The homozygous deletion found in the patients of the Dutch PARK7 family represents a natural knockout of DJ-1, indicating that the loss of function of DJ-1 is pathogenic. The L166P mutation probably also induces a loss of DJ-1 function because the mutant DJ-1L166P protein is unstable and rapidly degraded by the 20S/26S proteasome system, resulting in much lower steady-state levels in both transfected cells and patient lymphoblasts. The crystal structures confirmed that the residue mutated in the Italian PARK7 patients (L166) is located in a C-terminal a-helix and show that this helix is part of a hydrophobic core formed by three helices (two contributed by the C-terminal and one by the N-terminal part of the monomer), which is involved in the dimerization (37,38,41,43- 47). The L166P mutation appears to disrupt the C-terminal domain and the dimerization capability, suggesting that the dimerization is functionally important. Gel filtration studies suggest that the L166P mutant does not form dimers but either adopts a different higher order structure or complexes with other proteins or is severely misfolded. Interestingly, a deletion mutant lacking residues 173-189 is also reported to form higher aggregates. Taken as a whole, these findings suggest that the dimeric structure is important for the function of DJ-1.
Two other homozygous disease-linked missense mutations, M26I (48) and E64D (49), seem to have distinct molecular and biochemical properties. The highly conserved residue M26 is located in the N-terminal helix, which contributes to the same hydrophobic core and is spatially close to L166; furthermore, this N-terminal helix contributes to the putative active site of DJ-1 (34). The E64D protein is predicted to require a higher thermal energy for unfolding which might explain why available antibodies did not recognize the protein, however, whether this is relevant in vivo in unknown (45). In contrast to the L166P mutant, expression of M26I and E64D
mutant proteins is stable and they can form homodimers, although less efficient than WT DJ-1, and are not susceptible to proteasomal degradation (45,50). At this point the functional consequences of these mutations are therefore not understood.
In addition to having a low steady-state level, the subcellular localization of the mutant DJ-1L166P protein in transfection experiments is changed in comparison with the pattern seen with the wild type DJ-1. While the wild type DJ-1 shows uniform localization in the cytosol and nucleus, the DJ-1L166P mutant retains the nuclear localization but has lost the uniform cytosolic distribution in a fraction of the cells (between 10% and 50% of the cells depending on the study) and colocalizes with mitochondria (2,43,44,46). However, due to the high levels of expression in cell systems analyzed, it cannot be excluded that a fraction of wild type DJ-1 also localizes to mitochondria, and in fact there is evidence to indicate this (44). However, the mitochondrial localization has not yet been confirmed for endogenous protein in human brain tissue or primary mouse neurons. The observation that the mutant (or perhaps also the wild type) DJ-1 can colocalize with mitochondria suggests links to the function of these organelles, such as energy production, oxidative stress, and apoptosis.
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