RESUMEN
Pattern-recognition receptors including Toll-like receptors (TLRs) recognize invading pathogens and trigger an immune response in mammals. Here we show that mammalian ste20-like kinase 1/serine/threonine kinase 4 (MST1/STK4) functions as a negative regulator of lipopolysaccharide (LPS)-induced activation of the TLR4-NF-κB signaling pathway associated with inflammation. Myeloid-specific genetic ablation of MST1/STK4 increased the susceptibility of mice to LPS-induced septic shock. Ablation of MST1/STK4 also enhanced NF-κB activation triggered by LPS in bone marrow-derived macrophages (BMDMs), leading to increased production of proinflammatory cytokines by these cells. Furthermore, MST1/STK4 inhibited TRAF6 autoubiquitination as well as TRAF6-mediated downstream signaling induced by LPS. In addition, we found that TRAF6 mediates the LPS-induced activation of MST1/STK4 by catalyzing its ubiquitination, resulting in negative feedback regulation by MST1/STK4 of the LPS-induced pathway leading to cytokine production in macrophages. Together, our findings suggest that MST1/STK4 functions as a negative modulator of the LPS-induced NF-κB signaling pathway during macrophage activation.
Asunto(s)
Macrófagos/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Citocinas/sangre , Citocinas/genética , Citocinas/metabolismo , Células HEK293 , Humanos , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Sepsis/sangre , Sepsis/genética , Sepsis/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Factor 6 Asociado a Receptor de TNF/genética , Receptor Toll-Like 4/genética , Ubiquitinación/efectos de los fármacosRESUMEN
The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.
Asunto(s)
Fibroblastos/efectos de los fármacos , Macrófagos/efectos de los fármacos , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Fibroblastos/enzimología , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multienzimáticos , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
LC3-family member proteins play a critical role in autophagy, a cellular process responsible for the degradation of massive cellular components including intracellular pathogens. A variety of molecules involved in the autophagic pathway engage in specific interactions with a unique sequence motif referred to as the LIR (LC3-interacting region) motif. Although identification of conserved structural features of LIR motifs in complex with LC3-family members has established a canonical LIR motif, atypical conformations of LIR motifs have recently been revealed. Here, we determined the three-dimensional crystal structures of LC3B in complex with three different LIR motifs of RavZ from Legionella pneumophila, an intracellular pathogen that can manipulate the host autophagy system. The tandem LIR motifs located in the N-terminal region of RavZ adopt a novel ß-sheet conformation and thus provide specific ionic interactions with LC3B in addition to canonical hydrophobic plugged-in interactions. Consequently, these motifs possess higher binding affinity to LC3-family members than canonical LIR motifs, although the tandem repeats can only bind to one LC3 molecule. These findings broaden our understanding of the functional repertoire of LIR motifs in autophagy.
Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Autofagia , Proteínas Bacterianas/química , Humanos , Legionella pneumophila/química , Proteínas Asociadas a Microtúbulos/química , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de SecuenciaRESUMEN
MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1-/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4+ T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Factor de Crecimiento de Hepatocito/inmunología , Hipergammaglobulinemia/inmunología , Interleucina-4/inmunología , Proteínas Proto-Oncogénicas/inmunología , Animales , Factor de Crecimiento de Hepatocito/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/deficienciaRESUMEN
Post-transcriptional gene regulation is an important step in eukaryotic gene expression. The last step to govern production of nascent peptides is during the process of mRNA translation. mRNA translation is controlled by many translation initiation factors that are susceptible to post-translational modifications. Here we report that one of the translation initiation factors, eIF4E, is phosphorylated by Mammalian Ste20-like kinase (MST1). Upon phosphorylation, eIF4E weakly interacts with the 5' CAP to inhibit mRNA translation. Simultaneously, active polyribosome is more associated with long noncoding RNAs (lncRNAs). Moreover, the linc00689-derived micropeptide, STORM (Stress- and TNF-α-activated ORF Micropeptide), is triggered by TNF-α-induced and MST1-mediated eIF4E phosphorylation, which exhibits molecular mimicry of SRP19 and, thus, competes for 7SL RNA. Our findings have uncovered a novel function of MST1 in mRNA and lncRNA translation by direct phosphorylation of eIF4E. This novel signaling pathway will provide new platforms for regulation of mRNA translation via post-translational protein modification.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Fosforilación/fisiología , Biosíntesis de Proteínas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Ratones , Polirribosomas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Caperuzas de ARN/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Legionella/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fagocitos/metabolismo , Autofagia , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Fagosomas/metabolismo , Unión Proteica , Dominios Proteicos , Dispersión de Radiación , Resonancia por Plasmón de SuperficieRESUMEN
Reactive oxygen species (ROS) have many physiological and pathological effects on diverse cellular events. In particular, excessive ROS causes oxidative stress that leads to cell death. The mammalian STE20-like kinase-1 (MST1), a multifunctional serine-threonine kinase, plays a pivotal role in oxidative stress-induced cellular signaling events. Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is also known to be essential for oxidative stress-induced cell death. Here, we showed that H2O2 induced the physical interaction between TRAF2 and MST1, and that this interaction promoted the homodimerization as well as the activation of MST1. Furthermore, TRAF2 was required for MST1 to mediate the H2O2-induced stimulation of c-Jun N-terminal kinase and p38 kinase as well as apoptosis. Taken together, our results suggest that TRAF2 functions as a key activator of MST1 in oxidative stress-induced intracellular signaling processes.
Asunto(s)
Apoptosis , Factor de Crecimiento de Hepatocito/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Factor 2 Asociado a Receptor de TNF/fisiología , Animales , Células Cultivadas , Factor de Crecimiento de Hepatocito/química , Sistema de Señalización de MAP Quinasas , Ratones Noqueados , Estrés Oxidativo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Proto-Oncogénicas/química , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a conserved mechanism in all eukaryotes. When autophagy is induced, cellular components are sequestered within an autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the ATG12-ATG5 conjugate requires 2 different binding partners, ATG16L1 for autophagosome elongation and TECPR1 for lysosomal fusion. In our current study, we describe the crystal structures of human ATG5 in complex with an N-terminal domain of ATG16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar α-helical structure containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of the C-terminal unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell biological analyses. These results provide new insights into the molecular details of the autophagosome maturation process, from its elongation to its fusion with a lysosome.
Asunto(s)
Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteína 5 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Células COS , Proteínas Portadoras/metabolismo , Chlorocebus aethiops , Humanos , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación/genética , Fagosomas/ultraestructura , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismoRESUMEN
Son of sevenless 1 (SOS1) is a Ras-specific guanine-nucleotide-exchange factor (GEF) that mediates intracellular signaling processes induced by receptor tyrosine kinases. In this study, we show that CIIA (also known as VPS28) physically associates with SOS1 and thereby inhibits the GEF activity of SOS1 on Ras, which prevents the epidermal growth factor (EGF)-induced activation of the Ras-Erk1/2 pathway. Furthermore, CIIA inhibited cyclin D1 expression, as well as DNA, synthesis in response to EGF. Intriguingly, CIIA failed to inhibit the Ras-specific GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L and E846K). Taken together, our results suggest that CIIA functions as a negative modulator of the SOS1-Ras signaling events initiated by peptide growth factors including EGF.