In silico Studies of Parasporin Proteins: Structural and Functional Insights and Proposed Cancer Cell Killing Mechanism for Parasporin 5 and 6

Nasima Aktar^1,2, Muhammad Manjurul Karim¹, Shakila Nargis Khan¹, Mustafizur Rahman³, Anowara Begum¹, Md. Mozammel Hoq^1,*

Significance: Structural and functional insights of anti-cancer proteins from Bacillus thuringiensis.

Edited by: El-Sayed A. El-Sheikh, PhD . Plant Protection Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt.

Abstract

Background: Cancer is the leading cause of death in the world and the new types of cancer are diagnosed regularly but the advancement in their treatment is relatively slow and not to mention very costly. Parasporins (PS), parasporal inclusion proteins from Bacillus thuringiensis, possess specific cytotoxicity against different cancer cells which has suggested them to be potential for cancer treatment due to their specific binding to cancer cells. Methods: Computational investigation were performed to exploit their physicochemical characteristics, structural properties including three dimensional (3D) model, model quality analysis, phylogenetic assessment and functional analysis along with the cancer-cell killing mechanism of PS-5 and PS-6 proteins using standard tools of bioinformatics. Results: PS proteins were found to be slightly acidic based on their isoelectric points i.e., pI ranging from 5.12- 6.19, and the instability indices (29.03- 42.31) indicate their highly stable nature in test tubes and higher aliphatic indices (62.54-94.75) indicate their thermostability, a feature suitable for high-level industrial production. In silico analysis of cellular localization predicts that the parasporins are mostly located in the cytoplasm and few in the plasma membrane but devoid of any signal peptide. The generated 3D models of PS proteins upon verification by Ramachandran plot analysis confirmed that our prediction lies in the good quality model range and facilitated the understanding of the very protein folding, assembly into complexes and cell killing mechanisms. It could be hypothesized that the PS-5 protein might induce apoptosis or act as β- pore forming toxin to kill specific cancer cells while PS-6 might act simply as pore forming toxin. Conclusion: The theoretical overview of this research would facilitate the researchers with valuable insights of the PS protein structures, cancer cell killing mechanism of PS-5 and PS-6 proteins eventually in tumor micro-environment and their receptor molecules with a view to develop anti-cancer drugs.

Keywords: Parasporin, Domain, Motif, Cytotoxicity, Molecular docking.

Significance: Structural and functional insights of anti-cancer proteins from Bacillus thuringiensis.

Edited by: El-Sayed A. El-Sheikh, PhD

Plant Protection Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt.

Abbreviations: Bt, Bacillus thuringiensis; PS, Parasporin; β-PFT, β- Pore Forming Toxin; CD, Circular Dichroism; Cry, Crystal; pI, Isoelectric point; AI, Aliphatic Index; GRAVY, Grand average of hydropathy; PI, Phosphatidyl Inositol; GPI, Glycosyl Phosphatidyl Inositol; GOL, Glycerol; 13D, 1, 3- Diaminopropane; Br, Bromide; NGA, N- Acetyle- D- Galactosamine; U1, Uracil; E64, N- [N- [1- Hydroxycarboxyethyl- Carbonyl] Leucylamino- Butyl]- Guanidine; HEA, Hydroxyethylamine; MN, Manganese (II) ion.

References

Adang, M. J., Crickmore, N., & Jurat-Fuentes, J. L. (2014). Diversity of Bacillus thuringiensis Crystal Toxins and Mechanism of Action. In T. S. Dhadialla & S. S. Gill (Eds.), Advances in Insect Physiology (Insect Mid, Vol. 47, pp. 39–87). Oxford: Academic Press: Elsevier Ltd. https://doi.org/10.1016/B978-0-12-800197-4.00002-6

Akiba, T., Abe, Y., Kitada, S., Kusaka, Y., Ito, A., Ichimatsu, T., … Harata, K. (2009). Crystal structure of the parasporin-2 Bacillus thuringiensis toxin that recognizes cancer cells. Journal of Molecular Biology, 386(1), 121–33. https://doi.org/10.1016/j.jmb.2008.12.002

Aldeewan, A., Zhang, Y., & Su, L. (2014). Bacillus thuringiensis Parasporins Functions on Cancer Cells. International Journal Of Pure & Applied Bioscience, 2(4), 67–74.

Arnold, K., Bordoli, L., Kopp, J., & Schwede, T. (2006). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics (Oxford, England), 22(2), 195–201. https://doi.org/10.1093/bioinformatics/bti770

Beegle, C. C., & Yamamoto, T. (1992). History of Bacillus thuringiensis Berliner Research and Development. The Canadian Entomologist, 124(04), 587–616. https://doi.org/10.4039/Ent124587-4

Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., … Schwede, T. (2014). SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research, 42(Web Server issue), W252-8. https://doi.org/10.1093/nar/gku340

Daniotti, J. L., Vilcaes, A. A., Torres Demichelis, V., Ruggiero, F. M., & Rodriguez-Walker, M. (2013). Glycosylation of Glycolipids in Cancer: Basis for Development of Novel Therapeutic Approaches. Frontiers in Oncology, 3, 306. https://doi.org/10.3389/fonc.2013.00306

Eisenhaber, B., Bork, P., & Eisenhaber, F. (1998). Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase. Protein Engineering Design and Selection, 11(12), 1155–1161. https://doi.org/10.1093/protein/11.12.1155

Eisenhaber, B., Bork, P., & Eisenhaber, F. (1999). Prediction of potential GPI-modification sites in proprotein sequences. Journal of Molecular Biology, 292(3), 741–58. https://doi.org/10.1006/jmbi.1999.3069

Eisenhaber, B., Bork, P., Yuan, Y., Löffler, G., & Eisenhaber, F. (2000). Automated annotation of GPI anchor sites: case study C. elegans. Trends in Biochemical Sciences, 25(7), 340–1. https://doi.org/10.1016/S0968-0004(00)01601-7

Ekino, K., Okumura, S., Ishikawa, T., Kitada, S., Saitoh, H., Akao, T., … Mizuki, E. (2014). Cloning and characterization of a unique cytotoxic protein parasporin-5 produced by Bacillus thuringiensis a1100 strain. Toxins, 6(6). https://doi.org/10.3390/toxins6061882

Ferdous, U. T., Shishir, M. A., Khan, S. N., & Hoq, M. M. (2018). Bacillus spp.: Attractive Sources of Anti-cancer and Anti-proliferative Biomolecules. Microbial Bioactives, 1(1), E033–E045. https://doi.org/10.25163/microbbioacts.11005B0408130818

Finn, R. D., Coggill, P., Eberhardt, R. Y., Eddy, S. R., Mistry, J., Mitchell, A. L., … Bateman, A. (2016). The Pfam protein families database: towards a more sustainable future. Nucleic Acids Research, 44(D1), D279-85. https://doi.org/10.1093/nar/gkv1344

Guex, N., Peitsch, M. C., & Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis, 30 Suppl 1(S1), S162-73. https://doi.org/10.1002/elps.200900140

Guruprasad, K., Reddy, B. V, & Pandit, M. W. (1990). Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Engineering, 4(2), 155–61. https://doi.org/10.1088/1751-8113/44/8/085201

Hastowo, S., Lay, B. W., & Ohba, M. (1992). Naturally occurring Bacillus thuringiensis in Indonesia. Journal of Applied Bacteriology, 73(2), 108–113. https://doi.org/10.1111/j.1365-2672.1992.tb01695.x

Hirokawa, T., Boon-Chieng, S., & Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics. https://doi.org/10.1093/bioinformatics/14.4.378

Ikai, A. (1980). Thermostability and aliphatic index of globular proteins. Journal of Biochemistry, 88(6), 1895–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7462208

Källberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., & Xu, J. (2012). Template-based protein structure modeling using the RaptorX web server. Nature Protocols, 7(8), 1511–1522. https://doi.org/10.1038/nprot.2012.085

Kiefer, F., Arnold, K., Kunzli, M., Bordoli, L., & Schwede, T. (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research, 37(Database), D387–D392. https://doi.org/10.1093/nar/gkn750

Kitada, S., Abe, Y., Ito, A., Kuge, O., Akao, T., & Mizuki, E. (2005). Molecular Identification and Cytocidal Action of Parasporin, a Protein Group of Novel Crystal Toxins Targeting Human Cancer Cells. 6th Pacific Rim Conference on the Biotechnology of Bacillus Thuringiensis and Its Environmental Impact, 6–10.

Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 33(7), 1870–1874. https://doi.org/10.1093/molbev/msw054

Kyte, J., & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157(1), 105–32. https://doi.org/10.1016/0022-2836(82)90515-0

Li, W., Cowley, A., Uludag, M., Gur, T., McWilliam, H., Squizzato, S., … Lopez, R. (2015). The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Research, 43(W1), W580–W584. https://doi.org/10.1093/nar/gkv279

Martin, P. A., & Travers, R. S. (1989). Worldwide Abundance and Distribution of Bacillus thuringiensis Isolates. Applied and Environmental Microbiology, 55(10), 2437–42. Retrieved from http://aem.asm.org/content/55/10/2437

McWilliam, H., Li, W., Uludag, M., Squizzato, S., Park, Y. M., Buso, N., … Lopez, R. (2013). Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Research, 41(Web Server issue), W597-600. https://doi.org/10.1093/nar/gkt376

Meadows, M. P., Ellis, D. J., Butt, J., Jarrett, P., & Burges, H. D. (1992). Distribution, frequency, and diversity of Bacillus thuringiensis in an animal feed mill. Applied and Environmental Microbiology.

Mitaku, S., & Hirokawa, T. (1999). Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length. Protein Engineering, 12(11), 953–7. https://doi.org/10.1093/protein/12.11.953

Mitaku, S., Hirokawa, T., & Tsuji, T. (2002). Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces. Bioinformatics, 18(4), 608–616. https://doi.org/10.1093/bioinformatics/18.4.608

Mitchell, A., Chang, H.-Y., Daugherty, L., Fraser, M., Hunter, S., Lopez, R., … Finn, R. D. (2015). The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Research, 43(Database issue), D213-21. https://doi.org/10.1093/nar/gku1243

Mizuki, E., Ohba, M., Akao, T., Yamashita, S., Saitoh, H., & Park, Y. S. (1999). Unique activity associated with non-insecticidal Bacillus thuringiensis parasporal inclusions: in vitro cell-killing action on human cancer cells. Journal of Applied Microbiology, 86(3), 477–486. https://doi.org/10.1046/j.1365-2672.1999.00692.x

Mizuki, E., Ohba, M., Ichimatsu, T., Hwang, S. H., Higuchi, K., Saitoh, H., & Akao, T. (1998). Unique appendages associated with spores of Bacillus cereus isolates. Journal of Basic Microbiology, 38(1), 33–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9542106

Nagamatsu, Y., Okamura, S., Saitou, H., Akao, T., & Mizuki, E. (2010). Three Cry toxins in two types from Bacillus thuringiensis strain M019 preferentially kill human hepatocyte cancer and uterus cervix cancer cells. Bioscience, Biotechnology, and Biochemistry, 74(3), 494–8. https://doi.org/10.1271/bbb.90615

Nakai, K., & Horton, P. (1999). PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends in Biochemical Sciences, 24(1), 34–35. https://doi.org/10.1016/S0968-0004(98)01336-X

Ohba, M. (1996). Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries. Journal of Applied Bacteriology, 80(1), 56–64. https://doi.org/10.1111/j.1365-2672.1996.tb03190.x

Ohba, M., & Aizawa, K. (1986). Insect toxicity of Bacillus thuringiensis isolated from soils of Japan. Journal of Invertebrate Pathology. https://doi.org/10.1016/0022-2011(86)90158-8

Ohba, M., Mizuki, E., & Uemori, A. (2009). Parasporin, a new anticancer protein group from Bacillus thuringiensis. Anticancer Research, 29(1), 427–433.

Okassov, A., Nersesyan, A., Kitada, S., & Ilin, A. (2015). Parasporins as new natural anticancer agents: a review. Journal of Balkan Union of Oncology, 20(1), 5–16.

Okumura, S., Ohba, M., Mizuki, E., Crickmore, N., Côté, J.-C., Nagamatsu, Y., … Shin, T. (2010). Parasporin nomenclature. Retrieved December 10, 2017, from http://parasporin.fitc.pref.fukuoka.jp/

Okumura, S., Saitoh, H., Ishikawa, T., Inouye, K., & Mizuki, E. (2011). Mode of action of parasporin-4 , a cytocidal protein from Bacillus thuringiensis. Biochimica et Biophysica Acta, 1808(6), 1476–1482. https://doi.org/10.1016/j.bbamem.2010.11.003

Petersen, T. N., Brunak, S., von Heijne, G., & Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 8(10), 785–6. https://doi.org/10.1038/nmeth.1701

Poupon, A., & Janin, J. (2010). Analysis and Prediction of Protein Quaternary Structure. In Methods in molecular biology (Vol. 609, pp. 349–364). https://doi.org/10.1007/978-1-60327-241-4_20

Raymond W. Ruddon. (2003). What Makes a Cancer Cell a Cancer Cell? In D. W. Kufe, R. E. Pollock, R. R. Weichselbaum, R. C. Bast, T. S. Gansler, J. F. Holland, & E. Frei (Eds.), Holland-Frei Cancer Medicine (6th ed.). Hamilton (ON): BC Decker; 2003. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK12516/

Roh, J. Y., Park, H. W., Jin, B. R., Kim, H. S., Yu, Y. M., & Kang, S. K. (1996). Characterization of novel non-toxic Bacillus thuringiensis isolates from Korea. Letters in Applied Microbiology, 23(4), 249–252.

Shishir, A., Akter, A., Hassan, M. H., Kibria, G., Ilias, M., Khan, S. N., & Hoq, M. M. (2012). Characterization of locally isolated Bacillus thuringiensis for the Development of Eco-friendly Biopesticides in Bangladesh. Journal of Biopesticides, 5(Supplementary), 216–222.

Shishir, A., Bhowmik, A. A., Akanda, N. R., Al Mamun, A., Khan, S. N., & Hoq, M. M. (2015). Efficacy of Indigenous Bacillus thuringiensis Strains for Controlling Major Vegetable Pests in Bangladesh. Egyptian Journal of Biological Pest Control, 25(3), 729–734. Retrieved from http://search.ebscohost.com/login.aspx?direct=true&db=asx&AN=112957578&site=eds-live

Shishir, A., Roy, A., Islam, N., Rahman, A., Khan, S. N., & Hoq, M. M. (2014). Abundance and diversity of Bacillus thuringiensis in Bangladesh and their cry genes profile. Frontiers in Environmental Science, 2(June), 20. https://doi.org/10.3389/fenvs.2014.00020

Shishir, M. A., Akter, A., Bodiuzzaman, M., Aktar, N., Rahman, M., Khan, S. A., … Hoq, M. M. (2012). Molecular characterization of indigenous Bacillus thuringiensis kurstaki isolates with effective pesticidal activity against Bactrocera cucurbitae from Bangladesh. In R. Kada, J. Sasaki, C. R. Ahsan, & M. L. Bari (Eds.), Proceddings of 1st AFSA Conferences (Vol. 1, pp. 143–148). Osaka, Japan: Asian Food Safety & Security Association (AFSSA). Retrieved from http://www.afsahome.org/wp-content/uploads/2017/05/Food_Safety_and_Food_Security_2012-00.pdf

Shishir, M. A., Akter, A., Bodiuzzaman, M., Hossain, M. A., Alam, M. M., Khan, S. A., … Hoq, M. M. (2015). Novel Toxicity of Bacillus thuringiensis Strains against the Melon Fruit Fly, Bactrocera cucurbitae (Diptera: Tephritidae). Biocontrol Science, 20(2), 115–123. https://doi.org/10.4265/bio.20.115

Shishir, M. A., Pervin, S., Sultana, M., Khan, S. N., & Hoq, M. M. (2015). Genetic Diversity of Indigenous Bacillus thuringiensis Strains by RAPD-PCR to Combat Pest Resistance. Bt Research, 6(8), 1–16. https://doi.org/10.5376/bt.2015.06.0008

Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., … Higgins, D. G. (2014). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7(1), 539–539. https://doi.org/10.1038/msb.2011.75

Sunyaev, S. R., Eisenhaber, F., Rodchenkov, I. V., Eisenhaber, B., Tumanyan, V. G., & Kuznetsov, E. N. (1999). PSIC: profile extraction from sequence alignments with position-specific counts of independent observations. Protein Engineering, 12(5), 387–94. https://doi.org/10.1093/protein/12.5.387

Tyagi, A., Kapoor, P., Kumar, R., Chaudhary, K., Gautam, A., & Raghava, G. P. S. (2013). In silico models for designing and discovering novel anticancer peptides. Scientific Reports, 3(1), 2984. https://doi.org/10.1038/srep02984

WHO media center. (2017). Cancer fact sheet. Retrieved from http://www.who.int/mediacentre/factsheets/fs297/en/

Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., & Hochstrasser, D. F. (1999). Protein identification and analysis tools in the ExPASy server. Methods in Molecular Biology (Clifton, N.J.), 112, 531–52. https://doi.org/10.1088/1751-8113/44/8/085201

Yamashita, S., Katayama, H., Saitoh, H., Akao, T., Park, Y. S., Mizuki, E., … Ito, A. (2005). Typical three-domain cry proteins of Bacillus thuringiensis strain A1462 exhibit cytocidal activity on limited human cancer cells. Journal of Biochemistry, 138(6), 663–672. https://doi.org/10.1093/jb/mvi177