Role of Zinc in Parkin RING2 E3 Ubiquitin Ligase Ubiquitination

Abstract

In common neurodegenerative diseases among older adults, Parkinson’s disease (PD) ranks the second after Alzheimer’s disease. Symptoms of Parkinson’s disease are shaking, slowness of movement, rigidity of the extremities and neck, minimal facial expressions, short-walking steps, stopped posture, and arm swinging. Autosomal-Recessive Juvenile Parkinsonism (AR-JP) is the most frequent cause of familial PD, and results from mutations in the Parkin gene. The parkin protein acts as an E3 ubiquitin ligase and plays an important role in the ubiquitin proteasome system (UPS). Parkin’s role is to identify and tag proteins that are damaged or no longer needed. Mutations in Parkin represent ~50% of disease-causing defects in AR-JP. It is believed that the E3 ubiquitin ligase activity of the parkin protein may play a protective role in neurodegenerative disorders including Parkinson’s, Huntington’s, and Alzheimer’s diseases. Parkin belongs to a class of multiple RING (Really Interesting New Gene) domain proteins designated as RBR (RING, in between RING, RING) proteins. RING domains are rich in cysteine amino acids that act as ligands for binding zinc ions. Recent observations of the RING proteins suggest that binding of zinc ions may aid in maintaining their stability and solubility, thereby regulating their function. The main goal of work described in this thesis was to test the hypothesis that a “zinc switch” regulates the catalytic activity of an isolated RING2 domain of parkin which is responsible for the ubiquitinating activity of parkin. Using a protein construct containing the isolated RING2 domain of parkin, I show, using a combination of centrifugation and autoubiquitination assays, that the catalytic activity of the RING2 domain is strongly inhibited by small amounts of zinc ions in solution. The results support the idea that reversible binding of zinc to a low affinity site on the RING2 domain of parkin regulates the E3 ligase activity of parkin.