Depuis une vingtaine d’années, il est possible d’allonger la durée de vie génétiquement. Nombre d’études réalisées sur des espèces allant de la levure aux primates, ont permis d’identifier des cascades de signaux intracellulaires ayant un impact sur la longévité et la qualité du vieillissement. Il est important de noter que certaines de ces interventions réduisent considérablement l’incidence de cancers et de maladies liées au vieillissement chez les mammifères. Ceci témoigne des liens existant entre vieillissement et carcinogénèse et il probable que le développement de stratégies pharmacologiques ayant pour cible le vieillissement se révèlent efficaces contre les maladies du vieillissement comme le cancer, la maladie d’Alzheimer ou de Parkinson. Nous avons criblé la banque de mutants de Saccharomyces cerevisiae pour identifier des mutations génétiques qui augmentent la durée de vie. La plupart des gènes identifiés se sont révélés conservés puisqu’ils influencent aussi la longévité chez C. elegans. La protéine de liaison à l’acyl-CoA (ACBP) est une petite protéine (10 kDa) qui se lie avec une haute affinité aux chaîne d’acyl-CoA esters (moyennes et longues) et les transporte vers les sites de consommation de l'acyl-CoA. ACBP est hautement conservée parmi les espèces eucaryotes et joue un rôle important dans la biosynthèse des lipides et le trafic vésiculaire. Chez Saccharomyces cerevisiae, la délétion d’ACBP (ACB1) entraîne une augmentation de la longévité et favorise la résistance au stress. Pour tester si l’impact d’ACBP sur la longévité s'étend aux eucaryotes supérieurs, nous avons exploré le lien entre les gènes codant pour des ACBPs chez Caenorhabditis elegans et la longévité en utilisant l’ARN interfèrent. Chez C. elegans, sept paralogues ACBP ont été identifiés, qui sont exprimés dans différents tissus. Nous avons constaté que la réduction de l'expression de maa-1 (codant une ACBP associée aux membranes) prolonge la durée de vie des vers sauvages. Nos résultats démontrent que: 1) une perte de fonction de maa-1 entraîne une résistance au superoxyde, 2) et aux événements protéotoxiques telle que l'agrégation protéique associées aux maladies neurodégénératives comme la maladie de Huntington. Enfin, nous avons montré que l'activité du facteur de transcription HIF-1 (hypoxia inducible factor-1) contribue à la longévité causée par la mutation maa-1. En effet, la délétion du gène hif-1 annule complètement l’augmentation de la longévité causée par maa-1. / Understanding the aging process, its regulation, and how to delay it has become a priority for an increasing number of scientists worldwide. The principal reason for this is that it is becoming more and more evident that anti-aging interventions may be effective against age-related diseases such as cancer, cardiovascular, and neurodegenerative diseases. Simple model organisms such as Caenorhabditis elegans and Saccharomyces cerevisiae have been instrumental to identify the principal genes implicated in aging whose role has turned out to be conserved in mammals. The project presented here has originated from a genome-wide screen performed in S. cerevisiae that has led to discover several novel life span-regulatory genes whose deletion prevents aging. One of these genes encodes for Acyl-CoA binding protein (ACBP). ACBP is a small (10 kDa) protein that binds medium- and long-chain fatty acyl-CoA esters with high affinity and transports them to acyl-CoA consuming processes. ACBP is highly conserved among eukaryotic species and plays important roles in lipid biosynthesis and vesicle trafficking. In S. cerevisiae, lack of ACBP (Acb1) extends longevity and promotes stress resistance. To test whether the life span-regulatory role of ACBP extends to higher eukaryotes, we explored the link between the C. elegans ACBP genes and longevity by RNAi screening. In C. elegans, seven ACBP paralogs have been identified, which are expressed in different tissues. We found that reducing the expression of maa-1 (encoding a membrane associated ACBP) extended the longevity of wild-type worms. Our results show that 1) a loss of function maa-1 mutant is resistant to the superoxide-generating agent paraquat and 2) reduction of maa-1 expression increases resistance to the proteotoxicity associated with the aggregation of the Huntington's disease-associated polyQ peptide. The activity of the anti-aging transcription factor HIF-1 (hypoxia inducible factor-1) contributes to the extended longevity caused by lack of maa-1. The effect of MAA-1 loss on longevity was fully reverted by the deletion of the hif-1 gene.

With crude oil demand rising and supplies being depleted, alternative energy, specifically biofuels, are of intense scientific interest. Current plant crop based biofuels suffer from several problems, most importantly the use of land needed for food. Cyanobacteria offer a solution to this problem as they do not compete with land for food and produce hydrocarbons that can be used as biofuels. Upon examination of metabolic pathways competing with hydrocarbon synthesis, it appeared that cyanobacteria lacked the major fatty acid degradative metabolic pathway β-oxidation, generally thought to be a universally occurring pathway. Lack of this pathway in cyanobacteria was confirmed by employing a range of analytical techniques. Bioinformatic analysis suggested that potential enzymes with β-oxidation activity were involved in other metabolic pathways. A sensitive assay was set up to detect acyl- CoAs, the substrates of β-oxidation, using liquid chromatography triple quadrupole mass spectrometry. None could be detected in cyanobacteria. No enzymatic activity from the rate-limiting acyl-CoA dehydrogenase/oxidase could be detected in cyanobacterial extracts. It was found that radiolabeled fatty acids fed to cyanobacteria were utilised for lipid membranes as opposed to being converted to CO2 by respiration or into other compounds by the TCA cycle. An element of the β-oxidation pathway, E. coli acyl-CoA synthetase was ectopically expressed in a strain of cyanobacteria and implications of the introduction of acyl-CoA synthesis were assessed. Finally, the regulation of the fatty acid biosynthetic pathway was investigated. It was determined that under conditions of excess fatty acid, the transcription of acetyl-CoA carboxylase and enoyl-ACP reductase was repressed and acyl-ACP synthetase involved in fatty acid recycling was induced. These results were discussed in relation to fatty acid oxidation and hydrocarbon biosynthesis in other organisms.

Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is the most common inborn error of metabolism affecting the fatty acid oxidation pathway. The deficiency is caused by a defect in the medium-chain acyl-CoA dehydrogenase enzyme which catalyzes the first step in the oxidation of medium-chain fatty acids. Long-chain fatty acids, after being transported into the mitochondria and activated into long-chain acyl-CoAs, are sequentially broken down until they become medium-chain acyl-CoAs. Medium-chain acyl-CoAs are then broken down until they become short-chain acyl-CoAs. Short-chain acyl-CoAs are broken down until only acetyl-CoA remains. The block in the oxidation of fatty acids in those with MCADD happens once the long-chain acyl-CoAs have been oxidized to medium-chain acyl-CoAs. The medium-chain acyl-CoAs cannot be further oxidized and build up. Without the breakdown of fatty acids, individuals with MCADD cannot produce enough energy during times of increased metabolic demand. Thus, prolonged exercise, fasting, or fever can precipitate clinical symptoms once the body enters a hypoketotic hypoglycemic state. Those with MCADD typically present in the early months of life with fasting intolerance, vomiting, lethargy, and, in more serious cases, seizures. Adult presentation is rare, but should not be ruled out of a differential diagnosis, because early detection and intervention can prevent permanent brain damage and death. Because early detection can prevent the serious effects of metabolic decompensation, MCADD was added to the Newborn Screen and is tested through measuring levels of medium-chain acylcarnitines in dried blood smears by tandem mass spectrometry. Metabolic decompensation is manifested clinically through dehydration, vomiting, and acidosis. In serious cases, metabolic decompensation can progress to seizures, coma, and death. Introduction of the Newborn Screen has reduced the morbidity of the deficiency, but has not eliminated it. Those with MCADD need to be closely monitored and emergency glucose needs to be available to them in case of a hypoglycemic emergency. The Newborn Screen has been effective in finding mutations in the ACADM gene that produce a mild phenotype of MCADD. Before the Newborn Screen, the most common variant, K329E, was detected in clinically diagnosed patients. However, the screen has shown that there are about 150 variants leading to MCADD. The most common variant of the MCAD protein, K329E, has been studied and characterized in order to further understand the pathogenesis of MCADD. This mutation substitutes a lysine for a glutamic acid, introducing hindrance and the inability of the protein to form its fully functional tetrameric form. The mutant protein also has an increased sensitivity to heat denaturation. Currently, there are no pharmacological treatments for MCADD. The idea of pharmacological chaperones is explored by using the example of tetrahydrobiopterin and phenylketonuria. Future studies will need be done to find a treatment for MCADD that is curative rather than treating the symptoms of the deficiency; however, curative therapies which target the mutant enzyme may be problematic since there is a wide array of mutations that result in a defective enzyme in affected individuals.

Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. Triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although the use of molecular tools, including targeted disruption of either DGAT enzyme, has shed light on their metabolic functions, little is known about the mechanisms responsible for regulating DGAT activity. Several lines of evidence from previous studies have suggested that DGAT1, but not DGAT2, is subject to regulation by phosphorylation and that protein kinase A (PKA)-dependent pathways are likely involved. In this study, the role of PKA in regulating DGAT activity and triacylglycerol synthesis during lipolysis was investigated. By using 3T3-L1 adipocytes, in vitro DGAT activity was shown to increase 2 fold during lipolysis. This data suggests that PKA might phosphorylate and activate DGAT1 during lipolysis to promote the recycling/re-esterification of excessive free fatty acids into triacylglycerols before they reach toxic levels within the cell. Additionally, high-performance liquid chromatography electrospray ionization mass spectrometry/mass spectrometry was exploited to identify PKA phosphorylation sites of DGAT1, and serine-17, -20 and -25 were identified as potential PKA phosphorylation sites using this methodology. The functional importance of these three potential phosphorylation sites was examined. Mutations of these sites to alanines (to prevent phosphorylation) or aspartates (to mimic phosphorylation) gave rise to enzymes functioning similarly to wild-type DGAT1. These phosphorylation sites appeared to be functionally silent as they were not involved in regulating DGAT1 activity, multimer formation, or enzyme stability. However, PKA phosphorylation at these three sites seemed to play a role in affinity of DGAT1 for its diacylglycerol substrate. These results indicate the existence of other unidentified, functionally active PKA phosphorylation sites or phosphorylation sites of other kinases, which are involved in regulating DGAT1.

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. Triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although the use of molecular tools, including targeted disruption of either DGAT enzyme, has shed light on their metabolic functions, little is known about the mechanisms responsible for regulating DGAT activity. Several lines of evidence from previous studies have suggested that DGAT1, but not DGAT2, is subject to regulation by phosphorylation and that protein kinase A (PKA)-dependent pathways are likely involved. In this study, the role of PKA in regulating DGAT activity and triacylglycerol synthesis during lipolysis was investigated. By using 3T3-L1 adipocytes, in vitro DGAT activity was shown to increase 2 fold during lipolysis. This data suggests that PKA might phosphorylate and activate DGAT1 during lipolysis to promote the recycling/re-esterification of excessive free fatty acids into triacylglycerols before they reach toxic levels within the cell. Additionally, high-performance liquid chromatography electrospray ionization mass spectrometry/mass spectrometry was exploited to identify PKA phosphorylation sites of DGAT1, and serine-17, -20 and -25 were identified as potential PKA phosphorylation sites using this methodology. The functional importance of these three potential phosphorylation sites was examined. Mutations of these sites to alanines (to prevent phosphorylation) or aspartates (to mimic phosphorylation) gave rise to enzymes functioning similarly to wild-type DGAT1. These phosphorylation sites appeared to be functionally silent as they were not involved in regulating DGAT1 activity, multimer formation, or enzyme stability. However, PKA phosphorylation at these three sites seemed to play a role in affinity of DGAT1 for its diacylglycerol substrate. These results indicate the existence of other unidentified, functionally active PKA phosphorylation sites or phosphorylation sites of other kinases, which are involved in regulating DGAT1.

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

No description available.