Obtaining and Characterization of Recombinant Fluorescent Derivatives of Soluble Human Hb-Egf

Короткевич, Н. В.

doi:10.15407/biotech7.02.046

Cited by 3 publications

(6 citation statements)

References 8 publications

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“…We chose 4 different cell lines -L929a (mouse fibrosarcoma), Vero (immortalized kidney epithelial cells of green monkey), 3T3 (mouse embryonic fibroblasts) and A431 (human epidermoid carcinoma cells). The overall level of sHB-EGF receptors was investigated by flow cytometry using the fluorescently labeled derivative of rsHB-EGF (mCherry-rsHB-EGF) [17]. It was shown that binding of mCherry-rsHB-EGF to L929a and Vero cells was lower compared to 3T3 cells, and especially lower compared to A431 cells.…”

Section: Resultsmentioning

confidence: 99%

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Influence of human HB-EGF secreted form on cells with different EGFR and ErbB4 quantity

Krynina¹,

Korotkevych²,

Labyntsev³

et al. 2019

Ukr.Biochem.J.

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pivotal role in many cellular processes, but its effect differs from one cell type to another and remains not fully understood. the aim of this work was to investigate the dependence between the rate of hB-eGF mediated cell proliferation and activation of EGFR and ErbB4 receptors. Therefore, the effects of human recombinant sHB-EGF (rsHB-EGF) on the proliferation of cell lines with different EGFR and ErbB4 quantity and ratio, as well as activation of the marK-cascade p38 and erK1/2 (p42/44) kinases, were analyzed. For comparison, a similar study of the effect of native sHB-EGF secreted by human histiocytic lymphoma cells U937 during co-cultivation with different cell lines was performed. It was proved that cell proliferation in response to sHB-EGF depends not only on the quantity but also on the ratio of EGFR and ErbB4. It was shown that signaling through erbB4 is associated with activation of p38 kinase and signaling through eGFr associated with activation of ERK1/2 (p42/44) kinase. We assume the existence of two different mechanisms for sHB-eGF-mediated stimulation of cell proliferation, and the simultaneous launch of these mechanisms provides a maximal proliferative response. the results of this study support the feasibility of creating anti-proliferative drugs that target erbB4.

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Section: Resultsmentioning

confidence: 99%

“…recombinant proteins production. Human rsHB-EGF and its fluorescent derivative mCherry-rsHB-EGF were purified from e. coli BL21 Rosetta (DE3) that was obtained earlier [16,17]. Expression of recombinant proteins was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (Thermo Fisher, Dreieich, Germany).…”

mentioning

confidence: 99%

Influence of human HB-EGF secreted form on cells with different EGFR and ErbB4 quantity

Krynina¹,

Korotkevych²,

Labyntsev³

et al. 2019

Ukr.Biochem.J.

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“…Construction of the plasmids encoding recombinant soluble form of human HB-EGF (sHB-EGF) and mCherry-sHB-EGF was described earlier [ 18 , 19 ]. Briefly, both constructions were based on pET28a plasmid and E .…”

Section: Methodsmentioning

confidence: 99%

“…Bioactivity of recombinant proteins sHB-EGF and mCherry-sHB-EGF was previously shown [ 18 , 19 ]. In brief, mytogenic activity of human sHB-EGF and mCherry-sHB-EGF was evaluated with MTT proliferation assay on mouse fibroblasts BALB/c 3T3 (Cell lines bank of R.E.…”

Section: Methodsmentioning

confidence: 99%

The Soluble Heparin-Binding EGF-Like Growth Factor Stimulates EGF Receptor Trafficking to the Nucleus

Korotkevych

Labyntsev

et al. 2015

PLoS ONE

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Most ligands of epidermal growth factor receptor (EGFR) have the ability to induce EGFR translocation into the nucleus, where EGFR acts as an important transcriptional regulator. Soluble form of heparin-binding EGF-like growth factor (sHB-EGF) is one of the ligands for EGFR in many cell types; however, there is no evidence for the ability of sHB-EGF to induce EGFR nuclear importation. Here, we demonstrated that treatment of A431 cells with sHB-EGF resulted in nuclear localization of EGFR and such translocation occurs via retrograde pathway. It was shown by confocal microscopy and co-immunoprecipitation assay that the translocation complex consisted of both ligand and receptor. The chromatin immunoprecipitation assay showed the association of sHB-EGF–EGFR complex with promoter region of cyclin D1 in the cell nucleus and this association was prevented by application of EGFR kinase inhibitor AG-1478. The obtained data suggest that sHB-EGF acts similarly to other EGFR ligands and is capable to induce EGFR nuclear translocation as a part of ligand-receptor complex in a tyrosine phosphorylation-dependent manner.

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“…Earlier in our laboratory, it was designed recombinant full-size and truncated (with the lack of heparin-binding domain) forms of HB-EGF that were fluorescently-labeled with mCherry or EGFP. These proteins both had a proliferative effect on fibroblast cells and retained their ability to bind to EGFR, making them perfect tools for investigation of intracellular transport mechanisms [17]. The main goal of this work was to reveal a role of heparinbinding domain of HB-EGF in the process of its binding to receptors and trafficking to the cytoplasm.…”

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confidence: 99%

Role of the heparin-binding domain in intracellular trafficking of sHB-EGF

Krynina¹,

Manoilov²,

Dv³

et al. 2019

Ukr.Biochem.J

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Heparin-binding eGF-like growth factor (HB-eGF) is a member of the epidermal growth factor family that was proven as a potent mitogen and chemoattractant. HB-eGF mediated eGFr activation is a key event in the stimulation of gene expression, cell migration and proliferation during both normal and pathogenic physiological processes. The main goal of this research was to reveal the role of the heparin-binding domain of HB-eGF in the ligand-receptor formation and its further internalization to the cytoplasm. We used fluorescently-labeled recombinant derivative of soluble HB-EGF and its truncated form (sHB-EGF Δ84-106 ) with deletion of the heparin-binding domain. Firstly, the binding kinetics of two forms of sHB-eGF to its cell surface receptors was determined using flow cytometry. To determine how the absence of heparin-binding domain in the structure of HB-EGF affects its internalization, we analyzed the endocytosis process of EGFP-sHB-eGF Δ84-106 and eGFP-sHB-eGF complexes by confocal microscopy. It was found that the full-size form of HB-eGF is characterized by a lower intensity of translocation to the cytoplasm in comparison to HBDdeleted form. Thus, differences in the trafficking of the full-size or truncated forms of sHB-EGF in the cell cytoplasm may reflect the mechanisms of extracellular matrix influence on the biological activity of sHB-EGF. K e y w o r d s: heparin-binding eGF-like growth factor, epidermal growth factor receptor, endocytosis.

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Obtaining and Characterization of Recombinant Fluorescent Derivatives of Soluble Human Hb-Egf

Cited by 3 publications

References 8 publications

Influence of human HB-EGF secreted form on cells with different EGFR and ErbB4 quantity

Influence of human HB-EGF secreted form on cells with different EGFR and ErbB4 quantity

The Soluble Heparin-Binding EGF-Like Growth Factor Stimulates EGF Receptor Trafficking to the Nucleus

Role of the heparin-binding domain in intracellular trafficking of sHB-EGF

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