing very long chain fatty acids. We are currently working on obtaining EM pictures of this mutant and performing Hoechst stainings to further evaluate skin function. Contact : or Reference Elle et al. : ”Tissue- and paralogue –specific functions of acyl-CoA binding proteins in lipid metabolism in C. elegans”. Biochemical Journal, ** * * *** N2 acbp-1acbp-3acbp-2acbp-6acbp-4acbp-5 acbp-1;6;4;3 daf-2 *** N2+azide *** * acbp-1acbp-3acbp-2acbp-6acbp-4acbp-5 acbp-1;6;4;3 N2 ***


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