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Site-Directed Mutants of Parasporin PS2Aa1 with Enhanced Cytotoxic Activity in Colorectal Cancer Cell Lines
Miguel O Suárez-Barrera1,2,3, Lydia Visser1, Efraín H Pinzón-Reyes2
1Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9700 AB Groningen, The Netherlands.
Abstract:
Parasporin 2 has cytotoxic effects against numerous colon cancer cell lines, making it a viable alternative to traditional treatments. However, its mechanism of action and receptors remain unknown. In this study, site-directed mutagenesis was used to obtain PS2Aa1 mutants with variation in domain I at positions 256 and 257. Variants 015, 002, 3-3, 3-35, and 3-45 presented G256A, G256E, G257A, G257V, and G257E substitutions, respectively. Cytotoxicity tests were performed for the cell viability of cell lines SW480, SW620, and CaCo-2. Mutants 3-3, 3-35, and 3-45 efficiently killed the cell lines. It was found that the activated forms of caspase-3 and PARP were in higher abundance as well as increased production of γH2AX when 3-35 was used to treat CaCo-2 and SW480. To assess possible membrane-binding receptors involved in the interaction, an APN receptor blocking assay showed reduced activity of some parasporins. Hence, we performed molecular docking and molecular dynamics simulations to analyze the stability of possible interactions and identify the residues that could be involved in the protein-protein interaction of PS2Aa1 and APN. We found that residues 256 and 257 facilitate the interaction. Parasporin 3-35 is promising because it has higher cytotoxicity than PS2Aa1.
Insights
Parasporin 3-35 demonstrates enhanced colon cancer cell killing by inducing apoptosis. This study identifies key residues involved in parasporin-receptor interactions, paving the way for novel cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Parasporin 2 exhibits potent cytotoxicity against colon cancer cell lines.
- The precise mechanism of action and cellular receptors for parasporin 2 remain unidentified.
Purpose of the Study:
- To investigate the cytotoxic effects of modified parasporin 2 variants (PS2Aa1 mutants) on colon cancer cells.
- To elucidate the molecular interactions between PS2Aa1 and its potential receptors, specifically APN.
Main Methods:
- Site-directed mutagenesis was employed to create PS2Aa1 variants with substitutions at positions 256 and 257.
- Cytotoxicity assays were conducted on SW480, SW620, and CaCo-2 cell lines.
- Western blotting, APN receptor blocking assays, molecular docking, and molecular dynamics simulations were performed.
Main Results:
- Mutants 3-3, 3-35, and 3-45 showed significant colon cancer cell killing.
- Parasporin 3-35 treatment led to increased activation of caspase-3 and PARP, and elevated γH2AX levels.
- Residues 256 and 257 were identified as crucial for PS2Aa1-APN interaction.
Conclusions:
- Parasporin 3-35 exhibits superior cytotoxicity compared to wild-type PS2Aa1, mediated by apoptosis induction.
- The N-terminal domain, particularly residues 256 and 257, is critical for PS2Aa1 interaction with APN.
- These findings highlight the therapeutic potential of engineered parasporins for colon cancer treatment.
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