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The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.
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The HSP90-binding immunophilin FKBP51 is a soluble protein that shows high homology and structural similarity with FKBP52. Both immunophilins are functionally divergent and often show antagonistic actions. They were first described in steroid receptor complexes, their exchange in the complex being the earliest known event in steroid receptor activation upon ligand binding. In addition to steroid-related events, several pleiotropic actions of FKBP51 have emerged during the last years, ranging from cell differentiation and apoptosis to metabolic and psychiatric disorders. On the other hand, mitochondria play vital cellular roles in maintaining energy homeostasis, responding to stress conditions, and affecting cell cycle regulation, calcium signaling, redox homeostasis, and so forth. This is achieved by proteins that are encoded in both the nuclear genome and mitochondrial genes. This implies active nuclear-mitochondrial communication to maintain cell homeostasis. Such communication involves factors that regulate nuclear and mitochondrial gene expression affecting the synthesis and recruitment of mitochondrial and nonmitochondrial proteins, and/or changes in the functional state of the mitochondria itself, which enable mitochondria to recover from stress. FKBP51 has emerged as a serious candidate to participate in these regulatory roles since it has been unexpectedly found in mitochondria showing antiapoptotic effects. Such localization involves the tetratricopeptide repeats domains of the immunophilin and not its intrinsic enzymatic activity of peptidylprolyl-isomerase. Importantly, FKBP51 abandons the mitochondria and accumulates in the nucleus upon cell differentiation or during the onset of stress. Nuclear FKBP51 enhances the enzymatic activity of telomerase. The mitochondrial-nuclear trafficking is reversible, and certain situations such as viral infections promote the opposite trafficking, that is, FKBP51 abandons the nucleus and accumulates in mitochondria. In this article, we review the latest findings related to the mitochondrial-nuclear communication mediated by FKBP51 and speculate about the possible implications of this phenomenon.
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The immunophilin FKBP51 forms heterocomplexes with molecular chaperones, protein-kinases, protein-phosphatases, autophagy-related factors, and transcription factors. Like most scaffold proteins, FKBP51 can use a simple tethering mechanism to favor the efficiency of interactions with partner molecules, but it can also exert more complex allosteric controls over client factors, the immunophilin itself being a putative regulation target. One of the simplest strategies for regulating pathways and subcellular localization of proteins is phosphorylation. In this study, it is shown that scaffold immunophilin FKBP51 is resolved by resolutive electrophoresis in various phosphorylated isoforms. This was evidenced by their reactivity with specific anti-phosphoamino acid antibodies and their fade-out by treatment with alkaline phosphatase. Interestingly, stress situations such as exposure to oxidants or in vivo fasting favors FKBP51 translocation from mitochondria to the nucleus. While fasting involves phosphothreonine residues, oxidative stress involves tyrosine residues. Molecular modeling predicts the existence of potential targets located at the FK1 domain of the immunophilin. Thus, oxidative stress favors FKBP51 dephosphorylation and protein degradation by the proteasome, whereas FK506 binding protects the persistence of the post-translational modification in tyrosine, leading to FKBP51 stability under oxidative conditions. Therefore, FKBP51 is revealed as a phosphoprotein that undergoes differential phosphorylations according to the stimulus.
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Fosfoproteínas , Proteínas de Unión a Tacrolimus , Humanos , Fosfoproteínas/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Núcleo Celular/metabolismo , Mitocondrias/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Tirosina/metabolismoRESUMEN
Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.
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Autofagia , Caspasa 1 , Interleucina-1beta , Neutrófilos , Serina Proteasas , Autofagia/genética , Autofagia/inmunología , Caspasa 1/genética , Caspasa 1/metabolismo , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Serina Proteasas/genética , Serina Proteasas/inmunologíaRESUMEN
T and B cells employ integrin α4ß7 to migrate to intestine under homeostatic conditions. Whether those cells differentially rely on α4ß7 for homing during inflammatory conditions has not been fully examined. This may have implications for our understanding of the mode of action of anti-integrin therapies in inflammatory bowel disease (IBD). Here, we examined the role of α4ß7 integrin during chronic colitis using IL-10-/- mice, ß7-deficient IL-10-/-, IgA-deficient IL-10-/- mice, and antibody blockade of MAdCAM-1. We found that α4ß7 was predominantly expressed by B cells. ß7 deficiency and MAdCAM-1 blockade specifically depleted antibody secreting cells (ASC) (not T cells) from the colonic LP, leading to a fecal pan-immunoglobulin deficit, severe colitis, and alterations of microbiota composition. Colitis was not due to defective regulation, as dendritic cells (DC), regulatory T cells, retinaldehyde dehydrogenase (RALDH) expression, activity, and regulatory T/B-cell cytokines were all comparable between the strains/treatment. Finally, an IgA deficit closely recapitulated the clinical phenotype and altered microbiota composition of ß7-deficient IL-10-/- mice. Thus, a luminal IgA deficit contributes to accelerated colitis in the ß7-deficient state. Given the critical/nonredundant dependence of IgA ASC on α4ß7:MAdCAM-1 for intestinal homing, B cells may represent unappreciated targets of anti-integrin therapies.
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Células Productoras de Anticuerpos/inmunología , Moléculas de Adhesión Celular/metabolismo , Colitis/inmunología , Microbioma Gastrointestinal/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Integrina alfa4/metabolismo , Cadenas beta de Integrinas/metabolismo , Intestinos/fisiología , Mucoproteínas/metabolismo , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina A/metabolismo , Inmunomodulación , Cadenas beta de Integrinas/genética , Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Efficient IgA transcytosis is critical for the maintenance of a homeostatic microbiota. In the canonical model, locally-secreted dimeric (d)IgA reaches the polymeric immunoglobulin receptor (pIgR) on intestinal epithelium via simple diffusion. A role for integrin αE(CD103)ß7 during transcytosis has not been described, nor its expression by intestinal B cell lineage cells. We found that αE-deficient (αE-/-) mice have a luminal IgA deficit, despite normal antibody-secreting cells (ASC) recruitment, local IgA production and increased pIgR expression. This deficit was not due to dendritic cell (DC)-derived retinoic acid (RA) nor class-switching defects, as stool from RAG-/- mice reconstituted with αE-/- B cells was also IgA deficient. Flow cytometric, ultrastructural and transcriptional profiling showed that αEß7-expressing ASC represent an undescribed subset of terminally-differentiated intestinal plasma cells (PC) that establishes direct cell to cell contact with intestinal epithelium. We propose that IgA not only reaches pIgR through diffusion, but that αEß7+ PC dock with E-cadherin-expressing intestinal epithelium to directly relay IgA for transcytosis into the intestinal lumen.
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Inmunoglobulina A/inmunología , Integrinas/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Transcitosis/inmunología , Animales , Diferenciación Celular/inmunología , Expresión Génica , Regulación de la Expresión Génica , Inmunoglobulina A/metabolismo , Inmunoglobulina A Secretora/inmunología , Integrinas/deficiencia , Integrinas/metabolismo , Mucosa Intestinal/ultraestructura , Activación de Linfocitos , Ratones , Ratones Noqueados , Modelos Biológicos , Células Plasmáticas/citología , Células Plasmáticas/ultraestructuraRESUMEN
Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.
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Trampas Extracelulares/metabolismo , Macroautofagia , Proteínas de Unión al GTP Monoméricas/metabolismo , NADPH Oxidasas/metabolismo , Animales , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Calmodulina/metabolismo , Modelos Animales de Enfermedad , Espacio Intracelular/metabolismo , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al GTP Monoméricas/deficiencia , Activación Neutrófila , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Pseudomonas aeruginosa/fisiología , Especies Reactivas de Oxígeno/metabolismo , Salmonelosis Animal/microbiología , Salmonelosis Animal/patología , Salmonella typhimurium/fisiologíaRESUMEN
FKBP51 and FKBP52 are two iconic members of the family of peptidyl-prolyl-(cis/trans)-isomerases (EC: 5.2.1.8), which comprises proteins that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Originally, both proteins have been studied as molecular chaperones belonging to the steroid receptor heterocomplex, where they were first discovered. In addition to their expected role in receptor folding and chaperoning, FKBP51 and FKBP52 are also involved in many biological processes, such as signal transduction, transcriptional regulation, protein transport, cancer development, and cell differentiation, just to mention a few examples. Recent studies have revealed that both proteins are subject of post-translational modifications such as phosphorylation, SUMOlyation, and acetylation. In this work, we summarize recent advances in the study of these immunophilins portraying them as scaffolding proteins capable to organize protein heterocomplexes, describing some of their antagonistic properties in the physiology of the cell, and the putative regulation of their properties by those post-translational modifications.
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Procesamiento Proteico-Postraduccional , Proteínas de Unión a Tacrolimus/fisiología , Acetilación , Humanos , Fosforilación , Unión Proteica , Transporte de Proteínas , Sumoilación , Proteínas de Unión a Tacrolimus/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In this chapter, we summarize the birth of the field of nuclear receptors. These receptors exhibit a multitude of roles in cell biology and hence have attracted a great deal of interest in the drug discovery field. It is not certain whether these receptors evolved independently or an ancestral protein acquired various functions upon binding to preexisting small molecules, ligands. Currently, members of this receptor superfamily are categorized in six groups, including "orphan receptors." Research in the area has resulted in several clinically used drugs and continues to reveal further previously unknown roles for these receptors paving the road toward more valuable discoveries in the future.
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Receptores Nucleares Huérfanos/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal , Animales , Humanos , Ligandos , Receptores Nucleares Huérfanos/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides/fisiologíaRESUMEN
Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects.
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Proteínas HSP90 de Choque Térmico/metabolismo , Inmunofilinas/metabolismo , Animales , Proteínas HSP90 de Choque Térmico/química , Humanos , Inmunofilinas/química , Modelos Moleculares , Estructura Molecular , Unión ProteicaRESUMEN
In this article we summarize the birth of the field of nuclear receptors, the discovery of untransformed and transformed isoforms of ligand-binding macromolecules, the discovery of the three-domain structure of the receptors, and the properties of the Hsp90-based heterocomplex responsible for the overall structure of the oligomeric receptor and many aspects of the biological effects. The discovery and properties of the subfamily of receptors called orphan receptors is also outlined. Novel molecular aspects of the mechanism of action of nuclear receptors and challenges to resolve in the near future are discussed.
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Immunological interdependence between the two eyes has been reported for the cornea and the retina but not for the ocular mucosal surface. Intriguingly, patients frequently report ocular surface-related symptoms in the other eye after unilateral ocular surgery. Here we show how unilateral eye injuries in mice affect the mucosal immune response of the opposite ocular surface. We report that, despite the lack of lymphatic cross-drainage, a neurogenic inflammatory reflex in the contralateral conjunctiva is sufficient to increase, first, epithelial nuclear factor kappa B signaling, then, dendritic cell maturation, and finally, expansion of effector, instead of regulatory, T cells in the draining lymph node, leading to disrupted ocular mucosal tolerance. We also show that damage to ocular surface nerves is required. Using pharmacological inhibitors and agonists, we identified transient receptor potential vanilloid 1 (TRPV1) channel as the receptor sensing tissue damage in the injured eye and substance P released in the opposite ocular surface as the effector of the sympathetic response. Finally, blocking either step prevented subsequent ocular allergic reactions in the opposite eye in a unilateral corneal alkali burn model. This study demonstrates that both ocular surfaces are immunologically linked and suggests potential therapeutic targets for intervention.
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Ojo/inmunología , Inflamación/inmunología , Membrana Mucosa/inmunología , Sustancia P/inmunología , Canales Catiónicos TRPV/inmunología , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Hipersensibilidad/inmunología , Ganglios Linfáticos/inmunología , Melanoma , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
The ability to permit gene expression is managed by a set of relatively well known regulatory mechanisms. Nonetheless, this property can also be acquired during a life span as a consequence of environmental stimuli. Interestingly, some acquired information can be passed to the next generation of individuals without modifying gene information, but instead by the manner in which cells read and process such information. Molecular chaperones are classically related to the proper preservation of protein folding and anti-aggregation properties, but one of them, heat-shock protein 90 (Hsp90), is a refined sensor of protein function facilitating the biological activity of properly folded client proteins that already have a preserved tertiary structure. Interestingly, Hsp90 can also function as a critical switch able to regulate biological responses due to its association with key client proteins such as histone deacetylases or DNA methylases. Thus, a growing amount of evidence has connected the action of Hsp90 to post-translational modifications of soluble nuclear factors, DNA, and histones, which epigenetically affect gene expression upon the onset of an unfriendly environment. This response is commanded by the activation of the transcription factor heat-shock factor 1 (HSF1). Even though numerous stresses of diverse nature are known to trigger the stress response by activation of HSF1, it is still unknown whether there are different types of molecular sensors for each type of stimulus. In the present review, we will discuss various aspects of the regulatory action of HSF1 and Hsp90 on transcriptional regulation, and how this regulation may affect genetic assimilation mechanisms and the health of individuals.
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Regulación de la Expresión Génica/fisiología , Proteínas HSP90 de Choque Térmico/fisiología , Factores de Transcripción del Choque Térmico/fisiología , Animales , Variación Genética , Histona Desacetilasa 6/fisiología , Humanos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Estrés Fisiológico , Transcripción Genética/fisiologíaRESUMEN
FK506-binding proteins are members of the immunophilin family of proteins. Those immunophilins associated to the 90-kDa-heat-shock protein, Hsp90, have been proposed as potential modulators of signalling cascade factors chaperoned by Hsp90. FKBP51 and FKBP52 are the best characterized Hsp90-bound immunophilins first described associated to steroid-receptors. The reverse transcriptase subunit of telomerase, hTERT, is also an Hsp90 client-protein and is highly expressed in cancer cells, where it is required to compensate the loss of telomeric DNA after each successive cell division. Because FKBP51 is also a highly expressed protein in cancer tissues, we analyzed its potential association with hTERT·Hsp90 complexes and its possible biological role. In this study it is demonstrated that both immunophilins, FKBP51 and FKBP52, co-immunoprecipitate with hTERT. The Hsp90 inhibitor radicicol disrupts the heterocomplex and favors the partial cytoplasmic relocalization of hTERT in similar manner as the overexpression of the TPR-domain peptide of the immunophilin. While confocal microscopy images show that FKBP51 is primarily localized in mitochondria and hTERT is totally nuclear, upon the onset of oxidative stress, FKBP51 (but not FKBP52) becomes mostly nuclear colocalizing with hTERT, and longer exposure times to peroxide favors hTERT export to mitochondria. Importantly, telomerase activity of hTERT is significantly enhanced by FKBP51. These observations support the emerging role assigned to FKBP51 as antiapoptotic factor in cancer development and progression, and describe for the first time the potential role of this immunophilin favoring the clonal expansion by enhancing telomerase activity.