RESUMEN
We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.
Asunto(s)
Detergentes/química , Lípidos/química , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Micelas , Diacilglicerol Quinasa/química , Difusión , Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas Fluorescentes Verdes/química , Halobacteriaceae/química , Espectroscopía de Resonancia Magnética/métodos , Microscopía Electrónica de Transmisión/métodos , Proteínas de Transporte de Monosacáridos/química , Nanopartículas/química , Plásmidos/metabolismo , Rodopsinas Sensoriales/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Simportadores/químicaRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. RESULTS: A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. CONCLUSIONS: A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.