ABSTRACT
Mutations in the E3 ubiquitin ligase parkin are the most common known cause of autosomal recessive Parkinson's disease (PD), and parkin depletion may play a role in sporadic PD. Here, we sought to elucidate the mechanisms by which stress decreases parkin protein levels using cultured neuronal cells and the PD-relevant stressor, L-DOPA. We find that L-DOPA causes parkin loss through both oxidative stress-independent and oxidative stress-dependent pathways. Characterization of the latter reveals that it requires both the kinase PINK1 and parkin's interaction with phosphorylated ubiquitin (phospho-Ub) and is mediated by proteasomal degradation. Surprisingly, autoubiquitination and mitophagy do not appear to be required for such loss. In response to stress induced by hydrogen peroxide or CCCP, parkin degradation also requires its association with phospho-Ub, indicating that this mechanism is broadly generalizable. As oxidative stress, metabolic dysfunction and phospho-Ub levels are all elevated in PD, we suggest that these changes may contribute to a loss of parkin expression.
Subject(s)
Levodopa/pharmacology , Neurons/drug effects , Protein Kinases/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin/genetics , Animals , Carbonyl Cyanide m-Chlorophenyl Hydrazone/analogs & derivatives , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Differentiation , Cell Line, Tumor , Embryo, Mammalian , Gene Expression Regulation , Humans , Hydrogen Peroxide/pharmacology , Models, Biological , Neurons/metabolism , Neurons/pathology , PC12 Cells , Parkinsonian Disorders/genetics , Parkinsonian Disorders/metabolism , Parkinsonian Disorders/pathology , Phosphorylation/drug effects , Primary Cell Culture , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Kinases/metabolism , Proteolysis , Rats , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
The first example of aromatic cation-activated nucleophilic acyl substitution has been achieved. The conversion of carboxylic acids to their corresponding acid chlorides occurs rapidly in the presence of 3,3-dichlorocyclopropenes via the intermediacy of cyclopropenium carboxylate complexes. The effect of cyclopropene substituents on the rate of conversion is examined. The addition of tertiary amine base is found to dramatically accelerate reaction, and conditions were developed for the preparation of acid sensitive acid chlorides. Preparative scale peptide couplings of two N-Boc amino acids were achieved with this method.
