RESUMO
Borrowing some quotes from Harper Lee's novel "To Kill A Mockingbird" to help frame our manuscript, we discuss methods to profile local proteomes. We initially focus on chemical biology regimens that function in live organisms and use reactive biotin species for this purpose. We then consider ways to add new dimensions to these experimental regimens, principally by releasing less reactive (i. e., more selective) (preter)natural electrophiles. Although electrophile release methods may have lower resolution and label fewer proteins than biotinylation methods, their ability to probe simultaneously protein function and locale raises new and interesting possibilities for the field.
Assuntos
Biotina , Proteoma , BiotinilaçãoRESUMO
The Super-Conserved Receptors Expressed in the Brain (SREBs) form a subfamily of orphan G protein-coupled receptors, highly conserved in evolution and characterized by a predominant expression in the brain. The signaling pathways activated by these receptors (if any) are presently unclear. Given the strong conservation of their intracellular loops, we used a BioID2 proximity-labeling assay to identify protein partners of SREBs that would interact with these conserved domains. Using streptavidin pull-down followed by mass spectrometry analysis, we identified the amino acid transporter SLC3A2, the AKAP protein LRBA, and the 4.1 protein EPB41L2 as potential interactors of these GPCRs. Using co-immunoprecipitation experiments, we confirmed the physical association of these proteins with the receptors. We then studied the functional relevance of the interaction between EPB41L2 and SREB1. Immunofluorescence microscopy revealed that SREB1 and EPB41L2 co-localize at the plasma membrane and that SREB1 is enriched in the ß-catenin-positive cell membranes. siRNA knockdown experiments revealed that EPB41L2 promotes the localization of SREB1 at the plasma membrane and increases the solubilization of SREB1 when using detergents, suggesting a modification of its membrane microenvironment. Altogether, these data suggest that EPB41L2 could regulate the subcellular compartmentalization of SREBs and, as proposed for other GPCRs, could affect their stability or activation.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismoRESUMO
Here we discuss, how by applying chemical concepts to biological problems, methods have been developed to map spatiotemporal regulation of proteins and small-molecule modulation of proteome signaling responses. We outline why chemical-biology platforms are ideal for such purposes. We further discuss strengths and weaknesses of chemical-biology protocols, contrasting them against classical genetic and biochemical approaches. We make these evaluations based on three parameters: occupancy; functional information; and spatial restriction. We demonstrate how the specific choice of chemical reagent and experimental set-up unite to resolve biological problems. Potential improvements/extensions as well as specific controls that in our opinion are often overlooked or employed incorrectly are also considered. Finally, we discuss some of the latest emerging methods to illuminate how chemical-biology innovations provide a gateway toward information hitherto inaccessible by conventional genetic/biochemical means. Finally, we also caution against solely relying on chemical-biology strategies and urge the field to undertake orthogonal validations to ensure robustness of results.