title: Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder.
abstract: 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 (Ile326Thr) 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 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 I326T protein compared with that of wild-type protein. Analysis of the protein 3D-structure indicated that the Ile326 should be a core residue for folding kinetics and the substitution of Ile326 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.
authors: Department of Pediatrics, Gifu University School of Medicine, Gifu 501-1194, Japan.
authors: Kazuyuki
authors: K
authors: Hashimoto
authors: Zenichiro
authors: Z
authors: Kato
authors: Tomoko
authors: T
authors: Nagase
authors: Nobuyuki
authors: N
authors: Shimozawa
authors: Kazuo
authors: K
authors: Kuwata
authors: Kentaro
authors: K
authors: Omoya
authors: Ailian
authors: A
authors: Li
authors: Eiji
authors: E
authors: Matsukuma
authors: Yutaka
authors: Y
authors: Yamamoto
authors: Hidenori
authors: H
authors: Ohnishi
authors: Hidehito
authors: H
authors: Tochio
authors: Masahiro
authors: M
authors: Shirakawa
authors: Yasuyuki
authors: Y
authors: Suzuki
authors: Ronald J A
authors: RJ
authors: Wanders
authors: Naomi
authors: N
authors: Kondo
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D005347:Fibroblasts / Q000378:metabolism; D005838:Genotype; D006720:Homozygote; D006801:Humans; D007223:Infant; D007532:Isoleucine / Q000737:chemistry; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D018901:Peroxisomal Disorders / Q000175:diagnosis* / Q000235:genetics*; D010641:Phenotype; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D008565:Membrane Proteins; C101884:PEX13 protein, human; D010455:Peptides; D011994:Recombinant Proteins; D007532:Isoleucine; D014364:Tryptophan