Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder.

[neonatal adrenoleukodystrophy]

Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes , such as neonatal adrenoleukodystrophy (NALD ). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile 326 Thr ) within a SH 3 domain of PEX 13 protein (Pex 13 p ), 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-Pex 13 p antibody also revealed TS phenotype of the I 326 T mutant protein itself in the patient cells . Protease digestion of the recombinant Pex 13 p -SH 3 domain showed an increase of protease susceptibility , suggesting a problem of mutant protein fold . Conformational analyses against urea denaturation using urea gradient gel electrophoresis or fluorescence emission from tryptophan residue revealed that the mutant 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 their secondary structure with slightly different extent . The thermal unfolding profiles measured by CD showed a marked lower melting temperature for I 326 T protein compared with that of wild-type protein . Analysis of the protein 3 D-structure indicated that the Ile 326 should be a core residue for folding kinetics and the substitution of Ile 326 by threonine should directly alter the kinetic equilibrium , 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 correlations .