Lipid-protein interactions: Characterisation of the oxysterol mediated protein complexes by chemical proteomics

Lead Research Organisation: Swansea University


Cellular function relies on the coordinated communication and action of a collection of biomolecules including proteins and lipids. Lipids exhibit diverse biological effect via their interacting receptors. Proteins that contain a lipid binding domain can, upon binding of their lipid ligand, undergo a conformational change, which leads to interaction with new partners or translocation to another subcellular compartment. Such multicomponent complexes are transient, or dynamic under specific conditions, and difficult to investigate. Conventional biochemical methods to study lipid-protein interactions often require large amounts of material and are carried out on a 'one-at-a-time' basis. Thus, in this proposal we plan to develop a high throughput platform to capture in real time a picture of the spatial and temporal 'global' lipid-protein interaction network using a combination of cross-linking, lipid pull-down, and proteomics technologies. The identified interactions will then be examined for their effect on protein function and cellular activities using a quantitative proteomics approach. The class of lipids will be focused on in this study are oxysterols. Oxysterols are oxidized derivatives of cholesterol and their imbalance is implicated in atherosclerosis and neurodegenerative diseases. We will use the developed novel approach to identify oxysterol receptors and reveal their involvement in protein complexe assembly and disassembly. The results will potentially aid in the elucidation of biological process, the understanding of the importance of oxysterols in the healthy state and how their imbalance may be the cause of disease.

Technical Summary

This project is designed to tackle the analytical challenges encountered during the investigation of lipid-protein interactions and the reorganization of multicomponent complexes upon lipid binding. Firstly, we will produce a biotinylated photoreactive lipid bait which will also retain the physiochemical and biological properties of the authentic parent molecule i.e. a biomolecular mimic. The photoreactive group will covalently link proteins binding to the lipid (including weak binders), allowing their survival through subsequent purification steps, while an affinity tag will be used to enrich low abundance binding proteins. In this manner lipid binding proteins will be selectively separated/enriched from complex mixtures and will be identified by mass spectrometry. To investigate the reorganization of protein complexes induced by our lipid, we will take advantage of a non-cleavable crosslinker and a cleavable crosslinker to label protein complexes before and after addition of lipid mimics to cell culture. This will allow proteins involved in reorganization to be differentiated in subsequent elution steps. We will incorporate the stable isotope labelling by amino acids in cell culture (SILAC) strategy to quantitatively reveal the differences in protein complex organization under different condition. Finally the down stream effects of lipid-protein interactions will be assed using quantitative proteomics to examine their effect on global protein expression levels. We will employ SILAC, subcellular fractionation and a 2DLC-MS/MS approach to deep mine the proteome changes.


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Description We have successfully developed a biotinylated-oxysterol probe, which contains a tag but retains the physiological properties of the parent oxysterol, such as activation LXRs and inhibiting the gene expression of squalene synthase. This probe can be used to study oxysterol-protein interactions. The oxysterol-probe is made by linking biotin hydrazide to 24-ketocholesterol. We optimised the derivatisation and purification methods. We are able to achieve HPLC grade biotinylated-oxysterol in good yield.
By combination of affinity pull-down and mass spectrometry analysis, we identified candidate novel oxysterol binding proteins, including inhibitor of NF?B kinase (IKK). In quantitative proteomics analysis we found that M-CSF was induced by 24S,25-epoxycholesterol. M-CSF is a NF?B target gene and further study showed that this effect could be inhibited by Bay 11-7082, a IKK specific inhibitor. Together these results suggest that oxysterol activate NF?B pathway by direct interaction with IKK.
Exploitation Route Oxysterols is an emerging class of molecules which play important role in neuron development and survial and immunity. Thus the oxysterol probe can be used by other researchers from academia or pharmaceutical companies to explore the mechanisms of oxysterol activities.
Sectors Pharmaceuticals and Medical Biotechnology

Description Life Science Research Network Wales
Amount £50,000 (GBP)
Organisation Welsh Assembly 
Sector Public
Country United Kingdom
Start 03/2015 
End 02/2016
Description Life Sciences Research Network Wales Impact Award
Amount £50,000 (GBP)
Organisation Life Sciences Research Network Wales 
Sector Academic/University
Country United Kingdom
Start 06/2017 
End 04/2018
Description Karolinska 
Organisation Karolinska Institute
Country Sweden 
Sector Academic/University 
PI Contribution We developed analytical platform to identify endogenous ligands for LXR.
Collaborator Contribution They investiagted the ligand-receptor interactions and the effect of the ligands on neuron.
Impact This is a multidisciplinary collaboration between neuroscientists at Karolinska Institute and analytical and biochemists at Swansea University. This collaboration lead to two high impact publications in Nature Chemical Biology and The Journal of Clinical Investigation.
Start Year 2006
Description Soapbox science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Soapbox science Swansea 2016 was held in Swansea's shopping area in the city centre on Saturday September 10th,which buzzing with shoppers, families, tourists and other city dwellers. The event's mission is to raise the profile, and challenge the public's view, of women in science, technology, engineering, maths and medicine (STEMM). I was one of the presenters and talked my research on "cholesterol metabolism and human health".
Year(s) Of Engagement Activity 2016