Periodic Reporting for period 4 - ENABLE (Elucidating natural bilayer lipid environments)
Período documentado: 2020-12-01 hasta 2021-05-31
Previous developments in my laboratory had led to important new information concerning the role of lipids in mediating membrane protein interactions and function. Given the importance of membrane proteins in physiology, and therefore as potential drug targets, understanding their modulation by lipids would be a major step towards more effective drug development. However, given the transient nature of protein lipid interactions, the challenge was to develop a technology to study dynamic membrane proteins in their native lipid environment. This, in turn, would answer key questions about how lipids modulate protein interfaces, occupy different binding sites, fine-tune membrane protein structure and modify functions in vivo. The overall aim of the project was therefore to understand the effects of the natural lipid environment on membrane proteins. We were able to probe the dynamics of lipid binding, revealing they are exquisitely sensitive to established methods of extraction and solubilisation; this led us to develop a radically novel methodology to release intact membrane proteins directly from their native environments into the mass spectrometer with the least chemical intervention. Our methods are now being used to inform a wide variety of research questions from receptor biology to antimicrobial resistance.
Period 1:
We commenced work on elucidating the chemical synergy between membrane proteins and endogenous lipids, demonstrating for the first time how lipids can act as molecular ‘glues’ and are instrumental in regulating oligomeric assemblies of membrane proteins (Gupta et al, Nature, 2017: doi:10.1038/nature20820). We extended this research to investigate selected G-protein coupled receptors (GPCRs) and found that lipids also mediated critical interactions (Yen et al, Nature, 2018: doi.org/10.1038/s41586-018-0325-6). In parallel with this work we also commenced development of a native desorption electrospray ionisation (DESI) platform. Construction of this device enabled us to lift membrane proteins from planar targets, functionalised in-house, directly into the mass spectrometer for characterisation. Images of the DESI platform appeared on the inside back cover of Angewandte Chemie (Ambrose et al, Angewandte Chemie, 2017: doi: 10.1002/anie.201704849. The back cover image is attached to this report.
Period 2:
Building on the work undertaken in the first phase of the programme, we continued to expand our methodology and technology to explore new ways of extracting membrane proteins with associated lipids for study by mass spectrometry. The major achievement of Period 2 was an unexpected discovery made during our research: we discovered, by chance, a new chemical- and detergent-free approach which made it possible to study membrane protein complexes, and their interactions with lipids, directly from intact (unperturbed) native membranes. This represented a major breakthrough in the field and our novel approach, designated SoLVe-MS (Sonicated Lipid Vesicle-MS) led to a seminal paper in the journal Science (Chorev et al, Science, 2018: doi: 10.1126/science.aau0976). So novel was this approach that it was considered for the front cover of Science. We also needed to assist engagement and understanding of the complex science behind our breakthrough. We therefore commissioned an image (see attached image) which has subsequently been used in lectures and talks around the world.
Given that membrane protein complexes are key targets for drug discovery, we continued to develop the SoLVe-MS approach to a wide range of membrane proteins such as rhomboids (these drug candidates are implicated in malaria, cancer and Parkinson’s disease) and solute carriers which are present in Golgi, mitochondrial and plasma membranes and are widely considered the next major drug target. It was, however, necessary to validate data generated by this approach with complexes extracted using detergents. To this end, we commenced development of a series of tailor-made synthetic detergents (‘designer detergents’) to extract different complexes for validation.
Period 3:
To make the SoLVe approach accessible to the community we also published a step-by-step protocol, with troubleshooting for worked examples, detailing all the steps necessary to attain mass spectra from sonicated lipid vesicles (Chorev et al, Nature, 2020: https://doi.org/10.1038/s41596-020-0303-y). Subsequently, we have experienced broad take-up of our methods from academia to industry. We followed this with an in-depth review of the importance of the membrane for biophysical measurements (Chorev & Robinson, Nature Chemical Biology, 2020: https://doi.org/10.1038/s41589-020-0574-1).
We also realised that our SoLVe approach could be important for drug discovery but only if we could first could overcome one of the problems encountered whereby products remain ambiguous. To characterise the products of SoLVe we needed to perform further rounds of tandem MS combined with proteomics and lipidomics experiments. We managed to overcome this limitation with our nativeomics approach (Gault et al, Nature Methods, 2020: https://doi.org/10.1038/s41592-020-0821-0).
Concurrently, we continued developing novel detergents to complement our data and showed that it was possible to find new detergents that stabilise active conformations of GPCRs (Urner et al, Chemistry Europe, 2020: doi:10.1002/chem.202003991). These detegents enable us to carry out cross comparison of important lipids identified in the SoLVe strategy.
Period 4:
In the final 6 months of the grant we made the final ‘push’ to consolidate discoveries made throughout the previous period. Specifically, we demonstrated a signalling cascade across a native membrane, and showed how drugs that target receptors in the eye, can be studied in their native environments revealing different mechanisms of action. These, and our earlier findings, we prepared for publication and for a patent application.
We commenced work on elucidating the chemical synergy between membrane proteins and endogenous lipids, demonstrating for the first time how lipids can act as molecular ‘glues’ and are instrumental in regulating oligomeric assemblies of membrane proteins (Gupta et al, Nature, 2017: doi:10.1038/nature20820). We extended this research to investigate selected G-protein coupled receptors (GPCRs) and found that lipids also mediated critical interactions (Yen et al, Nature, 2018: doi.org/10.1038/s41586-018-0325-6). In parallel with this work we also commenced development of a native desorption electrospray ionisation (DESI) platform. Construction of this device enabled us to lift membrane proteins from planar targets, functionalised in-house, directly into the mass spectrometer for characterisation. Images of the DESI platform appeared on the inside back cover of Angewandte Chemie (Ambrose et al, Angewandte Chemie, 2017: doi: 10.1002/anie.201704849. The back cover image is attached to this report.
Period 2:
Building on the work undertaken in the first phase of the programme, we continued to expand our methodology and technology to explore new ways of extracting membrane proteins with associated lipids for study by mass spectrometry. The major achievement of Period 2 was an unexpected discovery made during our research: we discovered, by chance, a new chemical- and detergent-free approach which made it possible to study membrane protein complexes, and their interactions with lipids, directly from intact (unperturbed) native membranes. This represented a major breakthrough in the field and our novel approach, designated SoLVe-MS (Sonicated Lipid Vesicle-MS) led to a seminal paper in the journal Science (Chorev et al, Science, 2018: doi: 10.1126/science.aau0976). So novel was this approach that it was considered for the front cover of Science. We also needed to assist engagement and understanding of the complex science behind our breakthrough. We therefore commissioned an image (see attached image) which has subsequently been used in lectures and talks around the world.
Given that membrane protein complexes are key targets for drug discovery, we continued to develop the SoLVe-MS approach to a wide range of membrane proteins such as rhomboids (these drug candidates are implicated in malaria, cancer and Parkinson’s disease) and solute carriers which are present in Golgi, mitochondrial and plasma membranes and are widely considered the next major drug target. It was, however, necessary to validate data generated by this approach with complexes extracted using detergents. To this end, we commenced development of a series of tailor-made synthetic detergents (‘designer detergents’) to extract different complexes for validation.
Period 3:
To make the SoLVe approach accessible to the community we also published a step-by-step protocol, with troubleshooting for worked examples, detailing all the steps necessary to attain mass spectra from sonicated lipid vesicles (Chorev et al, Nature, 2020: https://doi.org/10.1038/s41596-020-0303-y). Subsequently, we have experienced broad take-up of our methods from academia to industry. We followed this with an in-depth review of the importance of the membrane for biophysical measurements (Chorev & Robinson, Nature Chemical Biology, 2020: https://doi.org/10.1038/s41589-020-0574-1).
We also realised that our SoLVe approach could be important for drug discovery but only if we could first could overcome one of the problems encountered whereby products remain ambiguous. To characterise the products of SoLVe we needed to perform further rounds of tandem MS combined with proteomics and lipidomics experiments. We managed to overcome this limitation with our nativeomics approach (Gault et al, Nature Methods, 2020: https://doi.org/10.1038/s41592-020-0821-0).
Concurrently, we continued developing novel detergents to complement our data and showed that it was possible to find new detergents that stabilise active conformations of GPCRs (Urner et al, Chemistry Europe, 2020: doi:10.1002/chem.202003991). These detegents enable us to carry out cross comparison of important lipids identified in the SoLVe strategy.
Period 4:
In the final 6 months of the grant we made the final ‘push’ to consolidate discoveries made throughout the previous period. Specifically, we demonstrated a signalling cascade across a native membrane, and showed how drugs that target receptors in the eye, can be studied in their native environments revealing different mechanisms of action. These, and our earlier findings, we prepared for publication and for a patent application.
Following on from our successful SoLVe-MS strategy we have successfully implemented an approach for ejecting receptors directly from native membranes to capture down-stream signaling partners and to study the roles of lipid bilayers in these processes. This work is currently under preparation for publication. We also showed the utility of our novel detergents in electron microscopy studies (manuscript in preparation). Our methods are also now being used to inform a wide variety of research questions from receptor biology to antimicrobial resistance.
In terms of drug discovery SoLVe is exciting since the addition of drugs directly to membranes and organelles, without a priori knowledge of the target, could be transformative. Most drug discovery takes place by first identifying the target, isolating it from the membrane environment and then chemical libraries are screened against the isolated protein target. Using our SoLVe methodology we would be able to add drugs directly to the intact membrane and use the mass spectrometer to define the target. In that way we would establish a new paradigm for drug discovery.
In terms of drug discovery SoLVe is exciting since the addition of drugs directly to membranes and organelles, without a priori knowledge of the target, could be transformative. Most drug discovery takes place by first identifying the target, isolating it from the membrane environment and then chemical libraries are screened against the isolated protein target. Using our SoLVe methodology we would be able to add drugs directly to the intact membrane and use the mass spectrometer to define the target. In that way we would establish a new paradigm for drug discovery.