Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenoty pes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a ...Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenoty pes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326-Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the I326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of p rotease susceptibility, suggesting a problem of mutant protein fold. Conformatio nal analyses against urea denaturation using urea gradient gel electrophoresis o r fluorescence emission from tryptophan residue revealed that themutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as th eir secondary structure with slightly different extent. The thermal unfolding pr ofiles measured by CD showed a marked lower melting temperature for I326T protei n compared with that of wild-type protein. Analysis of the protein 3D-structur e indicated that the Ile326 should be a core residue for folding kinetics and th e substitution of Ile326 by threonine should directly alter the kinetic equilibr ium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlat ions.展开更多
文摘Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenoty pes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326-Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the I326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of p rotease susceptibility, suggesting a problem of mutant protein fold. Conformatio nal analyses against urea denaturation using urea gradient gel electrophoresis o r fluorescence emission from tryptophan residue revealed that themutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as th eir secondary structure with slightly different extent. The thermal unfolding pr ofiles measured by CD showed a marked lower melting temperature for I326T protei n compared with that of wild-type protein. Analysis of the protein 3D-structur e indicated that the Ile326 should be a core residue for folding kinetics and th e substitution of Ile326 by threonine should directly alter the kinetic equilibr ium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlat ions.
