Differential Effects of Parkinson's Disease-associated Mutations on Stability and Folding of DJ-1

• Published in: The Journal of biological chemistry 2004-02, Vol.279 (8), p.6943-6951
• Main Authors: Görner, Karin, Holtorf, Eve, Odoy, Sabine, Nuscher, Brigitte, Yamamoto, Ayako, Regula, Jörg T., Beyer, Klaus, Haass, Christian, Kahle, Philipp J.
• Format: Article
• Language: English
• Subjects: Cell Line; Circular Dichroism; Cycloheximide; Cycloheximide - pharmacology; Cysteine - chemistry; Cysteine Endopeptidases - metabolism; Cysteine Proteinase Inhibitors - pharmacology; DJ-1 gene; DJ-1 protein; Enzyme Inhibitors - pharmacology; Escherichia coli; Escherichia coli - metabolism; Gene Deletion; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins - pharmacology; Mass Spectrometry; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Mutation; Neurons - metabolism; Oncogene Proteins - chemistry; Oncogene Proteins - genetics; PARK7 gene; Parkinson Disease - genetics; Point Mutation; Proteasome Endopeptidase Complex; Protein Conformation; Protein Deglycase DJ-1; Protein Folding; Protein Structure, Secondary; Protein Synthesis Inhibitors - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; Temperature; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M309204200

Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in β-strand and α-helix conformation. α-Helices were more susceptible to thermal denaturation than the β-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation.