Abstract 1734 Investigating the catalytic activity of PRSS23 as a novel serine protease in ovarian cancer

Introduction: Proteases play important roles in regulating protein activity and turnover, while their dysregulation can lead to disease. Among the >600 human proteases, many still lack defined functions. Serine Protease 23 (PRSS23) is a putative serine protease first discovered in mouse ovaries; its enzymatic and biological functions remain unknown. Our group has identified that high expression of PRSS23 in ovarian cancer patients is associated with poor survival. We have further found that PRSS23 induces resistance to anoikis, enabling cells to survive in suspension, and thus contributes to metastasis. Our studies suggest that PRSS23 can be a potential target for ovarian cancer therapy. Here, we aim to define the hypothesized catalytic activity of PRSS23 as a serine protease that plays a detrimental role in patients with ovarian cancer. Methods: PRSS23 was recombinantly expressed as a soluble fusion protein in the Arctic Express (DE3) strain of E. coli by utilizing the SUMO-tag as an N-terminal artificial pro-domain for the catalytic subunit. An S316A mutant version of the protein, which is predicted to be catalytically inactive, was generated and produced in parallel to provide a negative control for activity assays. The soluble recombinant fusion proteins were purified using Ni-NTA affinity chromatography. The SUMO-tag was removed using SUMO-protease digestion. Purity and digestion efficiency were assessed by Coomassie gel staining. Potential proteolytic activity was evaluated by EnzChek assay using green-fluorescent dye-quenched casein and with a series of colorimetric peptide substrates. Results: Recombinant SUMO-PRSS23 fusion protein was purified using Ni-NTA affinity chromatography, with >80-90% purity and a yield of 5 – 7mg/liter of E. coli culture. The fusion protein was processed with >90% efficiency by SUMO-protease to release the PRSS23 catalytic domain. PRSS23 catalyzed detectable cleavage of casein, although at a very slow rate. PRSS23 showed greatest proteolytic activity versus colorimetric peptide substrates with Arg in the P1 position; this specificity is consistent with a predicted role for Asp310, at a position potentially analogous to the specificity-determining Asp-189 of trypsin, in substrate recognition. PRSS23 showed optimum catalytic activity at pH 8.0, consistent with the pH dependence of other serine proteases. Future Directions: In ongoing work, we are optimizing the assay and characterizing the enzyme kinetics for the substrates identified. We hypothesize that pro-tumor effects in ovarian cancer are mediated through the proteolytic activity of PRSS23, and in future studies we will probe this hypothesis in biological models and seek to identify the biological substrates of PRSS23. We also aim to determine the X-ray crystal structure of PRSS23, which can serve as a basis for drug development studies. We acknowledge the support of the US-Israel Binational ScienceFoundation grant 2019303, the Mayo Clinic Proteomics Core, Mayo Graduate School of Biomedical Sciences and Dr. Evette Radisky's lab