In Vitro

The present work concerns the heterologous expression of the intracellular domain harbouring the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Protein expression was improved thanks to the deletion of a 13-amino acid peptide of the juxtamembrane region (JM). The recombinant proteins were produced as a glutathione S-transferase (GST) fusion in Escherichia coli, and the solubilisation was performed by sarkosyl addition during extraction. The produced proteins spontaneously dimerize allowing the activation of the tyrosine kinase domain in the presence of [γ-32P]ATP. The activity assay has revealed the autophosphorylation of EGFR proteins which was decreased in the presence of genistein. Our system could facilitate the screening of EGFR inhibitors without the need of adding an exogenous substrate

The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.Consejería de Educación, Juventud y Deportes–Comunidad de Madrid,Grant/Award Number: B2017/BMD‐36involving contributions from the EuropeanFunds for Regional Development (EFRD) andthe Social European Fund (SEF); ConsejoSuperior de Investigaciones Científicas, Grant/Award Number: COOPA20053;Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: SAF2014‐52048‐R; Agencia Española de Cooperación Internacional para el Desarrollo, Grant/Award Numbers: A/019018/08,A/5444/06, A/8197/0

A premature termination of human epidermal growth factor receptor transcription in Escherichia coli

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST) that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.This work was supported by the Ministry of Higher Education and Scientific Research of Tunisia and the Spanish Agency for International Cooperation and Development (AECID).Peer Reviewe

Targeting the calmodulin-regulated ErbB/Grb7 signaling axis in cancer therapy

Signal transduction pathways essential for the survival and viability of the cell and that frequently present aberrant expression or function in tumors are attractive targets for pharmacological intervention in human cancers. In this short review we will describe the regulation exerted by the calcium-receptor protein calmodulin (CaM) on signaling routes involving the family of ErbB receptors - highlighting the epidermal growth factor receptor (EGFR/ErbB1) and ErbB2 - and the adaptor protein Grb7, a downstream signaling component of these receptors. The signaling mechanism of the ErbB/Grb7 axis and the regulation exerted by CaM on this pathway will be described. We will present a brief overview of the current efforts to inhibit the hyperactivity of ErbB receptors and Grb7 in tumors. The currently available information on targeting the CaM-binding site of these signaling proteins will be analyzed, and the pros and cons of directly targeting CaM versus the CaM-binding domain of the ErbB receptors and Grb7 as potential anti-cancer therapy will be discussed.The work in the authors laboratory was funded in part by grants (to AV) from the Secretaría de Estado de Investigación Desarrollo e Innovación - SEIDEI (SAF2011-23494), the Consejería de Educación de la Comunidad de Madrid (S2010/BMD-2349), the Agencia Española de Cooperación Internacional para el Desarrollo - AECID (AP/040803/11), and the European Commission (contract PITN-GA-2011-289033). IG-P, SRS and KJ were respectively supported by a fellowship from the Ministerio de Educación Cultura y Deporte, a Madame Curie contract from the European Commission, and a grant from the AECID.Peer reviewe

Calmodulin-mediated regulation of the epidermal growth factor receptor

In this review, we first describe the mechanisms by which the epidermal growth factor receptor generates a Ca2+ signal and, subsequently, we compile the available experimental evidence regarding the role that the Ca 2+/calmodulin complex, formed after the rise in cytosolic free Ca2+ concentration, exerts on the receptor. We focus not only on the indirect action that Ca2+/calmodulin exerts on the epidermal growth factor receptor, as a result of the activation of distinct calmodulin-dependent kinases, but also, and more extensively, on the direct interaction of Ca 2+/calmodulin with the receptor. We also describe several mechanistic models that could account for the Ca2+/calmodulin-mediated regulation of epidermal growth factor receptor activity. The control exerted by calmodulin on distinct epidermal growth factor receptor-mediated cellular functions is also discussed. Finally, the phosphorylation of this Ca 2+ sensor by the epidermal growth factor receptor is highlighted. © 2009 FEBS.Research in the authors’ laboratory was financed by grants (to A.V.) from the Dirección General de Investigación, Ministerio de Ciencia e Innovación (SAF2008-00986), the Consejería de Educación de la Comunidad de Madrid (S-BIO-0170-2006), the Agencia Española de Cooperación Internacional para el Desarrollo (A/019018/08) and the European Commission (MRTN-CT-2005-19561). P.S.G. was supported by a predoctoral fellowship from the Junta de Ampliación de Estudios, CSIC, and K.J. was supported by an AECID grant.Peer Reviewe

Characteristics, Phytochemical Analysis and Biological Activities of Extracts from Tunisian Chetoui Olea europaea Variety

This study selected 10 extracts from Tunisian chetoui O. europaea variety for their total phenolics, flavonoids, and phytochemical analyses as well as for their antioxidant and antimicrobial activities determination. The in vitro antioxidant property was investigated using DPPH, ferric reducing antioxidant capacity (FRAP), oxygen reducing antioxidant capacity (ORAC), and β-carotene-linoleic acid bleaching assays while antimicrobial activity was evaluated using macrodilutions method. For all organs of chetoui O. europaea variety, the investigated activities were found to be higher in the polar extracts (ethyl acetate, methanol, and methanol/water). These activities were correlated with the presence of phenolic compounds. Phytochemical analyses revealed that the crude extracts contain triterpenoids, quinones, and flavonoids. High performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) confirmed the presence of phenolic compounds in the studied extracts

Effect of the amino chain length and the transformation into citric acid salts of aryl-diphenyl-butenes and ferrocenyl-diphenyl-butenes bearing two dimethylaminoalkyl chains on their antimicrobial activities

International audienceIn a previous work we have demonstrated the antimicrobial activity of ferrocenyl or phenyl derivatives of diphenyl butene series. This finding has opened a new area of applications of organometallic compounds. In order to improve these activities, we have synthesized new organic and organometallic diaryl butene compounds with different lengths of their amino chains. These new compounds, and also their ammonium salts, were tested against man pathogenic microorganisms Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538) and Enterococcus hirae (ATCC 10541). It emerged from the tests that the Gram+bacteria are more sensitive to the compounds than Gram-, and the compounds with 3 carbon amino chains have a better antimicrobial activity than the one having a chain of 2 or 4 carbons. The transformation of compounds to citrate salts was accompanied by a significant regression of antibacterial activity against Pseudomonas aeruginosa, for both organic and ferrocenic molecules. This resistance problem has been solved using hydrochlorides salts rather than citrates one