Differential effects of Parkinson's disease-associated mutations on stability and folding of DJ-1.
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 beta-strand and alpha-helix conformation. Alpha-helices were more susceptible to thermal denaturation than the beta-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.
Görner
Karin
K
Laboratory of Alzheimer's and Parkinson's Disease Research, Department of Biochemistry, Adolf Butenandt Institute, Ludwig Maximilians University, 80336 Munich, Germany.
Holtorf
Eve
E
Odoy
Sabine
S
Nuscher
Brigitte
B
Yamamoto
Ayako
A
Regula
Jörg T
JT
Beyer
Klaus
K
Haass
Christian
C
Kahle
Philipp J
PJ
D002460:Cell Line; D002942:Circular Dichroism; D003513:Cycloheximide / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins / Q000494:pharmacology; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics*; D010300:Parkinson Disease / Q000235:genetics*; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D000071617:Protein Deglycase DJ-1; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors
D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D009097:Multienzyme Complexes; D015513:Oncogene Proteins; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D003545:Cysteine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde