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

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.

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.