Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Kenya Moore - Dillard University
Co-Author(s): Arthur L. Haas, LSU School of Medicine, New Orleans, LA
Components of the ubiquitin proteasome system make up nearly 10% of the human genome. Prokaryotes have no molecule that is functionally analogous to ubiquitin. Ubiquitin plays a major role in the targeted degradation of proteins and cell regulation. Those processes function through the covalent conjugation of ubiquitin to cell proteins through a multi-enzyme pathway. In this pathway, the ubiquitin ligases recruit a charged E2~ubiquitin carrier protein and catalyzes the transfer of the activated ubiquitin from the E2 to a protein substrate. This project is centered on the ubiquitin ligase Parkin. Parkin is encoded by the PARK2 gene in humans and mutations in this gene are known to cause a familial form of Parkinson’s disease that disrupts regulation of mitophagy. Parkin is of particular interest as an example of a hybrid ligase. The RING (Really Interesting New Gene)-Between-RING family of E3 ligases functions with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT (Homologous to the E6AP Carboxyl Terminus) E3 ligases; therefore, it is identified as a RING/HECT hybrid. To better understand the enzyme, we expressed and purified Parkin with the long term goal of determining the mechanism of the ligase. Prior to these experiments, Parkin was subcloned into pGEX-Flag. Protein expression was conducted in Escherichia coli (BL21) cells. Induced with IPTG, the protein was purified by affinity chromatography using glutathione Sepharose. The molecular weight of the resulting GST-Parkin by SDS PAGE was 76 kDa, in good agreement with the expected value of 77 kDa. Once purified, biochemically defined assays demonstrate Parkin is active in forming a thioester intermediate with 125I-Ubiquitin using Ubc5B or UbcH7. We then cleaved GST from the fusion protein. Surprisingly, when the GST is cleaved, the enzyme loses activity. These results are consistent with other HECT ligases, for which the GST moiety promotes formation of the active oligomeric enzyme. This work establishes that recombinant Parkin shows robust activity in assembling 125I-polyubiquitin chains which can be used as a reporter function for future mechanistic studies.
This project is centered on the ubiquitin ligase Parkin. Parkin is encoded by the PARK2 gene in humans and mutations in this gene are known to cause a familial form of Parkinson?s disease that disrupts regulation of mitophagy. Parkin is of particular interest as an example of a hybrid ligase. The RING (Really Interesting New Gene)-Between-RING family of E3 ligases functions with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT (Homologous to the E6AP Carboxyl Terminus) E3 ligases; therefore, it is identified as a RING/HECT hybrid. To better understand the enzyme, we expressed and purified Parkin with the long term goal of determining the mechanism of the ligase.
Prior to these experiments, Parkin was subcloned into pGEX-Flag. Protein expression was conducted in Escherichia coli (BL21) cells. Induced with IPTG, the protein was purified by affinity chromatography using glutathione Sepharose. The molecular weight of the resulting GST-Parkin by SDS PAGE was 76 kDa, in good agreement with the expected value of 77 kDa. Once purified, biochemically defined assays demonstrate Parkin is active in forming a thioester intermediate with 125I-Ubiquitin using Ubc5B or UbcH7. We then cleaved GST from the fusion protein. Surprisingly, when the GST is cleaved, the enzyme loses activity. These results are consistent with other HECT ligases, for which the GST moiety promotes formation of the active oligomeric enzyme. This work establishes that recombinant Parkin shows robust activity in assembling 125I-polyubiquitin chains which can be used as a reporter function for future mechanistic studies.
Funder Acknowledgement(s): NIH
Faculty Advisor: Ruby Broadway, rbroadway@dillard.edu
Role: I did all of the research from the beginning to the end.