L166P Mutant DJ-1, Causative for Recessive Parkinson's Disease, Is Degraded through the Ubiquitin-Proteasome System

Abstract

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.

Familial Parkinson's Disease-associated L166P Mutation Disrupts DJ-1 Protein Folding and Function

“…By using gel filtration chromatography and co-immunoprecipitation analysis, we showed that DJ-1 exists as a dimer in solution, which is in agreement with recent reports (26,27,44). Our circular dichroism spectroscopic analyses revealed that, consistent with the crystal structure, the solution structure of DJ-1 contains substantial amount of ␣-helical structure.…”

“…The ubiquitin-proteasome pathway consists of two major steps: the conjugation of ubiquitin to the substrate and subsequent degradation of the ubiquitinated substrate protein by the 26 S proteasome. Although the L166P mutant was recently suggested to undergo degradation by the ubiquitin-proteasome pathway (44), it remains to be determined experimentally whether the L166P mutant is indeed ubiquitinated. To address this issue, we used a well established in vivo ubiquitination assay (39,45) to measure the ubiquitination of DJ-1 and its L166P mutant in mammalian cells.…”

Mitochondrial dysfunction in Parkinson's disease

“…61 The DJ1 mutations described are thought to affect either the active site or tertiary structure of the protein so as to reduce its half-life. 62 The mutations do not appear to modify its intracellular distribution. Cells lacking DJ1 were more sensitive to free radical damage and this was reversed by wild-type overexpression.…”

DJ-1 inhibits TRAIL-induced apoptosis by blocking pro-caspase-8 recruitment to FADD

“…To further verify the interactions between DJ-1 and FADD, we performed co-immunoprecipitation experiments using human embryonic kidney 293 (HEK 293) cells ( Figure 4B) and H1299 cells ( Figure 4C). Transfection of cells with equal amounts of plasmids containing either wild-type DJ-1 or L166P usually results in a lower expression level of L166P compared with wild-type DJ-1 because of the instability of L166P (Macedo et al, 2003;Miller et al, 2003;Moore et al, 2003). Considering this problem, we transfected the cells with increased amounts L166P plasmids to elevate L166P levels as described previously (Junn et al, 2005).…”