The DJ-1L166P mutant protein associated with early onset Parkinson's disease is unstable and forms higher-order protein complexes

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder that involves the selective degeneration of midbrain dopaminergic neurons. Recently DJ-1 mutations have been linked to autosomal-recessive earlyonset Parkinsonism in two European families. By using gel filtration assays under physiological conditions we demonstrate that DJ-1 protein forms a dimeric structure. Conversely, the DJ-1 L166P mutant protein shows a different elution profile as compared with DJ-1 WT both in overexpression cellular systems or in lymphoblasts cells, suggesting that it might form higher order protein structures. Furthermore we observed that the level of DJ-1 L166P mutant protein in the patient's lymphoblasts was very low as compared with the wildtype protein. We excluded a potential transcriptional impairment by performing quantitative RT-PCR on the patient's material. Pulse-chase experiments in transfected COS-1 cells and cycloheximide treatment in control and patient lymphoblasts indicated that the mutant protein was rapidly degraded. This rapid turnover and the structural changes of DJ-1 L166P mutant protein might be crucial in the disease pathogenesis.

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

“…Several loss-of-function mutations in the gene encoding DJ-1 have been identified in patients with familial PD (11,12 turnover of the protein (33)(34)(35). In contrast, other mutations have a less pronounced effect on DJ-1 stability, and it is unclear how they perturb DJ-1 function.…”

“…Data from various groups indicate that L166P has a pronounced protein folding defect, resulting in impaired homodimer formation, rapid protein turnover, and a high propensity to form large protein complexes (33)(34)(35). Clearly, pronounced structural defects lead to the compromised functionality of L166P.…”

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).…”

Aggresome-forming TTRAP mediates pro-apoptotic properties of Parkinson's disease-associated DJ-1 missense mutations

“…This mutant presents a structural rearrangement that interferes with dimer formation favoring protein instability. 20 However, mutants such as M26I are more stable than L166P, showing protein levels comparable or slightly lower than WT. Furthermore, recent computational and biochemical studies have shown that M26I dimerizes.…”

“…On the contrary, L166P is very unstable and its expression level lower than WT. [18][19][20][21] Together with a loss of function, DJ-1 missense mutations may be involved in pathological protein-protein interactions that may lead to a 'gain-of-function' behavior. The search for common protein partners that distinguish mutant DJ-1s from WT has been so far limited, with the important exception of an increased binding to parkin shared by M26I and L166P.…”