The Functional Study Of Fatty Acid 2-Hydroxylase In Caenorhabitis Elegans

Fatty acids(FAs)are one of the most common components of biological lipid species.FAs with diverse chemical structures are physiologically important in a variety of cellular functions including energy storage,membrane structure,trafficking and signaling.The hydroxylation of fatty acids at the C-2 position is the first step of fatty acid ?-oxidation and generates 2-hydroxy sphingolipids.Fatty acid 2-hydroxylase(FA2H)catalyzes the 2-hydroxylation of fatty acids to produce 2-hydroxy fatty acids.However,the precise roles of FA2H and fatty acid 2-hydroxylation in whole cell homeostasis still remain unclear.Here we use Caenorhabditis elegans as the model and investigate the physiological functions of FATH-1/C25A1.5,the worm homolog for mammalian FA2H enzyme.The methods used in this project are as follows:(1)We firstly analyzed the homology of C.elegans FATH-1 and mammalian FA2H,and constructed GFP fluorescent fusion protein for identifying the expression and localization of FATH-1 in C,elegans.(2)Global RNAi knockdown was used to record the phenotypes of fath-1 deficient C.elegans animals.Quantitative analysis of the changes of C.elegans body length and life span was performed to document developmental and aging defects caused by inactivated FATH-1 function.Tissue-specific RNAi knockdown offath-1 was used to determine in which tissue FATH-1 plays a key role.Exogenous 2-OH fatty acid rescue experiments were performed to reveal the functional metabolites of FATH-1.(3)The "click chemistry"method was employed to label 2-OH fatty acid,and colocalization analysis with other membranous organelles was used to confirm the subcellular location of FATH-1.(4)By inactivating fath-1 in the reporter strains that label a series of subcellular structures,organelles and life processes,we examined the functions of FATH-1 at the subcellular level.(5)Lipid analysis of the fath-1 deficient mutants are achieved by mass spectrometry,coupled with exogenous fatty acid rescue experiments to reveal the major downstream effector mediating the physiological roles of FATH-1 and the key functional metabolites of FATH-1.We obtained the results as follows:(1)Through analysis of FATH-1 protein sequences,we found that the major domains of FATH-1 in C.elegans is highly similar to mammalian FA2H.Translational fusion reporter showed that C.elegans FATH-1 is expressed at most developmental stages and in most tissues.(2)Loss of fath-1 expression results in severe growth retardation and shortened lifespan.Tissue-specific RNAi knockdown showed that FATH-1 function is crucially required in the intestine but not the epidermis.Exogenous 2-OH fatty acid rescuing experiment revealed that FATH-1 mostly functions through its catalytic product(R)-2-OH fatty acid with stereospecificity.(3)By using the "click chemistry" method,we successfully labeled 2-OH fatty acid,an exogenous analogue of the FATH-1 catalytic product in vivo.The staining showed that 2-OH fatty acid and derivatives are mainly enriched in membrane structures preferable to the apical side of the intestinal cells.(4)At the subcellular level,we found that loss of fath-1 expression inhibits lipid droplets formation,as well as selectively disrupts peroxisomes and apical endosomes.(5)Through lipid analysis of the fath-1 knockdown mutants using mass spectrometry,we observed a significant reduction in the content of heptadecenoic acid.Feeding of exogenous heptadecenoic acid,but not oleic acid,rescues the growth and subcellular defects of fath-1 knockdown worms.To summarize,we find that C.elegans FATH-1 is important for normal development and survival of the nematodes.Such functions are achieved mainly by regulating the dynamics of endosomes,peroxisomes and lipid droplets in the intestine.Our study revealed that FATH-1 and its catalytic products are highly specific in the context of chirality,C-chain length,spatial distribution,as well as the types of cellular organelles they affect.Such an unexpected degree of specificity for the synthesis and functions of hydroxylated FAs helps to regulate protein transport and fat metabolism,therefore maintaining the cellular homeostasis of the intestinal cells.These findings may help our understanding of FA2H functions across species,and offer potential therapeutical targets for treating FA2H-related diseases.