Mutations in PNKD causing paroxysmal dyskinesia alters protein cleavage and stability

Abstract

Paroxysmal non-kinesigenic dyskinesia (PNKD) is a rare autosomal dominant movement disorder triggered by stress, fatigue or consumption of either alcohol or caffeine. Attacks last 1–4 h and consist of dramatic dystonia and choreoathetosis in the limbs, trunk and face. The disease is associated with single amino acid changes (A7V or A9V) in PNKD, a protein of unknown function. Here we studied the stability, cellular localization and enzymatic activity of the PNKD protein in cultured cells and transgenic animals. The N-terminus of the wild-type (WT) long PNKD isoform (PNKD-L) undergoes a cleavage event in vitro, resistance to which is conferred by disease-associated mutations. Mutant PNKD-L protein is degraded faster than the WT protein. These results suggest that the disease mutations underlying PNKD may disrupt protein processing in vivo, a hypothesis supported by our observation of decreased cortical Pnkd-L levels in mutant transgenic mice. Pnkd is homologous to a superfamily of enzymes with conserved β-lactamase domains. It shares highest homology with glyoxalase II but does not catalyze the same reaction. Lower glutathione levels were found in cortex lysates from Pnkd knockout mice versus WT littermates. Taken together, our results suggest an important role for the Pnkd protein in maintaining cellular redox status.

Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis

“…Full-length cDNA of human PNKD long form was previously described (49). Double mutations (A7V/A9V) were introduced into the human PNKD cDNA sequence by QuikChange XL Site-Directed Mutagenesis Kit (Stratagene).…”

Genetics of Paroxysmal Dyskinesia

“…Studies of the subcellular localization of both wild-type and mutant MR1 isoforms pointed to a novel disease mechanism involving the mitochondrial targeting sequence [ 13 ]. Investigations of the stability, cellular localization, and enzymatic activity of the PNKD protein in cultured cells and transgenic animals indicated that MR1 mutations disrupt protein processing in vivo resulting in an impairment of cellular redox status due to reduced glutathione levels [ 14 ].…”

Familial paroxysmal nonkinesigenic dyskinesia: Clinical and genetic analysis of a Taiwanese family

“…2) [9]. The three disease-associated mutations localized within the cleaved sequences may make it cleavage resistant and promote protein degradation through a ubiquitinproteosome dependent pathway [14]. Thirdly, the BAC transgenic mice carrying both A7V-and A9V-encoding mutations in the mouse Pnkd locus equivalent to those found in patients expressed the human PNKD phenotype of dyskinesia after stress, and the animals showed dopamine dysregulation in the nigrostriatal system in response to caffeine and ethanol treatment [15].…”