Enhancing the Cytotoxicity and Apoptotic Efficacy of Parasporin-2-Derived Variants (Mpp46Aa1) on Cancer Cell Lines
Abstract
:1. Introduction
2. Results
2.1. Construction of the Library of Mutants from PS2Aa1
2.2. Protein Extraction
2.3. Cytotoxicity of PS2Aa1 and Variants in Colon Cancer and Leukemia Cells
2.4. PS2Aa1 Variants Induce Cell Death via Apoptotic Mechanisms in Colon Cancer and Leukemia Cells
2.5. Molecular Dynamics of PS2Aa1 (Mpp46Aa1) Interaction with the Possible Main Receptor, GPI–CD59
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Bacterial Strains and Culture Condition
5.2. Cell Culture Conditions
5.3. Generation of Parasporin Mutants from Site-Directed Mutagenesis
5.4. Extraction and Activation of Parasporins
5.5. Cytotoxicity Assay and Selectivity Calculation
5.6. Apoptosis Detection via Annexin V-Cy3/6-DFDA Assay
5.7. Caspases 3/7 and 9 Assay
5.8. Determination of Mitochondrial Permeability Potential
5.9. Molecular Dynamic Analysis
5.10. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Nomenclature | Type/Location of the Mutation | Notation of the Mutation |
---|---|---|
PS2Aa1 | PS2Aa1 (PDB 2ZTB) | - |
0–15 | SUS/256 | G256A |
3–35 | SUS/257 | G257V |
N65 | SUS/92,175,218 | N92D, K175R, S218G |
Parasporin/Cell Line | IC50 of Parasporins in µg/mL (95% CI) | ||||||
---|---|---|---|---|---|---|---|
SW480 | SW620 | CaCO-2 | MOLT-4 | Jurkat | CHOK1 | NCM460 | |
0–15 | 2.9 (2.2–3.4) | 2 (1.6–2.5) | 73.32 (11.6–122.4) | 1.7 (0.8–3.6) | 1.2 (0.6–2.6) | 20.2 (20–20.3) | 77.7 (71.6–8) |
3–35 | 0.9 (0.6–1.1) | 1.2 (0.9–1.5) | 0.88 (0.81–0.9) | 1.0 (0.6–1.8) | 1.9 (1.2–3.2) | 39.9 (39.8–4) | 78.8 (70.5–89.6) |
N65 | 1.2 (1–1.3) | 1.0 (0.9–1.2) | 0.51 (0.427–0.6) | 0.6 (0.4–0.9) | 0.7 (0.5–1) | 40 (39.8–40.2) | 84.2 (79.2–9) |
PS2Aa1 | 2.1 (1.5–2.8) | 2.3 (1.7–2.9) | 2.57 (1.84–4.1) | 4.7 (3.6–6.5) | 1.6 (1.2–2.2) | 40.1 (40–40.3) | 67.7 (63.6–72.1) |
Parasporin/Cell Lines | SW480 | SW620 | CaCo-2 | MOLT-4 | Jurkat |
---|---|---|---|---|---|
0–15 | 27.0 | 38.2 | 1.1 | 45.7 | 63.7 |
N65 | 67.4 | 77.1 | 165.1 | 131.3 | 107.9 |
3–35 | 97.8 | 72.5 | 89.5 | 78.7 | 43.4 |
PS2Aa1 | 31.9 | 29.5 | 26.3 | 14.2 | 42.3 |
MD | Contact Residues (>70%) | |
---|---|---|
PS2Aa1 Residues—CD59 | PS2Aa1 Residues—GPI | |
Replicate 1 | ALA105, GLN106, TYR107, GLY108, TYR110 | ARG252 |
Replicate 2 | TYR57, ASN92, PRO101, ALA105, GLN106, TYR107, GLY108, TYR110 | ARG252 |
Replicate 3 | TYR57, ASN92, PRO101, ALA105, GLN106, TYR107, GLY108, TYR110 | ARG252 |
Comparison | PS2Aa1 Residues—CD59 |
---|---|
ALA105 | 5.6 Å |
GLN106 | 5.7 Å |
TYR107 | 3.6 Å |
GLY108 | 4.8 Å |
TYR110 | 3.8 Å |
TYR57 | 5.4 Å |
ASN92 | 5.6 Å |
PRO101 | 3.6 Å |
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Alarcón-Aldana, J.S.; Visser, L.; Rueda-Forero, N.J.; Pinzón-Reyes, E.H.; Rondón-Villarreal, P.; Suárez-Barrera, M.O. Enhancing the Cytotoxicity and Apoptotic Efficacy of Parasporin-2-Derived Variants (Mpp46Aa1) on Cancer Cell Lines. Toxins 2024, 16, 415. https://doi.org/10.3390/toxins16100415
Alarcón-Aldana JS, Visser L, Rueda-Forero NJ, Pinzón-Reyes EH, Rondón-Villarreal P, Suárez-Barrera MO. Enhancing the Cytotoxicity and Apoptotic Efficacy of Parasporin-2-Derived Variants (Mpp46Aa1) on Cancer Cell Lines. Toxins. 2024; 16(10):415. https://doi.org/10.3390/toxins16100415
Chicago/Turabian StyleAlarcón-Aldana, Juan S., Lydia Visser, Nohora J. Rueda-Forero, Efraín H. Pinzón-Reyes, Paola Rondón-Villarreal, and Miguel O. Suárez-Barrera. 2024. "Enhancing the Cytotoxicity and Apoptotic Efficacy of Parasporin-2-Derived Variants (Mpp46Aa1) on Cancer Cell Lines" Toxins 16, no. 10: 415. https://doi.org/10.3390/toxins16100415