Periodic Reporting for period 1 - EGFR-Activ (Epidermal growth factor receptor (EGFR) activation by cytohesins - Structural Insights)
Période du rapport: 2015-05-04 au 2017-05-03
In humans, EGFR’s incorrect signalling is associated with neurodegenerative diseases and a number of cancers. Thus, mechanisms that modulate the activation of these receptors are high profile drug targets. However, many of the drugs in use cause a variety of skin toxicities and some types of cancer seem to be resistant. Furthermore, achieving the high level of structural information required for the study of these systems is a complex task. Therefore, new approaches to study and fight EGF receptor-dependent malignancies are urgently needed.
The overall objectives of the projects focused on a better understanding of cytoplasmic components that may play a, yet not well characterized, role in EGFR activation. Better insights into these factors may help to develop new therapeutic strategies to target various EGFR-related pathogenic signalling pathways. To obtain reliable information of these molecular details, it is of fundamental importance to also apply high-resolution approaches that on the one hand provide structural information in a site-resolved manner and on the other hand are applicable to environmental conditions that sufficiently mimic native conditions. For this reason, mainly high-resolution solution-state NMR was used at temperature, buffer and environmental conditions such as detergent free membrane mimetics at physiological pH and temperature. The structural NMR studies in particular focused on the role of its juxtamembrane domain (JM), the membrane environment and cytoplasmic co-factors (i.e. the Sec7 of cytohesin-2). Overall, we succeeded in identifying interactions and characterizing structural details of the JM-Sec7 binding as well as JM-membrane association. For the latter, we also identified a dependence of the lipid composition of the membrane mimetic.
The obtained data may be transferred towards academic and non-academic environments and contribute to the global knowledge of EGFR-signalling and to the discovery of novel therapeutic agents with the potential to save human lives.
The overall objectives of the projects focused on a better understanding of cytoplasmic components that may play a, yet not well characterized, role in EGFR activation. Better insights into these factors may help to develop new therapeutic strategies to target various EGFR-related pathogenic signalling pathways. To obtain reliable information of these molecular details, it is of fundamental importance to also apply high-resolution approaches that on the one hand provide structural information in a site-resolved manner and on the other hand are applicable to environmental conditions that sufficiently mimic native conditions. For this reason, mainly high-resolution solution-state NMR was used at temperature, buffer and environmental conditions such as detergent free membrane mimetics at physiological pH and temperature. The structural NMR studies in particular focused on the role of its juxtamembrane domain (JM), the membrane environment and cytoplasmic co-factors (i.e. the Sec7 of cytohesin-2). Overall, we succeeded in identifying interactions and characterizing structural details of the JM-Sec7 binding as well as JM-membrane association. For the latter, we also identified a dependence of the lipid composition of the membrane mimetic.
The obtained data may be transferred towards academic and non-academic environments and contribute to the global knowledge of EGFR-signalling and to the discovery of novel therapeutic agents with the potential to save human lives.
The performed research focused on the understanding of how membrane environment and cytoplasmic factors (cytohesins) modulate EGFR activation. For this we have used Nuclear Magnetic Resonance (NMR) to study the interaction between the Juxtamembrane (JM) segment of EGFR and the Sec7 domain of cytohesin-2 (ARNO), postulated to act as an EGFR activating factor. Furthermore, we also studied the influence of the composition of the cell membrane. The first step was to perform the backbone assignment of JM and Sec7. This was done using a standard triple resonance approach. The data for Sec7 revealed a well folded structure, composed by 10 alpha-helices, in good agreement with previously determined structures. For JM the data showed that, in solution, it’s structure is mainly random coil.
The next step involved the study of the interaction of JM with Sec7 and the identification of the residues involved in this interaction. For this we used NMR chemical shift perturbations by performing two different titrations: first, we titrated Sec7 with JM and then we titrated JM with Sec7. The data clearly showed that JM and Sec7 interact and allowed to precisely identify the residues involved in binding from both partners. Interaction of JM with Sec7 was also confirmed by using Saturation Transfer Difference NMR (STD-NMR).
JM-membrane interactions were investigated using lipid bilayer nanodiscs with defined lipid composition. Different membrane binding modes of the JM domain were identified depending on lipid charge content.
Overall our results help to clarify, so far unfamiliar and controversially discussed, mechanism of cytohesin-mediated EGFR signalling and open the door to the possible therapeutic exploitation of this mechanism.
In the process of this work, new NMR methodologies were developed that are tailored to the study of complex systems and have the potential to improve the amount and quality of the acquired data, thus facilitating the study of these systems. Additionally, the usage of detergent-free membrane mimetics for NMR-based structural studies was carefully evaluated. Large parts of the results of this project have already been communicated to the scientific community via three publications in peer-reviewed international journals (Viegas A, et al., 2016, Biological Chemistry, 397 (12), 1335-1354; Viennet T, Viegas A, et al., 2016, Ang. Chem. Int. Ed., 55, 10746 –10750; Viegas A, et al., 2016, JBNMR, 64, 9–15), two invited oral presentations (EUROMAR, 2015 – Prague, Czech Republic; Instituto de Tecnologia Química e Biológica, 2016 – Lisbon, Portugal) and two poster presentations (EUROMAR, 2015 – Prague, Czech Republic; 38th FGMR Discussion Meeting, 2016 – Düsseldorf, Germany). Additional publications, including all so far not published results, are currently in preparation.
The next step involved the study of the interaction of JM with Sec7 and the identification of the residues involved in this interaction. For this we used NMR chemical shift perturbations by performing two different titrations: first, we titrated Sec7 with JM and then we titrated JM with Sec7. The data clearly showed that JM and Sec7 interact and allowed to precisely identify the residues involved in binding from both partners. Interaction of JM with Sec7 was also confirmed by using Saturation Transfer Difference NMR (STD-NMR).
JM-membrane interactions were investigated using lipid bilayer nanodiscs with defined lipid composition. Different membrane binding modes of the JM domain were identified depending on lipid charge content.
Overall our results help to clarify, so far unfamiliar and controversially discussed, mechanism of cytohesin-mediated EGFR signalling and open the door to the possible therapeutic exploitation of this mechanism.
In the process of this work, new NMR methodologies were developed that are tailored to the study of complex systems and have the potential to improve the amount and quality of the acquired data, thus facilitating the study of these systems. Additionally, the usage of detergent-free membrane mimetics for NMR-based structural studies was carefully evaluated. Large parts of the results of this project have already been communicated to the scientific community via three publications in peer-reviewed international journals (Viegas A, et al., 2016, Biological Chemistry, 397 (12), 1335-1354; Viennet T, Viegas A, et al., 2016, Ang. Chem. Int. Ed., 55, 10746 –10750; Viegas A, et al., 2016, JBNMR, 64, 9–15), two invited oral presentations (EUROMAR, 2015 – Prague, Czech Republic; Instituto de Tecnologia Química e Biológica, 2016 – Lisbon, Portugal) and two poster presentations (EUROMAR, 2015 – Prague, Czech Republic; 38th FGMR Discussion Meeting, 2016 – Düsseldorf, Germany). Additional publications, including all so far not published results, are currently in preparation.
The results of this project have an impact on how we understand EGFR signalling and help understand molecular details of factors that can play a role in its signalling regulation. Specifically, this study identified and characterized the interaction of a key segment of EGFR (the juxtamembrane) with a cytoplasmic factor (the Sec7 domain of Cytohesin-2). Moreover, it provided insights into the importance of the composition of the cell membrane for EGFR membrane association, which also has the potential to modulate its signalling. In the process, we developed new NMR methodology aimed at the study and characterization of complex systems. The new tools developed during the course of this project are also applicable to the scientific community and can be applied to a wide range of other systems involved in different aspects of human health.
It can be anticipated that the cytoplasmic factors, including the ones that were characterized in this project, will play an increasingly important role in the development of an urgently needed new generation of therapeutic strategies targeting the EGFR signalling pathway.
It can be anticipated that the cytoplasmic factors, including the ones that were characterized in this project, will play an increasingly important role in the development of an urgently needed new generation of therapeutic strategies targeting the EGFR signalling pathway.