Clonage de l'ADNc de la 3-phosphoglycérate-déshydrogénase de foie de rat et étude de la régulation de son expression par les hormones et les nutriments
(2000)
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- The main subject of this thesis is the dietary control of the hepatic expression of 3-phophoglycerta-dehydrogenase (3-PGDH), the enzyme that catalyses the first step in the pathways of serine biosynthesis. <BR> The introduction (Chapter 1) is devoted to a description of serine metabolism, with particular emphasis on 3-PGDH, and of the nutritional regulation of gene expression. 3-PGDH is widely distributed in the living world. The Escherichia coli enzyme has been crystallised and its three-dimensional structure established by X-ray diffraction studies. It is a homotetrameric enzyme that is allostericaly inhibited by serine, but not by phosphohydroxypyruvate, and in which each monomer contains three domains binding the substrate, the nucleotide and the allosteric effector, respectively. By contrast, the mammalian enzyme is inhibited by phosphohydroxypyruvate but not by serine. In rat liver, the activity of 3-PGDH strongly depends on the nutritional status: it is low in animals fed a normal diet and increase mire than 30-fold upon feeding a low protein, carbohydrate-rich diet, a situation analogous to kwashiorkor in man. Such an effect of the diet is most likely mediated through changes in the blood concentration of amino acids or hormones such as insulin and glucagon. It may involve changes in gene transcription, mRNA stability and/or translation. These mechanisms are briefly reviewed. Finally several examples of genes undergoing dietary regulation are provided: serine dehydratase, which illustrates cAMP-mediated regulation, asparagine synthetase, which involves control by aminoacyl-tRANs and alpha-1 type I collagen, which is an example of control by the amino acid cysteine. <BR> Chapter 2 reports the cloning of a cDNA encoding rat 3- PGDH. The similarity of this enzyme with 3-PGDH from other species is more apparent in the substrate- and NAD-binding domains than in the C-terminal domain, which is known to mediate allosteric inhibition by serine in the E. coli enzyme. The full length rat 3-PGDH and a shorter version lacking the C-terminal domain were expressed in E. coli, purified and shown to have kinetic properties similar to those of the enzyme purified from rat liver, including inhibition by 3-phosphohydroxypyruvate. This result indicated that the C-terminal domain is not implicated in the latter property. Northern blots indicated the presence of a 2.1 kb mRNA in the livers of rats fed only with starch, though not in the livers of control rats. The abundance of the 2.1 kb mRNA was independent of the nutritional status in other tissues. <BR> In Chapter 3, we show that cysteine is the amino acid whose concentration decreases the most when rats are fed only with starch and that its concentration can be restored to normal by refeeding a control diet, or by administration of cysteine or methionine. In starch-fed rats, the concentration of the 3-PGDH mRNA in liver decreases following refeeding a control diet, or following administration of glucagon, cysteineor methionine. Glucagon, as well as cysteine (tested with methionine) decrease the amount of the 3-PGDH mRNA in hepatocytes in primary culture, whereas insulin has a positive effect. Cysteine but not methionine decreases the amount of the 3_PGDH mRNA in hepatoma cells. Experiments with a transcription inhibitor and run-on assays in hepatocytes in primary culture indicate thatglucagon acts on the transcription of the gene whereas cysteine acts on mRNA stability. <BR> Chapter 4 reports experiments showing that the mRNA encoding phosphoserine transaminase, the enzyme catalysing the second step of the serine biosynthesis pathway, increases in the liver when rats are fed starch and that administration if cysteine causes, after 16h, its almost complete desappearence. <BR> In Chapter 5, we report that the rat gene encoding 3-PGDN contains 13 exons and is located on chromosome 2q34. 5’-RACE experiments indicated that the same promoter is used in liver as in other tissues but that a second promoter, about 2000 bp more upstream, is also used in testis. Transfection of hepatoma cells with various portions of the putative ubiquitous promoter showed that it drove the expression of a reporter gene, but in a cysteine-insensitive manner. <BR> Chapter 6 is devoted to a general discussion of our work. We first recall the arguments allowing us to conclude that the 3-PGDH cDNA that we have cloned encodes the enzyme that is present in liver and in other tissues. We also discuss the potential regulatory role of the C-terminal region, which we rule out on the basis of the experiments performed with the truncated protein. We then try to identify the dietary factors and hormones that could participate in the control of the expression of 3-PGDH and stress the role of cysteine, whose plasmatic concentration rapidly decreases during protein starvation. We also take into consideration the role of glucagon and insulin, which have opposite effects on the concentration of the 3-PGDH mRNA in liver and the plasmatic concentration of which is modified according to the composition of the diet. We conclude that the mechanism by which a diet consisting only of carbohydrate increased the expression of 3-PGDH, involves a decrease in the concentrations of glucagon and cysteine, two agents that decrease the amount of the mRNA, one by inhibiting transcription and the other by decreasing mRNA stability. It may also involve an increase in the concentration of insulin, which positively affects the expression of 3-PGDH
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Achouri, Y. (2000). Clonage de l’ADNc de la 3-phosphoglycérate-déshydrogénase de foie de rat et étude de la régulation de son expression par les hormones et les nutriments. https://hdl.handle.net/2078.5/111089