RESUMEN
Caspases are a family of conserved cysteine proteases that play key roles in programmed cell death and inflammation. In multicellular organisms, caspases are activated via macromolecular signaling complexes that bring inactive procaspases together and promote their proximity-induced autoactivation and proteolytic processing. Activation of caspases ultimately results in programmed execution of cell death, and the nature of this cell death is determined by the specific caspases involved. Pioneering new research has unraveled distinct roles and cross talk of caspases in the regulation of programmed cell death, inflammation, and innate immune responses. In-depth understanding of these mechanisms is essential to foster the development of precise therapeutic targets to treat autoinflammatory disorders, infectious diseases, and cancer. This review focuses on mechanisms governing caspase activation and programmed cell death with special emphasis on the recent progress in caspase cross talk and caspase-driven gasdermin D-induced pyroptosis.
Asunto(s)
Caspasas/metabolismo , Muerte Celular , Inflamación/etiología , Inflamación/metabolismo , Proteínas de Neoplasias/genética , Piroptosis/genética , Animales , Apoptosis , Biomarcadores , Caspasas/genética , Muerte Celular/genética , Susceptibilidad a Enfermedades , Activación Enzimática , Humanos , Inflamación/patología , Proteínas de Neoplasias/metabolismo , Transducción de SeñalRESUMEN
The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.
Asunto(s)
Serina Endopeptidasas , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Humanos , Cristalografía por Rayos X , Coronavirus/metabolismo , Coronavirus/química , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Modelos Moleculares , Unión Proteica , Células HEK293 , Animales , Activación Enzimática , Internalización del VirusRESUMEN
The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.
Asunto(s)
Hematopoyesis , Sistema Inmunológico , Proteína Quinasa C/metabolismo , Animales , Coenzimas/metabolismo , Activación Enzimática/inmunología , Humanos , Proteína Quinasa C/inmunología , Transducción de SeñalRESUMEN
We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.
Asunto(s)
Técnicas Biosensibles , Péptidos/química , Imagen Individual de Molécula , Animales , Adhesión Celular , Línea Celular , Supervivencia Celular , Embrión de Mamíferos/citología , Activación Enzimática , Fibroblastos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Ratones , Nanopartículas/química , Conformación Proteica , Familia-src Quinasas/metabolismoRESUMEN
Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.
Asunto(s)
Condensados Biomoleculares/metabolismo , Gránulos Citoplasmáticos/metabolismo , Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación Enzimática , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Células HEK293 , Humanos , Proteína SOS1/metabolismo , Transducción de SeñalRESUMEN
Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Citoprotección , Células Ganglionares de la Retina/patología , Visión Ocular , Animales , Axones/efectos de los fármacos , Axones/patología , Encéfalo/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Glaucoma/genética , Glaucoma/patología , Ratones Endogámicos C57BL , Neurotoxinas/toxicidad , Traumatismos del Nervio Óptico/patología , Transducción de SeñalRESUMEN
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
Asunto(s)
Fagocitosis , Fosfofructoquinasa-1 Tipo Hepático/metabolismo , Estallido Respiratorio , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Proteínas de Unión a Fosfato/metabolismo , Fosfofructoquinasa-1 Tipo Hepático/antagonistas & inhibidores , Fosfofructoquinasa-1 Tipo Hepático/ultraestructura , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/aislamiento & purificación , Estallido Respiratorio/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT.
Asunto(s)
Ácido Araquidónico/análisis , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Eicosanoides/metabolismo , Animales , Ácido Araquidónico/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Citosol/metabolismo , Eicosanoides/fisiología , Activación Enzimática , Femenino , Humanos , Metabolismo de los Lípidos/fisiología , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/fisiología , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasas A2/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.
Asunto(s)
Ribosomas/metabolismo , Estrés Fisiológico , Transportadoras de Casetes de Unión a ATP/metabolismo , Anisomicina/farmacología , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de la radiación , Activación Enzimática , Humanos , Quinasas Quinasa Quinasa PAM/deficiencia , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Fosforilación , Polirribosomas/metabolismo , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Rayos Ultravioleta , eIF-2 Quinasa/metabolismoRESUMEN
The immune system has evolved to mount an effective defense against pathogens and to minimize deleterious immune-mediated inflammation caused by commensal microorganisms, immune responses against self and environmental antigens, and metabolic inflammatory disorders. Regulatory T (Treg) cell-mediated suppression serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and autoinflammatory disorders, allergy, acute and chronic infections, cancer, and metabolic inflammation. The discovery that Foxp3 is the transcription factor that specifies the Treg cell lineage facilitated recent progress in understanding the biology of regulatory T cells. In this review, we discuss cellular and molecular mechanisms in the differentiation and function of these cells.
Asunto(s)
Linfocitos T Reguladores/inmunología , Animales , Diferenciación Celular , Citocinas/inmunología , Citocinas/metabolismo , Activación Enzimática , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Homeostasis/inmunología , Humanos , Tolerancia Inmunológica , MicroARNs/inmunología , MicroARNs/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Timo/inmunología , Timo/metabolismo , Transcripción GenéticaRESUMEN
mTOR is an evolutionarily conserved serine/threonine kinase that plays a central role in integrating environmental cues in the form of growth factors, amino acids, and energy. In the study of the immune system, mTOR is emerging as a critical regulator of immune function because of its role in sensing and integrating cues from the immune microenvironment. With the greater appreciation of cellular metabolism as an important regulator of immune cell function, mTOR is proving to be a vital link between immune function and metabolism. In this review, we discuss the ability of mTOR to direct the adaptive immune response. Specifically, we focus on the role of mTOR in promoting differentiation, activation, and function in T cells, B cells, and antigen-presenting cells.
Asunto(s)
Inmunidad , Serina-Treonina Quinasas TOR/metabolismo , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Activación Enzimática , Humanos , Inmunosupresores/farmacología , Activación de Linfocitos/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Anisotropía , Sitios de Unión/fisiología , Activación Enzimática , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional/fisiología , Proteolisis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/fisiología , Ubiquitina/metabolismoRESUMEN
Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.
Asunto(s)
Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Treonina/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Células HCT116 , Humanos , Fosforilación , Dominios Homólogos a Pleckstrina , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-akt/química , Serina/química , Transducción de Señal , Treonina/químicaRESUMEN
Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.
Asunto(s)
Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patología , Eritropoyetina/genética , Mutación Missense , Transducción de Señal , Anemia de Diamond-Blackfan/terapia , Niño , Consanguinidad , Activación Enzimática , Eritropoyesis , Eritropoyetina/química , Femenino , Humanos , Janus Quinasa 2/metabolismo , Cinética , Masculino , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismoRESUMEN
In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.
Asunto(s)
Proteínas de Unión al ADN , Endodesoxirribonucleasas , Endonucleasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Endonucleasas/metabolismo , Endonucleasas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/química , Humanos , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Modelos Moleculares , Fosforilación , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Roturas del ADN de Doble Cadena , Ácido Anhídrido Hidrolasas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Mutación , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Reparación del ADN , Activación EnzimáticaRESUMEN
N-myristoyltransferase (NMT) attaches the fatty acid myristate to the N-terminal glycine of proteins to sort them into soluble and membrane-bound fractions. Function of the energy-sensing AMP-activated protein kinase, AMPK, is myristoylation dependent. In rheumatoid arthritis (RA), pathogenic T cells shift glucose away from adenosine tri-phosphate production toward synthetic and proliferative programs, promoting proliferation, cytokine production, and tissue invasion. We found that RA T cells had a defect in NMT1 function, which prevented AMPK activation and enabled unopposed mTORC1 signaling. Lack of the myristate lipid tail disrupted the lysosomal translocation and activation of AMPK. Instead, myristoylation-incompetent RA T cells hyperactivated the mTORC1 pathway and differentiated into pro-inflammatory TH1 and TH17 helper T cells. In vivo, NMT1 loss caused robust synovial tissue inflammation, whereas forced NMT1 overexpression rescued AMPK activation and suppressed synovitis. Thus, NMT1 has tissue-protective functions by facilitating lysosomal recruitment of AMPK and dampening mTORC1 signaling.
Asunto(s)
Proteínas Quinasas Activadas por AMP/inmunología , Aciltransferasas/inmunología , Artritis Reumatoide/inmunología , Sinovitis/inmunología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adulto , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Células Cultivadas , Activación Enzimática/inmunología , Femenino , Humanos , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Interferencia de ARN , Sinovitis/genética , Sinovitis/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Adulto JovenRESUMEN
B cell antigen receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to optimize selection for high-affinity B cells. In GCBC, BCR signals are constrained, but the mechanisms are not well understood. Here we describe a GC-specific, AKT-kinase-driven negative feedback loop that attenuates BCR signaling. Mass spectrometry revealed that AKT target activity was altered in GCBCs compared with naive B cells. Retargeting was linked to differential AKT T308 and S473 phosphorylation, in turn controlled by GC-specific upregulation of phosphoinositide-dependent protein kinase PDK1 and the phosphatase PTEN. In GCBCs, AKT preferentially targeted CSK, SHP-1 and HPK1, which are negative regulators of BCR signaling. We found that phosphorylation enhances enzymatic activity of these proteins, creating a negative feedback loop that dampens upstream BCR signaling. AKT inhibition relieved this negative feedback and enhanced activation of BCR-proximal kinase LYN, as well as downstream BCR signaling molecules in GCBCs.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Centro Germinal/inmunología , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Biomarcadores , Biología Computacional/métodos , Activación Enzimática , Técnicas de Inactivación de Genes , Humanos , Ratones Noqueados , Fosforilación , Especificidad por SustratoRESUMEN
Resting CD4+ T cells are highly resistant to the production of human immunodeficiency virus type 1 (HIV-1). However, the mechanism by which resting CD4+ T cells restrict such production in the late viral replication phase of infection has remained unclear. In this study, we found that the cell membrane metalloprotease TRAB domain-containing protein 2A (TRABD2A) inhibited this production in resting CD4+ T cells by degrading the virion structural precursor polyprotein Gag at the plasma membrane. Depletion or inhibition of metalloprotease activity by TRABD2A profoundly enhanced HIV-1 production in resting CD4+ T cells. TRABD2A expression was much higher in resting CD4+ T cells than in activated CD4+ T cells and was considerably reduced by T cell activation. Moreover, reexpressing TRABD2A reinforced the resistance of activated CD4+ T cells to the production of HIV-1 progeny. Collectively, these results elucidate the molecular mechanism employed by resting CD4+ T cells to potently restrict the assembly and production of HIV-1 progeny.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Metaloproteasas/genética , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Cationes , Línea Celular , Activación Enzimática , Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas de la Membrana/metabolismo , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Familia de Multigenes , Proteolisis , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Carga ViralRESUMEN
Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.
Asunto(s)
Caspasas/inmunología , Mediadores de Inflamación/inmunología , Inflamación/inmunología , Serpinas/inmunología , Animales , Caspasas/genética , Caspasas/metabolismo , Línea Celular , Células Cultivadas , Activación Enzimática/inmunología , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Células RAW 264.7 , Interferencia de ARN , Serina Proteasas/inmunología , Serina Proteasas/metabolismo , Serpinas/genética , Serpinas/metabolismo , Células THP-1 , Células U937RESUMEN
Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11, however, are not cytosolic, and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here, we identify outer membrane vesicles (OMVs) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMVs are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMVs, reveals the importance of OMVs in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMVs in enabling the cytosolic entry of LPS and, consequently, caspase-11 activation during Gram-negative bacterial infections.