RESUMEN
The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Sinapsis Inmunológicas/metabolismo , Optogenética/métodos , Proteómica/métodos , Receptores de Superficie Celular/inmunología , Anticuerpos/química , Células Presentadoras de Antígenos/citología , Linfocitos B/inmunología , Linfocitos B/patología , Productos Biológicos/química , Linfocitos T CD8-positivos/citología , Comunicación Celular , Línea Celular Tumoral , Cromatografía Liquida , Expresión Génica , Células HL-60 , Humanos , Ligandos , Luz , Activación de Linfocitos , Optogenética/instrumentación , Medicina de Precisión/instrumentación , Medicina de Precisión/métodos , Unión Proteica , Proteómica/instrumentación , Receptores de Superficie Celular/genética , Transducción de Señal , Oxígeno Singlete/química , Oxígeno Singlete/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Espectrometría de Masas en Tándem , Virión/químicaRESUMEN
With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5'-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis in Escherichia coli.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico Activo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformación ProteicaRESUMEN
Antimicrobial resistance among Gram-negative bacteria is a growing problem, fueled by the paucity of new antibiotics that target these microorganisms. One novel family of macrocyclic ß-hairpin-shaped peptidomimetics was recently shown to act specifically against Pseudomonas spp. by a novel mechanism of action, targeting the outer membrane protein LptD, which mediates lipopolysaccharide transport to the cell surface during outer membrane biogenesis. Here we explore the mode of binding of one of these ß-hairpin peptidomimetics to LptD in Pseudomonas aeruginosa, by examining the effects on antimicrobial activity following N-methylation of individual peptide bonds. An N-methyl scan of the cyclic peptide revealed that residues on both sides of the ß-hairpin structure at a non-hydrogen bonding position likely mediate hydrogen-bonding interactions with the target LptD. Structural analyses by NMR spectroscopy further reinforce the conclusion that the folded ß-hairpin structure of the peptidomimetic is critical for binding to the target LptD. Finally, new NMe analogues with potent activity have been identified, which opens new avenues for optimization in this family of antimicrobial peptides.