Autophagy is the process that degrades cytosolic constituents into products that can be recycled for use in energy generation and other processes. The endoplasmic reticulum is responsible for the bulk synthesis of the phospholipid phosphatidylethanolamine (PE) via the CDP-ethanolamine pathway. The aim of the present study was to determine the role of PE synthesis and the CDP-ethanolamine pathway in autophagy. This objective was examined through the use of two novel models deficient in Pcyt2, a gene that encodes the rate-limiting enzyme CTP-ethanolamine cytidyltransferase (ET) in the CDP-ethanolamine pathway. PCYT2 knockdown in human fibroblast cells did not respond normally to starvation conditions that activate autophagy. Similarly, Pcyt2 knockout in mice showed differences in autophagy induction in/between muscle, liver, and adipose tissue based on metabolic state (fasting/feeding). Pcyt2 knockout mice display evidence of metabolic syndrome at an older age and experiments with these mice determined that there was an effect of age (healthy young mice versus obese older mice) on autophagy induction. It was concluded based on in vitro and in vivo studies that autophagy induction is affected by impairment to the CDP-ethanolamine pathway and subsequent PE synthesis.

The human genome project has allowed for the rapid identification of a large number of protein families based on similarities in their genetic sequences. In the present study, I report the functional characterization of a 44 kDa protein that functions in cardiolipin synthesis and remodeling. Although it is present in most tissues, it is abundant in multiple brain regions including olfactory bulbs, hippocampus, cerebellum, cortex, and brain stem, and is detectable in both primary neurons and glial cells. In assays performed in vitro, this protein significantly increased the incorporation of [14C]oleoyl-CoA into phosphatidylinositol and CL using either lysophosphatidylinositol, or monolysocardiolipin or dilysocardiolipin as acyl acceptors, respectively. This protein did not display significant acyltransferase activity with a number of other lysophospholipid acyl acceptors. Overexpressing this enzyme in HEK-293 cells increased total CL content, but did not significantly affect levels of other glycerophospholipids. Analysis of the fatty acyl profile of CL from cells overexpressing this protein indicated increased total saturated fatty acids, particularly stearate, palmitate, and myristate, and increased levels of n-3 polyunsaturated fatty acids α-linoleic acid (18:3n-3), eicosatrienoic acid (20:3n-3), and eicosapentanoic acid (20:5n-3). In accordance with its observed role in CL remodeling, subcellular localization of this protein was predominately mitochondrial. This protein is also regulated during embryogenesis, and in varying metabolic states.

Cell and organelle membranes consist of a complex mixture of phospholipids that determine their size, shape, and function. Among the distinct types of phospholipids found in membranes of living organisms, phosphatidylcholine (PC) is the most abundant. The rate-limiting step of the predominant pathway for PC synthesis in eukaryotic cells is catalysed by the enzyme, CTP: phosphocholine cytidylyltransferase α (CCTα or PCYT1A). CCTα has a critical role in lipid metabolism and also has direct clinical relevance as mutations in CCTα result in an interesting spectrum of human diseases, such as lipodystrophy with fatty liver, growth plate dysplasia and cone-rod related dystrophy. Numerous biochemical and structural studies on purified CCTα have revealed its membrane-bound activation and suggested that it acts as a lipid compositional sensor, yet the in vivo mechanism of how CCTα senses and regulates PC levels in membranes remains unclear. Here I show that in budding yeast Saccharomyces cerevisiae, Pct1, the yeast homolog of CCTα, is intranuclear and translocates to the nuclear membrane in response to changes in membrane properties and the need for membrane PC synthesis. By aligning imaging with lipidomic analysis and data-driven modelling, Pct1 membrane association is demonstrated to correlate with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous Pct1. These data suggest an ancient mechanism by which CCTα senses lipid packing defects and regulates phospholipid homeostasis from the nucleus. Additionally, I identified the importance of mammalian CCTα in early adipogenesis and investigated the enzymatic function of PCYT1A mutants in fibroblasts from lipodystrophic patients. The allele Val142Met is evaluated to be the main cause of loss-of-function in the compound heterozygous mutations by using yeast survival assay. These results collectively provide preliminary evidence for the pathogenicity of PCYT1A mutations in adipose tissue. From yeast to humans, this study uncovers the critical role of Pct1/CCTα in maintaining the internal membrane environment.

High-value industrial oils are essential for many processes and have great economic and environmental impacts. The tung tree produces a high-value seed oil. Approximately 80% of tung oil is α-eleostearic acid, which has a high degree of unsaturation thus giving it properties as a drying oil. The identification of the biological components in tung is imperative to further the knowledge of its processes. Four unique tung acyl-CoA binding proteins, VfACBP3a, VfACBP3b, VfACBP4, and VfACBP6 were identified and the genes encoding them were cloned and analyzed to determine their biological roles. The VfACBPs were observed to be similar to other organisms' ACBPs, especially Arabidopsis thaliana. In addition, each gene was expressed in all tung tissues. They were shown to interact with VfDGAT1 and VfDGAT2, two known components of tung lipid metabolism. Finally, VfACBP3a and VfACBP6 were expressed in the seeds of transgenic plants to study the effects of VfACBP expression on seed lipid fatty acid content.

Soybean (Glycine max (L.) Merr) is the largest oil and protein crop in the world and it is grown for both oil and protein. To address the needs of both the edible oil market and industrial applications of soybean oil, fatty acid modification has been a focus of soybean breeding programs. Natural variation, mutagenesis and genetic engineering have been used to alter the fatty acid profile. Several genes, mostly desaturases, have been associated with altered fatty acid profiles but enzymes in the Kennedy Pathway have yet to be studied as another source of genetic variation for altering the fatty acid profiles. The Kennedy Pathway is also known as the oil producing pathway and consists of four enzymes: glycerol-3-phosphate acyltransferase (G3PAT); lysophosphatidic acid acyltransferase (LPAAT); phosphatidic acid phosphatase (PAP); and diacylglycerol acyltransferase 1 (DGAT1). The starting material for this pathway is glycerol-3-phosphate, which is produced from glycerol by glycerol kinase (GK), and the product of this pathway is triacylglycerol (TAG). The overall objective of this study was to elucidate the role that the Kennedy Pathway plays in determining the fatty acid profile in two ways: (1) sequencing the transcribed region of the genomic genes encoding the enzymes of GK, G3PAT, LPAAT, and DGAT1 in soybean genotypes with altered fatty acid profiles; and (2) studying their expression over seed development, across three growing temperatures. The genetic material for the study consisted of four soybean genotypes with altered fatty acid profile: RG2, RG7, RG10, and SV64-53. Results from sequencing showed that the mutations identified in G3PAT, LPAAT, and DGAT1 in the four soybean genotypes did not explain the differences in the fatty acid profiles. The expression of G3PAT, LPAAT, and DGAT1 over seed development showed that G3PAT had the lowest levels, followed by LPAAT, then DGAT1, across the growing temperatures. The differences in expression among genotypes corresponded to differences in fatty acid accumulation, suggesting that expression rather than genetic mutations in the transcribed region of the genes influenced the fatty acid profile of the genotypes in this study. In conclusion, the enzymes of the Kennedy Pathway appear to contribute to the altered fatty acid profiles observed in the soybean mutant genotypes. / Ontario Ministry of Economic Development and Innovation (formerly Ontario Ministry of Research and Innovation), BioCar Initiative, Grain Farmers of Ontario, SeCan

The human genome project has allowed for the rapid identification of a large number of protein families based on similarities in their genetic sequences. The acyl-glycerol phosphate acyltransferase (AGPAT) family of enzymes have been largely identified through sequence homology, with eleven isoforms identified in both mice and humans. Interestingly, very little work has been done on the characterization of AGPAT isoform 4. In the present study, I report the functional characterization of AGPAT4 as an acyl-CoA: lysocardiolipin acyltransferase (ALCAT), which we have renamed ALCAT2. Although ALCAT2 is present in most tissues, it is abundant in multiple brain regions including olfactory bulbs, hippocampus, cerebellum, cortex, and brain stem, and is detectable in both primary neurons and glial cells. In assays performed in vitro, ALCAT2 significantly increased the incorporation of [14C]oleoyl-CoA into phosphatidylinositol and CL using either lysophosphatidylinositol, or monolysocardiolipin or dilysocardiolipin as acyl acceptors, respectively. ALCAT2 did not display significant acyltransferase activity with lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, or lysophosphatidylglycerol acyl acceptors. Overexpressing ALCAT2 in HEK-293 cells increased the total CL content, but did not significantly affect levels of other glycerophospholipids including phosphatidylinositol. Analysis of the fatty acyl profile of CL from ALCAT2-overexpressing cells indicated increased total saturated fatty acids, particularly stearate, palmitate, and myristate, and increased levels of n-3 polyunsaturated fatty acids α-linolenic acid (18:3n-3), eicosatrienoic acid (20:3n-3), and eicosapentanoic acid (20:5n-3). In accordance with its observed role in cardiolipin remodeling, ALCAT2 localized predominately to the mitochondria. ALCAT2 was also regulated during embryogenesis, and in varying metabolic states. In summary, ALCAT2 is a new enzyme in CL remodeling with a potential role in mitochondrial function.