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Investigación en terapias específicas de mutación metabólicas hereditarias

AutorAguado, Cristina
DirectorRuiz, Lourdes
Palabras claveEnfermedades hereditarias metabólicas
Fecha de publicación2008
EditorUniversidad Autónoma de Madrid
ResumenIn this work, we have investigated specific mutation therapies in metabolic genetic diseases, specifically we have analyzed the mechanisms underlying cofactor and coenzyme responsiveness in phenylketonuria (PKU), propionic acidemia (PA), methylcrotonylglycinuria (MCG) and methylmalonic aciemia (MMA) and splicing therapies in PA. We have identified the mutations present in Spanish PKU patients who respond to BH4 and we have selected some mutations associated to responsiveness for expression analysis. We have analyzed in total 14 missense mutations using different prokaryote, eukaryote and cell‐free systems. Five mutations were expressed in vitro in E.coli as MBP‐PAH fusion proteins, we have determined PAH steady‐state kinetic properties in order to detect possible defects in BH4 affinity. The majority of the mutations affected kinetic and regulatory properties. Only one mutation showed slightly reduced affinity for the cofactor under steady‐state conditions. We have also measured the degradation rates of 11 mutant PAH proteins expressed in a cell‐free system, using pulse‐chase analysis and presence or absence of BH4 and determined the amount of three of the mutant proteins after transfection in Hep3B as FLAG‐PAH fusion proteins in presence of different levels of BH4. The results show that high concentrations of BH4 protect some mutant proteins against degradation and result in an increase in the steady‐state amounts of protein in hepatoma cells. In the hepatoma cellular model, BH4 levels don’t affect the expression of the PAH gene. In MMA cblB type we have selected two mutations previously associated with responsiveness to OHcobalamin. We have expressed the ATR protein in E. coli to determine steady‐state kinetic properties in order to detect possible defects in cobalamin affinity. The results suggest a stability defect for one of the mutant proteins, with reduced enzymatic activity but with K(m) for ATP and Kd for cob(I)alamin similar to wild‐type values. The last coenzyme analyzed in this work was biotin, for which there is no consensus in the literature if it affects the expression of biotin‐dependent carboxylases. In Hep3B cells we have analyzed the effect of different levels of biotin in the activity, amount of functional active protein and mRNA levels of two carboxylases, PCC and MCC, that when defective cause PA and MCG, respectively. In our model, biotin does not affect the expression of the enzymes but favours the conversion of apocarboxylase to holocarboxylase. Finally, we describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in PA. The two point mutations described in deep intronic regions increase the splicing score of pseudoexonic regions or generate consensus binding motifs for splicing factors, such as SRp40, which favour the intronic inclusion in PCCA (r.1284ins84) and PCCB (r.654ins72) mRNAs. AMOs were targeted to the 5´ and 3´ cryptic splice sites of the pseudoexons to block the access of the splicing machinery. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was efficiently translated and PCC activities were rescued in patients´fibroblasts. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease‐causing mutations in these patients and that the use of AMOs is an effective therapeutic strategy in this genetic disorder.
DescripciónTesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-04-08
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