Deciphering the lipid code for lysosomal channels and transporters in inflammatory, metabolic, and neurological disorders

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Solute carrier transporters (SLC) are integral membrane proteins that mediate the uptake, extrusion and exchange of small molecules across biological membranes. SLCs represent the second largest family of membrane proteins in animal and plant genomes and are linked to numerous diseases. In recent years mounting evidence has indicated an important but unclear role for lipids in mediating the function of SLC proteins and linking disease phenotypes to dysregulation of lipid interactions in the cell. Recent discoveries from our group, funded through UKRI grants, have revealed that phospholipids can regulate oligomeric state, control on/off states and regulate trafficking in the cell. However, a mechanistic understanding of lipid regulated functions within the SLC family remain elusive. Our project, in partnership with OMass therapeutics, will directly address this question through a unique combination of in vitro and in vivo biochemistry, structural studies using cryo-EM and native mass spectrometry (MS) coupled with lipidomics. Our aim is to create the first interaction map for lipid-SLC interactions in plant and animal cells. Specifically, the student will use a range of synthetic nanobodies targeted to SLC proteins that reside in specific locations in the cell and use these binders to affinity purify the target proteins for subsequent lipid analysis using native MS. These include vacuolar amino acid transporters from plant cells and their counterparts in the lysosome in animal cells. The aim is to understand which lipids are associated with these proteins to generate an initial lipid interaction map. These studies will be complemented with both in vivo nanodisc reconstitutions (i.e., without prior purification), which will enable native-like purification of the proteins for single particle cryo-EM analysis, followed up with computational analyses using molecular dynamics. It is expected that through discussion with the student the balance of workload between laboratory and computational work will be decided as the project progresses. Insights gained from the native MS and computational data will be used to assign lipid densities in the cryo-EM maps, whilst also serving to inform and direct biochemical transport assays in liposomes of defined composition. The liposome assays have already been established, as have the molecular dynamics pipelines for cholesterol by a current DTP student. In summary, the resulting data will be used to develop a blueprint for understanding the importance of specific lipid types (phospholipid vs. cholesterol) in SLC biology for plant and animal cells and advance our understanding of this neglected aspect of molecular membrane biology.BBSRC theme - Mechanistic Molecular and Cellular Bioscience