Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in PINK1 (PTEN-induced Kinase 1) are the second most common cause of early-onset Parkinson’s Disease (PD). PINK1 is believed to maintain mitochondrial integrity by orchestrating mitophagy of dysfunctional mitochondria through phosphorylation of its substrate, Parkin. However, the effects of PD-associated mutations remain unclear.
To investigate this, a PINK1 orthologue, Tribolium castaneum PINK1 (TcPINK1), was genetically engineered and purified for biochemical studies. Then, TcPINK1 was reacted against the Ubiquitin-like domain (UBL1-76) of Parkin and other proteins with a similar beta-grasp fold including Ubiquitin, ATG8, NEDD8, and SUMO using an in vitro radioisotopic filter-based kinase assay. The data revealed that TcPINK1’s preferred substrate with the highest amount of activity was UBL followed by Ubiquitin, NEDD8, and SUMO, with no activity against ATG8, which lacks a Serine residue equivalent to the phosphorylated residue in UBL. NEDD8 and SUMO were phosphorylated even though they are not substrates which suggests that PINK1 is capable of nonspecific phosphorylation of proteins with a similar fold to UBL. In addition, it is possible that the phosphorylation of Ubiquitin as reported in the literature may be nonspecific as well.
TcPINK1 point mutations equivalent to the PD-associated human PINK1 mutations were genetically engineered, purified, and reacted against UBL. The P374L mutant showed a similar activity to wild type, and the A194D, G285D, and S289M mutants showed a significant decrease in activity. Since P374 resides in the C-lobe of the kinase away from the active site, the data suggest that this residue may not be involved with catalysis or with UBL binding. As A194, G285, and S289 all reside in the N-lobe near the active site, the data suggest that these point mutations may be involved with catalysis.
In conclusion, the data suggest that PINK1 specificity for Parkin may involve binding outside of the UBL domain. / 2024-05-26
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/29303 |
| Date | 05 1900 |
| Creators | Engel, Victoria Alexe' |
| Contributors | Hoang, Quyen Q., Harrington, Maureen A., Johnson, Steven M., Wang, Mu, Yamamoto, Bryan K. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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