Regulation of DJ-1 structure and function: Implications for Parkinson's disease

John D Hulleman, Purdue University

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by oxidative stress and protein aggregation. Both toxic phenomena are mitigated by DJ-1, a homodimeric protein with proposed antioxidant and chaperone activities. The neuroprotective function of DJ-1 is modulated by oxidation of cysteine 106, a residue that may act as an oxidative stress 'sensor'. Loss-of-function mutations in the DJ-1 gene have been linked to early onset PD, and age-dependent over-oxidation of DJ-1 is thought to contribute to sporadic PD. The familial mutant L166P fails to dimerize and is rapidly degraded, suggesting that protein destabilization accounts for dysfunction of this mutant. However, the effects of other familial substitutions (e.g. M26I and E64D) as well as oxidation on DJ-1 structure and function remain unclear. First we investigated how the structure and stability of DJ-1 are impacted by the two pathogenic substitutions, M26I and E64D, and by over-oxidation with H2O2. Whereas the recombinant wild-type protein and E64D both adopted a dimeric structure and had similar thermodynamic stabilities, M26I showed an increased propensity to aggregate and decreased secondary structure. Similar to M26I, over-oxidized wild-type DJ-1 exhibited reduced secondary structure, and this property correlated with destabilization of the dimer. The engineered mutant C106A had a greater thermodynamic stability and was more resistant to oxidation-induced destabilization than the wild-type protein. These results suggest that (i) the M26I substitution and over-oxidation destabilize dimeric DJ-1, and (ii) the oxidation of cysteine 106 contributes to DJ-1 destabilization. We also provide evidence that the familial mutant M26I is selectively destabilized by exposure of the protein to metal ions, including Ni +2 and Fe +3. The presence of these metal ions causes a significant reduction in M26I dimer formation, as well as an increased propensity of M26I to aggregate, compared to the wild-type protein. Furthermore, variants of wild-type DJ-1 and M26I purified in the presence of Ni +2 have a significantly attenuated ability to suppress α-synuclein (αS) aggregation via a chaperone function. We next identified key regulatory residues of DJ-1 chaperone function and clarified the mechanisms by which DJ-1 reduces αS aggregation. Wild-type DJ-1 forms a stable, SDS-resistant complex with both insoluble and potentially toxic, protofibrillar αS. The interactions between DJ-1 and αS are dependent on the oxidation state of DJ-1 at C106. While wild-type DJ-1 and the C53A mutant prevent fibrillization and interact with both insoluble and protofibrillar αS, C106A does not prevent fibrillization, nor does it form a complex with αS. Interestingly, it appears that controlled oxidation at C106 (to the sulfinic acid) is necessary for the prevention of αS fibrillization. Thus, the oxidation state of C106 not only regulates the structure of DJ-1, but also its chaperone function. Overall, these data have revealed new targets for slowing the progression of familial and sporadic PD. More specifically, our results indicate that stimulating DJ-1 function may be a reasonable therapeutic strategy, and this may be achieved via: (i) stabilization of the DJ-1 homodimer, (ii) chelation of metals, and/or (iii) targeted upregulation of DJ-1 in neurons vulnerable to PD-related insults.

Degree

Ph.D.

Advisors

Rochet, Purdue University.

Subject Area

Neurosciences

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