Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities

2019

Adv Pharm Bull

Self Cite

Purpose: Interferon beta (IFN-β) is used to combat multiple sclerosis (MS) disease. Creating R27T and V101F mutations (mHuIFN-β-27 and mHuIFN-β-101) is one of the tasks performed to improve human interferon beta (HuIFN-β) half-life, function and expression. In this work, the impact of R27T and V101F mutations in recombinant IFN-β on its binding to interferon receptors were studied by molecular docking. Methods: This work was performed through in silico study. The simulation of mutation was performed using the online Rosetta Backrub software and checked using server verify3D. Comparison of access to the solvent of the amino acids in the structures created was performed using the asaview online server. Also, the effect of mutations on the fold of the protein was reviewed by the online HOPE server. The molecular docking was performed between HuIFN-β and the external region of IFNAR receptor using the online ClusPro2 protein-protein docking server. Results: The comparison of the values of the negative binding energy (ΔGbind) obtained from protein-protein molecular docking between IFNAR receptor and HuIFN-β, mHuIFN-β-27, mHuIFN-β-101 and mHuIFN-β-27-101 ligands did not show a significant difference, and these differences do not see any meaningful relationship between them (P > 0.9999). Conclusion: Regarding these results, it can be concluded that these mutations do not have a negative effect on the composition of the complex rHuIFN-β/IFNAR. So, they do not interfere with the binding of the IFN-β to the receptor. It is concluded that the quality of the rHuIFN-β is improved by introducing these two mutations.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The Comparison Between the Mutated HuIFN-β 27-101 and the WildType Interferon β: the Comprehensive In Silico Study to Evaluate theEffect of Mutations on IFN-β

Balkhi

2019

Adv Pharm Bull

Self Cite

In vitro and computational studies on the effects of ARE deletion and targeted mutations on the expression of interferon beta-1a in HEK293T cells

Norouzi

Appl Microbiol Biotechnol

Dehbashi

2018

Interferon beta (IFNβ) is transiently expressed in response to viral infections and widely used to treat relapsing-remitting multiple sclerosis (MS). We introduced mutations in the IFNβ gene (in the 27th and 101st codons and in the Kozak sequence, and also deletion of 3' and 5' unstable, untranslated region, UTR) with the aim of increasing the expression of IFNβ. Computational analyses of mutant and wild-type RNAs and proteins of IFNβ by RNAfold, ASAView, HOPE and Ramachandran plot, and iStable web servers showed that the mutations could decrease RNA stability, protein solvent accessibility, and protein stability but could not change correct folding. Two recombinant IFNβ101 and IFNβ101+27 constructs were designed by site-directed mutagenesis. The wild-type IFNβ gene also was used as a control. In vitro experiments by quantitative real-time PCR, dot blot, SDS-PAGE, and Western blot assays showed an increased expression level of recombinant IFNβs. 79.9-fold, 99.7-fold, and 190-fold elevations in the mRNA expression of IFNβw, IFNβ101, and IFNβ101+27 were seen, respectively, in comparison with the endogenous IFNβ mRNA in untransfected HEK293T cells. The IFNβ mRNA expression was increased 2.38-fold and 1.25-fold for 101+27 and 101 mutated forms, respectively, in comparison with the IFNβ wild-type construct. An elevation in IFNβ protein production was also clearly detected in the transfected HEK293T cell containing recombinant IFNβ101 and IFNβ101+27 constructs. Finally, these directed mutations in the IFNβ gene successfully elevated protein and mRNA production but in silico analyses of mutant mRNAs showed decreased mRNA stability, unlike previous studies, in comparison with the wild-type mRNA.

In Silico and In Vitro Investigations On Mutated IFNβ-scFv Against Human Collagen Type II

Alimohammadi

Ganjalikhani‐Hakemi

2021

Preprint

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Current medications for rheumatoid arthritis (RA), a common synovial autoimmune disease, are associated with adverse effects. Interestingly, interferon beta (IFNβ), effective in multiple sclerosis (MS) treatment, also can help decreasing articular destruction in RA. Here, a novel fusion protein was introduced containing human mutated IFNβ (with mutations in 27th and 101th residues; IFNβ27+101) fused to a single chain fragment variable (scFv) antibody against human collagen type II for decreasing IFNβ27+101 off-targets (according to drug targeting benefits) in future in vivo and clinical experiments. After designing, bioinformatic analyses and the recombinant vector transfection into HEK293 cells, the mutated IFNβ-scFv protein confirmation and function were assessed by SDS-PAGE, western blotting, ELISA, and real-time PCR. The fusion protein secondary and tertiary structures had proper folding. Also, the recombinant mRNA secondary structure considered stable. 2.35 fold difference between the test and negative control groups confirmed the scFv attachment to human collagen type II (p= 0.046). MxA 25.68 fold overexpression in peripheral blood mononuclear cells (PBMCs) treated with the recombinant protein compared with the non-treated sample (p= 0.0001), demonstrated IFNβ27+101 bioactivity as the fusion protein. In vitro and in silico studies verified function of mutated IFNβ-scFv, however in vivo studies are proposed for further validation.