In silico mutational analysis and identification of stability centers in human interleukin-4

Document Type : Original article

Authors

Department of Bioinformatics, G.G.D.S.D. College, Chandigarh, India

Abstract

Interleukin-4 (IL-4) is a multifunctional cytokine that plays a critical role in apoptosis, differentiation and proliferation. The intensity of IL4 response depends upon binding to its receptor, IL-4R. The therapeutic efficiency of interleukins can be increased by generating structural mutants having greater stability. In the present work, attempts were made to increase the stability of human IL-4 using in-silico site directed mutagenesis. Different orthologous sequences of IL4 from Pan troglodytes, Aotusnigriceps, Macacamulatta, Papiohamadryas, Chlorocebusaethiops, Vicugnapacos, Susscrofa and Homo sapiens were aligned using Clustal Omega that revealed the conserved and non-conserved positions. For each non-conserved position, possible favorable and stabilizing mutations were found using CUPSAT with predicted ΔΔG (kcal/mol). The one with highest ΔΔG (kcal/mol) among all possible mutations, for each non-conserved position was selected and introduced manually in human IL-4 sequence resulting in multiple mutants of IL-4. Mutant proteins were modeled using structure of IL4 (PDB ID: 2B8U) as a template by SWISS MODEL. The mutants A49L and Q106T were identified to have stability centre using SCide. Molecular dynamics and docking analysis also confirmed the mutants stability and binding respectively. Mutants A49L and Q106T had -7.580079 kcal/mol and -39.418124 kcal/mol respectively lesser energy value than the wild type IL4. The result suggested that, the stability of human IL-4 has been increased by mutation.

Keywords

References

1. Zhang JM, An J. Cytokines, Inflammation and Pain. International anesthesiology clinics. 2007;45:27-37.
2. Gulati K, Guhathakurta S, Joshi J, Rai N, Ray A. Cytokines and their Role in Health and Disease: A Brief Overview. MOJ Immunol 2016;4:00121.
3.Vazquez LR, Roome B, Christ D. Molecular engineering of therapeutic cytokines. Antibodies 2013;2:426-451.
4. Charles A D, Mier JW. Interleukins. Ann Rev Med 1986;37:173-178.
5. Akdis M, Aab A, Altunbulakli C, Azkur K, Costa RA, Crameri R, Duan S, Eiwegger T, Eljaszewicz A, Ferstl R, Frei R, Garbani M, Globinska A, Hess L, Huitema C, Kubo T, Komlosi Z, Konieczna P, Kovacs N, Kucuksezer UC, Meyer N, Morita H, Olzhausen J, O'Mahony L, Pezer M, Prati M, Rebane A, Rhyner C, Rinaldi A, Sokolowska M, Stanic B, Sugita K, Treis A, van de Veen W, Wanke K, Wawrzyniak M,  Wawrzyniak P, Wirz OF, Zakzuk JS, Akdis CA. Interleukins from IL-1 to IL-38, interferons, transforming growth factor β, and TNF-α: Receptors, functions, and roles in diseases. J Allergy Clin Immunol 2016;138:984-1010.
6. Thompson D, Matsumoto A, Nebert DW, Vasiliou V.Evolutionary divergence and functions of the human interleukin (IL) gene family. Hum Genomics 2010;5:30-55.
7. Peters M. Actions of cytokines on the immune response and viral interactions: An overview. Hepatology 1996;23:909-916.
8. Shaikh PZ. Cytokines & their physiologic and pharmacologic functions in inflammation: A review. Int J Pharm Life Sci 2011;2:1247-1263.
9. Zamorano J, Rivas MD, Perez GM. Interleukin-4: A multifunctional cytokine. Inmunologia 2003;22:215-24.
10. Gadani SP, Cronk JC, Norris GT, Kipnis J. Interleukin-4: A Cytokine to Remember. J Immunol (Baltimore, Md : 1950) 2012;189:4213-4219.
11. Yu SJ, Kim HS, Cho SW, Sohn J. IL-4 inhibits proliferation of renal carcinoma cells by increasing the expression of p21 WAF1 and IRF-1. Exp Mol Med 2004;36:372-379.
12. Hu-Li J, Shevach EM, Mizuguchi J, Ohara J, Mosmann T, Paul WE. B cell stimulatory factor 1 (interleukin 4) is a potent costimulant for normal resting T lymphocytes. J Exp Med 1987;165:157-172.
13. Mandler R, Finkelman FD, Levine AD, Snapper CM. IL-4 induction of IgE class switching by lipopolysaccharide-activated murine B cells occurs predominantly through sequential switching. J Immunol 1993;150:407-418.
14. Anthony RM, Rutitzky L, Urban JF, Stadecker MJ, Gause WC. Protective immune mechanisms in helminth infection. Nat Rev Immunol 2007;7:975-987.
15. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999;17:701-738.
16. Izuhara K, Heike T, Otsuka T, Yamaoka K, Mayumi M, Imamura T, Niho Y, Harada N. Signal transduction pathway of interleukin-4 and interleukin-13 in human B cells derived from X-linked severe combined immunodeficiency patients. J Biol Chem 1996;271:619-622.
17. Tayal V, Kalra BS. Cytokines and anti-cytokines as therapeutics--an update. Eur J Pharmacol 2008;579:1-12.
18. Schooltink H, Rose-John S. Cytokines as therapeutic drugs. J Interferon Cytokine Res 2002;22:505-516.
19. Antony GK, Dudek AZ. Interleukin 2 in cancer therapy. Curr Med Chem 2010;17:3297-3302.
20. Razavi GSE, Allen T. Emerging Role of Interleukins in Cancer Treatment. Immunome Res 2015;S2:006.
21. Spangler JB, Moraga I, Mendoza JL, Garcia KC. Insights into cytokine-receptor interactions from cytokine engineering. Annu Rev Immunol 2015;33:139-167.
22. Zanotti G, Folli C, Cendron L, Alfieri B, Nishida SK, Gliubich F, Pasquato N, Negro A, Berni R. Structural and mutational analyses of protein-protein interactions between transthyretin and retinol-binding protein. FEBS J 2008;275:5841-5854.
23. Vignesh SN, Narayanan S, Sivanandham M. Mutational analysis on human granulocyte macrophage-colony stimulating factor stability using computational approaches. Trends Bioinform 2015;8:1-13.
24. Banerjee A, Ray S. Molecular modeling, mutational analysis and conformational switching in IL27: An in silico structural insight towards AIDS research. Gene 2016;576:72-78.
25. Dakshinamurthi AK, Chidambaram MV, Manivel VA, Detchanamurthy S. Site directed mutagenesis of human Interleukin-2 gene to increase the stability of the gene product- A bioinformatics approach. Int J Bioinformatics Res 2009;1:4-13.
26. Lakshmipathy D, Ramasubban G, Therese L, Vetrivel U, Sivashanmugam M, Rajendiran S, Sridhar R, Madhavan HN, Meenakshi N. In silico Analysis of novel mutation ala102pro targeting pncA gene of M. Tuberculosis. J Comput Sci Syst Biol 2013;6:83-87.
27. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The protein data bank. Nucleic Acids Res 2000;28:235-242.
28. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-410.
29. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011;7:539.
30. Parthiban V, Gromiha MM, Schomburg D. CUPSAT: prediction of protein stability upon point mutations. Nucleic Acids Res 2006;34:W239-242.
31. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 2014;42:W252-258.
32. Dosztanyi Z, Magyar C, Tusnády G, Simon I. SCide: identification of stabilization centers in proteins. Bioinformatics 2003;19:899-900.
33. Dassault Systemes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systemes, 2016.
34. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M. CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem 1983;4:187-217.
35. HyperChem (TM) Professional 7.51, Hypercube, Inc., 1115 NW 4th Street, Gainesville, Florida 32601, USA.
36. Ponder JW, Richards FM. An efficient newton-like method for molecular mechanics energy minimization of large molecules. J Comput Chem 1987;8:1016–1024.
37. Stich I, Car R, Parrinello M, Baroni S. Conjugate gradient minimization of the energy functional: A new method for electronic structure calculation. Phys Rev B Condens Matter 1989;39:4997-5004.
38. Ritchie D, Hex 8.0.0 user manual, 1996, http://hex.loria.fr/ manual800/hex manual.pdf
39. Michalovich D, Overington J, Fagan R.Protein sequence analysis in silico: application of structure-based bioinformatics to genomic initiatives. Curr Opin Pharmacol 2002;2:574-580.
40. Kanehisa M, Bork P. Bioinformatics in the post-sequence era. Nat Genet 2003;33Suppl: 305-310.
41. Jankun-Kelly T, Lindeman AD, Bridges SM. Exploratory visual analysis of conserved domains on multiple sequence alignments. BMC Bioinformatics 2009;10(Suppl 11):S7.
42. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. Multiple sequence alignment with the clustal series of programs. Nucleic Acids Res 2003;31: 3497-500.
43. Marini NJ, Thomas PD, Rine J. The use of orthologous sequences to predict the impact of amino acid substitutions on protein function. PLoS Genet 2010;6:e1000968.
44. Zhang Z, Wang L, Gao Y, Zhang J, Zhenirovskyy M, Alexov E. Predicting folding free energy changes upon single point mutations. Bioinformatics 2012;28:664-671.
45. Chiu TL, Goldstein R. Optimizing energy potentials for success in protein tertiary structure prediction. Fold Des 1998;3:223-228.
46. Matthews BW, Nicholson H, Becktel WJ. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA 1987;84: 6663-6667.
47. Kaffe-Abramovich T, Unger R.A simple model for evolution of proteins towards the global minimum of free energy. Fold Des 1998;3:389-99.
48. Monzon AM, Zea DJ, Fornasari MS, Saldano TE, Fernandez-Alberti S, Tosatto SC, Parisi G. Conformational diversity analysis reveals three functional mechanisms in proteins. PLoS Comput Biol 2017;13:e1005398.
49. Damm KL, Carlson HA. Gaussian-weighted RMSD superposition of proteins: A structural comparison for flexible proteins and predicted protein structures. Biophys J 2006;90:4558-4573.
50. Rueda AJV, Monzon AM, Ardanaz SM, Iglesias LE, Parisi G. Large scale analysis of protein conformational transitions from aqueous to non-aqueous media. BMC Bioinformatics 2018;19:27.
51. Baase WA, Eriksson AE, Zhang XJ, Heinz DW, Sauer U, Blaber M, Baldwin EP, Wozniak JA, Matthews BW. Dissection of protein structure and folding by directed mutagenesis. Faraday Discuss 1992;93:173-181.
52. Wang W, Malcolm BA. Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis. Biotechniques 1999;26:680-682.
53. Socha RD, Tokuriki N. Modulating protein stability-directed evolution strategies for improved protein function. FEBS J 2013;280:5582-5595.
54. Faryal MA, Ayesha O, Aqsa I, Hussnain AJ. Mutation-structure-function relationship based integrated strategy reveals the potential impact of deleterious missense mutations in autophagy related proteins on hepatocellular carcinoma (HCC): A comprehensive informatics approach. Int J Mol Sci 2017;18:139.
55. Abida A, Rashda A, Christian MKS, Muhammad A, Nafees A. In silico analysis of mutations in PITX3 gene. International Conference on Systems Biology 2014;ISB. 10.1109/ISB.2014.6990430.
56. Rao BM, Girvin AT, Ciardelli T, Lauffenburger DA, Wittrup KD. Interleukin-2 mutants with enhanced alpha-receptor subunit binding affinity. Protein Eng 2003;16:1081-1087.
57. Lopez AF, Shannon MF, Barry S, Phillips JA, Cambareri B, Dottore M, Simmons P, Vadas M A. A human interleukin 3 analog with increased biological and binding activities. Proc Natl Acad Sci USA 1992;89:11842-11846
58. Pereira E, Goldblatt J, Rye P, Sanderson C, Le Souef P. Mutation analysis of interleukin-5 in an asthmatic cohort. Hum Mutat 1998;11:51-54.
59. Nacheva G, Lilkova E, Petkov P, Petkov P, Ilieva N, Ivanov I and Litov L. In silico studies on the stability of human interferon-gamma mutants. Biotechnol Biotechnol Equip 2014;26:200-204.
60. Pandey AV. Bioinformatics tools and databases for the study of human growth hormone. Endocr Dev 2012;23:71-85.
61. Urmi R. Structural modeling of tumor necrosis factor: A protein of immunological importance. Biotechnol Appl Biochem 2017:64:454-463.
62. Jiang SJ, Liou JW, Chang CC, Chung Y, Lin LF, Hsu HJ. Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1. Sci Rep 2015;5:18638.
63. Iqbal H, Sarfaraz T, Anjum F, Anwar Z, Mir A. Identification of missense mutation (I12T) in the BSND gene and bioinformatics analysis. J Biomed Biotechnol 2011;2011:304612.
64. Scotti C, Olivieri C, Boeri L, Canzonieri C, Ornati F, Buscarini E, Pagella F, Danesino C. Bioinformatic analysis of pathogenic missense mutations of activin receptor like kinase 1 ectodomain. PLoS One 2011;6:e26431.
65. N N, Zhu H, Liu J, V K, C GP, Chakraborty C, Chen L. Analysing the effect of mutation on protein function and discovering potential inhibitors of CDK4: Molecular modelling and dynamics sdtudies. PLoS One 2015;10:e0133969.
66. Dasgupta J, Sen U, Dattagupta JK. In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein. Protein Eng 2003;16:489-496.
67. Maher DW, Davis I, Boyd AW, Morstyn G. Human interleukin-4: an immunomodulator with potential therapeutic applications. Prog Growth Factor Res 1991;3:43-56.
68. Lundin J, Kimby E, Bergmann L, Karakas T, Mellstedt H, Osterborg A. Interleukin 4 therapy for patients with chronic lymphocytic leukaemia: a phase I/II study. Br J Haematol 2001;112:155-160.
69. Taylor CW, LeBlanc M, Fisher RI, Moore DF Sr, Roach RW, Elias L, Miller TP. Phase II evaluation of interleukin-4 in patients with non-Hodgkin's lymphoma: a Southwest oncology group trial. Anticancer Drugs 2000;11:695-700.
70. Seldin DC, Leder P. Mutational analysis of a critical signaling domain of the human interleukin 4 receptor. Proc Natl Acad Sci USA 1994;91:2140-2144.
71. Pan PY, Rothman P. IL-4 receptor mutations.Curr Opin Immunol 1999;11:615-620.
72. Akash MSH, Rehman K, Tariq M, Chen S. Development of therapeutic proteins: advances and challenges. Turk J Biol 2015;39:343-358.
73. Zhang Z, Miteva MA, Wang L, Alexov E. Analyzing effects of naturally occurring missense mutations. Comput Math Methods Med 2012:805827.

Files

Share

How to cite

Statistics