Stuart Smith

Biosynthesis of Fatty Acids by Lactating Human Breast Epithelial Cells: An Evaluation of the Contribution to the Overall Composition of Human Milk Fat

ABSTRACT: The objective of this study was to characterize the fatty acid biosynthetic pathway of the lactating human breast. Mixed cell populations, obtained by centrifugation of human milk, were enriched in breast epithelial cells by a selective adsorption procedure. Confirmation of the identity of the breast epithelial cells was obtained immunohistochemically. These viable breast epithelial cells incorporated radioactively labeled acetate predominantly into fatty acids with less than 16C atoms. The presence of the two key enzymes characteristic of the medium-chain fatty acid biosynthetic pathway of nonruminants, fatty acid synthetase, and thioesterase II, was demonstrated both qualitatively, by immunohistochemistry, and quantitatively, by enzyme assay. The results indicate that the lipogenic system of the human breast is qualitatively very similar to that of rats, mice, and rabbits, which also secrete milk fats containing medium-chain fatty acids. Quantitatively, however, the mammary fatty acid biosynthetic pathway appears to be less active in humans than in these other species.

Cloning, Expression, Characterization, and Interaction of Two Components of a Human Mitochondrial Fatty Acid Synthase MALONYLTRANSFERASE AND ACYL CARRIER PROTEIN

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.

Fatty acid synthesis in developing mouse liver

Abstract Fatty acid synthesis in developing mouse liver has been studied by measuring incorporation of labeled acetate and pyruvate into fatty acids by tissue slices (lipogenic capacity) in conjunction with the determination of the activities of several enzymes involved in lipogenesis. Hepatic lipogenic capacity, which is normally low in suckling pups, can be prematurely increased by weaning onto a fat-free diet on or after Day 16 postpartum. Furthermore, pups ingesting a linoleate-deficient milk during Days 6 to 15 postpartum showed greater hepatic lipogenic capacities than pups ingesting a linoleate-rich milk. The data have been interpreted to indicate that hepatic lipogenesis in suckling and weanling mice can be regulated by the dietary linoleate content. The increase in hepatic lipogenic capacity in mouse pups weaned onto a fat-free diet and the accompanying increase in the activities of citrate cleavage enzyme, acetyl CoA carboxylase, fatty acid synthetase, and malic enzyme are dependent on the synthesis of new protein and new RNA. The increases in activities of these enzymes at weaning follow different time courses; fatty acid synthetase was observed to reach maximum activity earliest.

Coupling of the de Novo Fatty Acid Biosynthesis and Lipoylation Pathways in Mammalian Mitochondria

The objective of this study was to identify the products and possible role of a putative pathway for de novo fatty acid synthesis in mammalian mitochondria. Bovine heart mitochondrial matrix preparations were prepared free from contamination by proteins from other subcellular components and, using a combination of radioisotopic labeling and mass spectrometry, were shown to contain all of the enzymes required for the extension of a 2-carbon precursor by malonyl moieties to saturated acyl-ACP thioesters containing up to 14 carbon atoms. A major product was octanoyl-ACP and, in the presence of the apo-H-protein of the glycine cleavage complex, the newly synthesized octanoyl moieties were translocated to the lipoylation site on the acceptor protein. These studies demonstrate that one of the functions of the de novo fatty acid biosynthetic pathway in mammalian mitochondria is to provide the octanoyl precursor required for the essential protein lipoylation pathway.

Down-regulation of Mitochondrial Acyl Carrier Protein in Mammalian Cells Compromises Protein Lipoylation and Respiratory Complex I and Results in Cell Death

The objective of this study was to evaluate the physiological importance of the mitochondrial fatty acid synthesis pathway in mammalian cells using the RNA interference strategy. Transfection of HEK293T cells with small interfering RNAs targeting the acyl carrier protein (ACP) component reduced ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic changes observed were a marked decrease in the proportion of post-translationally lipoylated mitochondrial proteins recognized by anti-lipoate antibodies and a reduction in their catalytic activity, and a slowing of the cell growth rate. Later effects observed included a reduction in the specific activity of respiratory complex I, lowered mitochondrial membrane potential, the development of cytoplasmic membrane blebs containing high levels of reactive oxygen species and ultimately, cell death. Supplementation of the culture medium with lipoic acid offered some protection against oxidative damage but did not reverse the protein lipoylation defect. These observations are consistent with a dual role for ACP in mammalian mitochondrial function. First, as a key component of the mitochondrial fatty acid biosynthetic pathway, ACP plays an essential role in providing the octanoyl-ACP precursor required for the protein lipoylation pathway. Second, as one of the subunits of complex I, ACP is required for the efficient functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential.

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have investigated the possibility that the nonrandom association of fatty acids in rat milk triglycerides results from specificity of the acyl transferases in the glycerolphosphate pathway. Subcellular fractionation of lactating rat mammary gland revealed that the microsomal fraction was the most active in acylation of 3-sn-[U-14C] glycerolphosphate with various acyl-CoAs. The major products were diacylglycerolphosphate and diglyceride; no monoacylglycerolphosphate was detected. Maximum rate of acylation occurred at or below the critical micelle concentration for each acyl-CoA, indicating that only the monomeric substrate molecules were acceptable by the enzyme system. The observed acyl specificity, 16∶0>18∶0≏14∶0>12∶0>10∶0>8∶0 is consistent with the concept that, in general, milk triglycerides are synthesized by insertion of a short or medium chain fatty acid into a long chain diglyceride.

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase☆

Abstract The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C 16 C 22 ), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C 16 thioester and that further elongation to C 18 , C 20 , and C 22 is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.

Studies on the immunological cross-reactivity and physical properties of fatty acid synthetases.

Abstract Fatty acid synthetase enzymes were purified from the liver, mammary gland, and adipose tissue of rats and the liver and mammary gland of mice. The enzymes from the liver and mammary gland of the same species have similar molecular weights and and dissociate into subunits at comparable rates. Rabbit antisera were prepared against the fatty acid synthetase from the lactating rat mammary gland. Cross-reactivity between different fatty acid synthetases was determined by immunodiffusion and immunoprecipitin tests. No differences in immunological cross-reactivity could be detected in liver, mammary gland, and adipose enzymes from the same species; fatty acid synthetases from the rat and mouse gave reactions of incomplete identity. Partially purified fatty acid synthetases from pigeon liver and rabbit mammary gland did not react with the antiserum. It is concluded that the immunochemical approach is useful in determining the degree of resemblance between fatty acid synthetases from different species. Within a given species, the liver and mammary gland fatty acid synthetases seem to be very similar, if not identical, proteins.

Purification and properties of fatty acid synthetase from a human breast cell line

A human mammary epithelial cell line (SKBr3) has been identified in which fatty acid synthetase constitutes up to 28%, by weight of the cytosolic proteins. The enzymes has been purified to near homogeneity from this cell line and some of its properties studied. In common with fatty acid synthetases from other animal tissues, the enzyme is a 480 000 dalton dimer of similar molecular weight subunits, it synthesizes predominantly palmitic acid and is inactive in the absence of free coenzyme A. The kinetic properties and amino acid composition of the enzyme are also similar to those of fatty acid synthetases from various tissues of other animals. Appreciable structural resemblance between human and rodent fatty acid synthetases is indicated by studies on the immunological cross-reactivities of these enzymes.

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

Background: The source of malonyl-CoA required for de novo fatty acid synthesis in mammalian mitochondria is unknown. Results: Mammalian ACSF3 protein is a mitochondrial malonyl-CoA synthetase. Conclusion: Free malonate is a precursor for mitochondrial malonyl-CoA. Significance: Mammalian mitochondria are unusual in utilizing a malonyl-CoA synthetase as a surrogate for the acetyl-CoA carboxylase usually employed in fatty acid synthesis. The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.