Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.
In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.
Department of Biology, University College London, Gower Street, London WC1E 6BT, UK.
Michael J
MJ
Lumb
Graeme M
GM
Birdsey
Christopher J
CJ
Danpure
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004795:Enzyme Stability; D015316:Genetic Therapy; D014644:Genetic Variation; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000201:enzymology* / Q000235:genetics*; D007669:Kidney Calculi / Q000201:enzymology* / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D000637:Transaminases; C019635:Alanine-glyoxylate transaminase