RESUMO
HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Membrana Celular/metabolismo , HIV-1/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Família da Proteína 8 Relacionada à Autofagia/química , Sítios de Ligação , Extratos Celulares , Sequência Conservada , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismoRESUMO
Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.