Fatty Acid Metabolism And Sex Determination In C. Elegans

Metabolism has become an intensely studied research field in recent years by scientists using both biochemical and genetic approaches mainly because metabolism-related human diseases and health conditions, such as obesity, has drastically increases in past decades. Obesity, which arises when energy intake exceeds energy expenditure, is a significant risk factor for human health as it can trigger Type II diabetes and cardiovascular diseases. Genetic predisposition is a significant contributor to obesity, making genetic analysis an effective approach to tackle metabolic problems.Energy is largelly stored as fat or triglycerides that contain three fatty acid side chains. The study on fatty acid metabolism is thus an important part of obesity-related research. ACSs (Acyl-CoA synthetase) are critical enzymes of fatty acid metabolism pathways. ACSs catalyze the reaction between free fatty acids and CoA to form Acyl-CoA molecules, which "activates" fatty acids for further biochemical reactions. Specifically, all the free fatty acids need to be activated by the ACSs before they can enter anabolism (such as incorporation into high-order lipids or elongation into longer fatty acids) or catabolism (such as fatty acid β oxidation and degradation). Genetic studies have shown that the mutations of ACSs result in certain disorders of fat metabolism.Acyl-CoA molecules are involved in many cellular activities beyond their roles in lipid anabolism or catabolism. For example, certain acyl-CoAs form direct interactions with the membrane, acting as the modulator of energy metabolisms and vacuolar trafficking. Acyl-CoAs are also the substrates of acylation transferases that are involved in acylation of proteins. Fatty acid modification of proteins often play important role in modulating the cellular functions of these proteins (e.g., a subunit of G protein and tyrosine kinase Src).Understanding the roles of ACSs in cellular processes during cellular development and other physiological functions will be important for us to understand the mechanisms of lipid-involved cell signaling processes and related metabolic diseases. In this paper, I used the nematode C. elegans as an animal model to analyze the functions of an acyl-CoA synthetase, ACS-4. The study was initiated by identifying and characterizing the genetic phenotypes of loss of acs-4functions, but much of the effort also focused on uncovering the molecular mechanism by which this enzyme modulate signal transduction and developmental decisions.Hermaphrodites that have lost acs-4function by either mutation or RNA interference (RNAi)[called acs-4(-) thereafter] are sterile because they produce no oocytes and sperms. All the germ cells are differentiated into primary spermatocytes at the cost of oogenesis. Phenotype characterization of sperms deficiency indicated that the primary spermatocytes do not divide into spermatids. Therefore, acs-4(-) results in the masculinization of germline (Mog) and blocks the cytokinesis of primary spermatocyte. In this thesis, most of the analysis focused on the Mog phenotype. ACS-4is expressed in the intestine, neurons, the germline and the somatic gonad. Loss of acs-4function in the germline was found to be responsible for the Mog phenotype. acs-4(-) in the germline may affect sex determination in the germline favoring the generation of spermatocytes. Genetic analysis indicated loss-of-function (lf) mutation in fog-1,fog-3, fem-3, fem-2, and fem-1and gain-of-function (gf) mutation in tra-1can suppress masculinization of germline, suggesting that these genes may act downstream of acs-4.As an acyl-CoA synthetase, ACS-4may impact the sex determination pathway by regulating fat metabolism. However, analysis of fatty acid and lipid profiles using mass spectrometry and gas chromatography did not show prominent difference between the acs-4(-) mutant and the control. To search downstream activity of acs-4, I investigated transferases usually act at the next step of ACSs. An RNAi screen found that loss of function of an N-miristorylation transferase gene, nmt-1, in the germline displayed the germline masculinization phenotype (Mog). Dysfunction of another myritoylation-involved gene, map-2, also resulted in the Mog. Therefore, myristoylatoin appears to be involved in sex determination and acs-4may conceivably provide C14-CoA substrate to NMT-1for the myristoylation. To test this hypothesis, I found that expression of acs-4in the yeast could activate C14and C16fatty acids. In addition, acs-4and acs-17are partially redundant in their function since acs-4(-); acs-17(-) double mutants, but not single mutant of each, are lethal, which is similar to that of strains subjected to nmt-1(RNAi) or map-1(RNAi); map-2(RNAi). In addition, like acs-4, acs-17can also activate C14and C16free fatty acid. Based on these data, I propose that in wild type animals, acs-4provides substrate to NMT-1for myristoylation. acs-4(-) drastically reduces C14-CoA level and thus severely reduces myristoylation of some proteins that critically function in the sex determination pathway for the generation of primary spermatocytes. This hypothesis predicts that artificially increase the level of C14-CoA should suppress the oocytes deficiency of acs-4(-). Indeed, I showed that microinjection of exogenous C14-CoA into acs-4(-)germline recovered oocytes. Results of following tests are also consistent with the notion that increasing C14-CoA overcame the loss of acs-4function. First, supplementing acs-4(-) mutant with free fatty acid could rescue the oocytes defect and the rescue was dependent on acs-17. Second, prx-5(lf) and prx-6(lf) mutations, which disrupt peroxisomal functions and cause an increase in cellular C14-CoA level, also suppressed oocytes deficiency in the acs-4(-) mutant. Finally, RNAi against elo-1and elo-2to block elongation and formation of poly-unsaturated fatty acid could also recover oocytes defect in the acs-4(-) mutants. The next step is to identify the protein targets of acs-4-invo\ved myristoylation for the sex determination function. FEM-3became a leading candidate as I found through an immunofluorescence assay that FEM-3could not cluster on the cell membrane in acs-4(-) mutant.I propose a hypothesis that the loss of acs-4function decreases the level of C14-CoA specialized for nmt-1to modify certain protein targets, likely including FEM-3. In the acs-4(-) mutant, inability of myristoylation of FEM-3may alter the subcellular localization of FEM-3and in turn disrupt the activity of the sex determination pathway required for generating primary spermatocytes. In this thesis, a totally new regulation mechanism of sex determination pathway is found. Also a new mechanism, by which fatty acids metabolism impacts signaling pathway, is proposed.