2024-2025 Grant in Aid of Research
Exploring the Potential Overlap in Function of CPO and GCPII.
Konstantin Zubkov
Carboxypeptidases are enzymes that modify the carboxyl ends of proteins and peptides. Human Carboxypeptidase O is an enzyme that is highly expressed in the brain and intestine and displays a high preference for acidic residues. Although CPO’s biochemical substrate has been characterized, its physiological substrate remains unreported. In this study, I will investigate the hypothesis that CPO cleaves the same substrate as GCPII, an enzyme that also cleaves acidic residue. To address this, I will first express CPO in insect Sf9 cells and subsequently purify CPO by using affinity chromatography. Then, I will test the effect of GCPII inhibitors on CPO by measuring the cleavage of an artificial, glutamate-containing substrate in a spectrophotometer. Additionally, the localization of CPO in neural cells will be assessed by expressing CPO in a neural PC12 cell line and visualizing CPO localization in those same cells using immunocytochemistry. Lastly, the effect of CPO on NAAG will be determined by expressing CPO in a neural cell line that synthesizes NAAG, SH-SY5Y. The levels of glutamate, which will indicate NAAG cleavage, will be compared between cells expressing CPO and cells that do not. Additionally, immunocytochemistry will be performed to visualize CPO localization within those cells.