Abstract
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.
Highlights
Hereditary parkinsonism is very rare compared with sporadic Parkinson’s disease (PD),1 the identification of PARK genes has greatly expanded the molecular understand
Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in -strand and ␣-helix conformation. ␣-Helices were more susceptible to thermal denaturation than the -sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1
We found that the solution structures of [WT]DJ-1 and [E64D]DJ-1 were rich in ␣-helix and -sheet, but ␣-helical elements were more susceptible to thermal denaturation in [WT]DJ-1 than in [E64D]DJ-1
Summary
Hereditary parkinsonism is very rare compared with sporadic Parkinson’s disease (PD), the identification of PARK genes has greatly expanded the molecular understand-. Complete loss of functional DJ-1 was predicted for a young-onset PD patient with compound frameshift and splice mutations [7]. It remains to be shown what physiological function of DJ-1 is depleted. The homozygous index patient had early-onset PD along with significant depletion of striatal dopamine receptors, as evidenced by the reduced [18F]FP-CIT uptake. To elucidate the molecular basis of the deficits of DJ-1 point mutations, the expression, processing, and turnover of WT and mutant DJ-1 were examined. Expression levels of mutant [L166P]DJ-1 were significantly reduced compared with [WT]DJ-1 [20, 21] as well as [E64D]DJ-1, both in prokaryotic and eukaryotic cells.
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