Arginine-170 is Important in Stabilizing the Active Parkin Oligomer
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Parkinson’s disease (PD) is the second most common neurodegenerative disease, with more than 10 million people affected worldwide and 60,000 new cases reported in the United States annually. It is estimated that 5-10% of these cases are linked to underlying genetic causes. The predominant autosomal recessive forms of PD are associated with mutations in the gene PARK2, which encodes the enzyme Parkin. Parkin is a ubiquitin ligase required for the degradation of mitochondrial proteins damaged by oxidative stress. With its loss of function, the neural cell death associated with PD occurs. This targeted degradation of damaged mitochondrial proteins occurs through the ubiquitin proteasome system in which ubiquitin ligases recruit a charged E2~ubiquitin carrier protein and catalyze the transfer of activated ubiquitin from the E2 to the protein substrate. Polyubiquitin chains are subsequently formed on the protein substrate that are recognized by the proteasome and result in the degradation of the targeted protein. Despite information available on Parkin and its relation to PD pathogenesis, Parkin’s mechanism of action is not well understood. Parkin is an RBG ligase similar in catalytic function to HECT domain ligases. The Haas lab has recently extended earlier work on HECT domain ligases to RBG ligases with current experiments aimed to explore the mechanism of human Parkin conjugation. Previous kinetic studies show that Parkin exhibits cooperativity with a Hill value of 2, suggesting that its active form is a dimer. Prior to these experiments, a synthetic gene for human Parkin (variant 1; accession number BAA25751) was optimized for bacterial codon usage and the resulting synthetic Parkin coding region was subcloned into the pGEX-4T3-6A expression plasmid. Recombinant GST-Parkin was expressed in Escherichia coli BL21 (DE3) cells at 16°C for 14 hours after induction with 0.40 mM IPTG. The resulting protein was then purified by affinity chromatography using glutathione Sepharose. The GST-Parkin was processed using thrombin and was resolved from processed GST using glutathione Sepharose. The resulting free, unbound Parkin was used in subsequent experiments. A similar approach was used for the generation of GST-PINK1, with the thrombin processing step omitted. Parkin was then activated by incubation with GST-PINK1 under optimized conditions to phosphorylate the Ser-65 residue of the former. GST-PINK1 was removed by passing the mixture through glutathione Sepharose. Final experiments were designed to test the hypothesis that the active form of Parkin is a dimer stabilized by the Arg- 170 residue using kinetic assays. Guanidine HCl was used in the assays to determine if inhibition of Arg-170 would affect polyubiquitin chain formation. Results of the experiments indicated Parkin functions as a dimer stabilized by side chain interactions between Arg-170 and Lys-220. Thus, Guanidine HCl represents a potential lead compound for the development of drugs targeting Parkin activity.
Recommended Citation
Hossain, Kariza N., "Arginine-170 is Important in Stabilizing the Active Parkin Oligomer" (2019). Summer Research Internship Program. 17.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/17
Arginine-170 is Important in Stabilizing the Active Parkin Oligomer
LSU Health Medical Education Building
Parkinson’s disease (PD) is the second most common neurodegenerative disease, with more than 10 million people affected worldwide and 60,000 new cases reported in the United States annually. It is estimated that 5-10% of these cases are linked to underlying genetic causes. The predominant autosomal recessive forms of PD are associated with mutations in the gene PARK2, which encodes the enzyme Parkin. Parkin is a ubiquitin ligase required for the degradation of mitochondrial proteins damaged by oxidative stress. With its loss of function, the neural cell death associated with PD occurs. This targeted degradation of damaged mitochondrial proteins occurs through the ubiquitin proteasome system in which ubiquitin ligases recruit a charged E2~ubiquitin carrier protein and catalyze the transfer of activated ubiquitin from the E2 to the protein substrate. Polyubiquitin chains are subsequently formed on the protein substrate that are recognized by the proteasome and result in the degradation of the targeted protein. Despite information available on Parkin and its relation to PD pathogenesis, Parkin’s mechanism of action is not well understood. Parkin is an RBG ligase similar in catalytic function to HECT domain ligases. The Haas lab has recently extended earlier work on HECT domain ligases to RBG ligases with current experiments aimed to explore the mechanism of human Parkin conjugation. Previous kinetic studies show that Parkin exhibits cooperativity with a Hill value of 2, suggesting that its active form is a dimer. Prior to these experiments, a synthetic gene for human Parkin (variant 1; accession number BAA25751) was optimized for bacterial codon usage and the resulting synthetic Parkin coding region was subcloned into the pGEX-4T3-6A expression plasmid. Recombinant GST-Parkin was expressed in Escherichia coli BL21 (DE3) cells at 16°C for 14 hours after induction with 0.40 mM IPTG. The resulting protein was then purified by affinity chromatography using glutathione Sepharose. The GST-Parkin was processed using thrombin and was resolved from processed GST using glutathione Sepharose. The resulting free, unbound Parkin was used in subsequent experiments. A similar approach was used for the generation of GST-PINK1, with the thrombin processing step omitted. Parkin was then activated by incubation with GST-PINK1 under optimized conditions to phosphorylate the Ser-65 residue of the former. GST-PINK1 was removed by passing the mixture through glutathione Sepharose. Final experiments were designed to test the hypothesis that the active form of Parkin is a dimer stabilized by the Arg- 170 residue using kinetic assays. Guanidine HCl was used in the assays to determine if inhibition of Arg-170 would affect polyubiquitin chain formation. Results of the experiments indicated Parkin functions as a dimer stabilized by side chain interactions between Arg-170 and Lys-220. Thus, Guanidine HCl represents a potential lead compound for the development of drugs targeting Parkin activity.
Comments
Mentors: Jennifer Klein and Dr. Arthur L. Haas, Department of Biochemistry & Molecular Biology