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Structural and biophysical studies of the parkinsonism-associated protein DJ-1
Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. DJ-1 is a small (20kDa), homodimeric conserved protein whose mutation causes a rare form of inherited parkinsonism. DJ-1 is cytoprotective, particularly against oxidative stress and mitochondrial damage, and this activity depends on a highly conserved and oxidative-prone cysteine residue (Cys106). Cys106-sulfinic acid (Cys106-SO2-) is stabilized through a hydrogen bonding network involving a protonated Glu18 residue. As discussed in chapter 4, the use of multiple biophysical techniques established that several Glu18 mutations (E18N, E18D, E18Q and E18A) alter the oxidative propensity of Cys106 and are fully dimeric. ^ Several parkinsonian point mutations in DJ-1 have been identified, some of which profoundly disrupt DJ-1 structure. Others, such as M26I, are structurally benign and thus the origin of their pathogenicity is unknown. M26I DJ-1 has diminished protein levels in the cell, however its crystal structure is similar to that of wild-type DJ-1. As discussed in chapter 5, we have found that slow protein conformational dynamics are increased in M26I DJ-1 near physiological temperature (37°C), particularly within the hydrophobic core of the protein. The increased flexibility of M26I DJ-1 at 37°C causes instability and results in a temperature-sensitive loss-of-function phenotype that explains this mutation's pathogenic nature. ^ Isocyanide hydratase (ICH) is an enzyme of the DJ-1/PfpI superfamily that hydrates toxic isocyanide species to yield the N-substituted formamide. Like other members of this superfamily of proteins, ICH requires a highly conserved,cysteine (Cys101) for catalysis. As discussed in chapter 6, we aimed to understand the mechanism of ICH catalysis. The ICH structure showed that Cys101 interacts with Asp17, Thr102, and an ordered water molecule to facilitate catalysis. Through the use of kinetic assays, T102 was found to be an important residue, but it is not essential for ICH catalysis. ^ Collectively, this dissertation aimed to further understand the biophysical behavior of a pathogenic mutation in the human protein DJ-1 and a related superfamily member, ICH.^
Milkovic, Nicole Marie, "Structural and biophysical studies of the parkinsonism-associated protein DJ-1" (2014). ETD collection for University of Nebraska - Lincoln. AAI3667137.