RESUMEN
The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Paxillin/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Quinasas p21 Activadas/fisiología , Proteínas ADAM/sangre , Proteína ADAM10 , Proteína ADAM17 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/sangre , Estudios de Casos y Controles , Activación Enzimática , Células HEK293 , Infecciones por VIH/sangre , Infecciones por VIH/enzimología , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Humanos , Melanoma/sangre , Melanoma/enzimología , Microdominios de Membrana/enzimología , Proteínas de la Membrana/sangre , Mutagénesis Sitio-Dirigida , Paxillin/genética , Paxillin/metabolismo , Fosforilación , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Proteína Quinasa C-delta/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ribonucleoproteínas/metabolismo , Vesículas Secretoras/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
The effect of nitrogen regulation on the level of transcriptional control has been investigated in a variety of bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, and Streptomyces coelicolor; however, until now there have been no data for mycobacteria. In this study, we found that the OmpR-type regulator protein GlnR controls nitrogen-dependent transcription regulation in Mycobacterium smegmatis. Based on RNA hybridization experiments with a wild-type strain and a corresponding mutant strain, real-time reverse transcription-PCR analyses, and DNA binding studies using cell extract and purified protein, the glnA (msmeg_4290) gene, which codes for glutamine synthetase, and the amtB (msmeg_2425) and amt1 (msmeg_6259) genes, which encode ammonium permeases, are controlled by GlnR. Furthermore, since glnK (msmeg_2426), encoding a PII-type signal transduction protein, and glnD (msmeg_2427), coding for a putative uridylyltransferase, are in an operon together with amtB, these genes are part of the GlnR regulon as well. The GlnR protein binds specifically to the corresponding promoter sequences and functions as an activator of transcription when cells are subjected to nitrogen starvation.