Aging is a well regulated biological process involving oxidative stress and macromolecular damages. Three main pathways have been shown to influence lifespan, the insulin/insulin-like growth factor-1 pathway, the silent regulator pathway, and the target of rapamycin pathway. Among many proteins influencing lifespan, two transcription factors, FOXO and the heat shock factor, have been shown to be involved in the aging process and in small heat shock proteins (sHsps) expression following stress and during lifespan. We have recently shown that overexpressing the mitochondrial Hsp22 increases Drosophila melanogaster lifespan by 32% and resistance to oxidative stress. Here we show that flies that are not expressing this mitochondrial small Hsp22 have a 40% decrease in lifespan. These flies die faster than their matched control and display a decrease of 30% in locomotor activity compared with controls. The absence of Hsp22 also sensitizes flies to mild stress. These data support a key role of sHsps in aging and underline the importance of mitochondrial sHsps in this process.
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.
1Deborah Research Institute, 2Browns Mills, NJ, and Laboratoire de Génétique Cellulaire et développementale, Université Laval, Ste Foy, Québec, Canada.
As a step towards the cloning of the gene for fumarylacetoacetate hydrolase (FAH), we have purified the FAH mRNA from rat liver by specific immunoadsorption of polysomes. The relative abundance of this mRNA has been estimated to be 0.14%. The major in vitro translation product of the purified mRNA preparation is specifically precipitated by a rabbit anti-rat FAH antiserum and it is, furthermore, undistinguishable by criteria of mass and charge from purified rat FAH.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
