RESUMEN
BACKGROUND AND PURPOSE: The transient receptor potential vanilloid 4 (TRPV4) cation channel participates in multiple physiological processes and is also at the core of different diseases, making this channel an interesting pharmacological target with therapeutic potential. However, little is known about the structural elements governing its inhibition. EXPERIMENTAL APPROACH: We have now combined in silico drug discovery and molecular dynamics simulation based on Xenopus tropicalis xTRPV4 structure with functional studies measuring cell Ca2+ influx mediated by human TRPV4 channel to characterize the binding site of known TRPV4 inhibitors and to identify novel small molecule channel modulators. KEY RESULTS: We have found that the inhibitor HC067047 binds to a pocket conformed by residues from S2-S3 linker (xTRPV4-D542), S4 (xTRPV4-M583 and Y587 and S5 (xTRPV4-D609 and F613). This pocket was also used for structure-based virtual screening in the search of novel channel modulators. Forty potential hits were selected based on the lower docking scores (from ~250,000 compounds) and their effect upon TRPV4 functionally tested. Three were further analysed for stability using molecular dynamics simulation and functionally tested on TRPV4 channels carrying mutations in the binding pocket. Compound NSC151066, shown to require residue xTRPV4-M583 for its inhibitory effect, presented an IC50 of 145 nM and demonstrated to be an effective antiviral against Zika virus with a potency similar to HC067047. CONCLUSION AND IMPLICATIONS: Together, we propose structural insights into the inhibition of TRPV4 and how this information can be used for the design of novel channel modulators. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.
Asunto(s)
Canales de Potencial de Receptor Transitorio , Infección por el Virus Zika , Virus Zika , Animales , Antivirales/farmacología , Sitios de Unión , Humanos , Canales Catiónicos TRPV/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Xenopus/metabolismo , Virus Zika/metabolismoRESUMEN
Mechanical forces are exerted throughout cytokinesis, the final step of cell division. Yet, how forces are transduced and affect the signaling dynamics of cytokinetic proteins remains poorly characterized. We now show that the mechanosensitive Piezo1 channel is activated at the intercellular bridge (ICB) connecting daughter cells to regulate abscission. Inhibition of Piezo1 caused multinucleation both in vitro and in vivo. Piezo1 positioning at the ICB during cytokinesis depends on Pacsin3. Pharmacological and genetic inhibition of Piezo1 or Pacsin3 resulted in mislocation of Rab11-family-interacting protein 3 (Rab11-FIP3) endosomes, apoptosis-linked gene 2-interacting protein X (ALIX), and endosomal sorting complex required for transport III (ESCRT-III). Furthermore, we identified FIP3 as the link between Piezo1-generated Ca2+ signals and ALIX delivery to the ICB, where ALIX recruits the ESCRT-III component charged multivesicular body protein 4B, which promotes abscission. These results provide a different view of how mechanical forces participate in cytokinesis and identify Piezo1 as a key modulator of endosome trafficking.
RESUMEN
Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.
Asunto(s)
Tamaño de la Célula , Canales de Cloruro/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas , Muerte Celular , Supervivencia Celular , Células HeLa , Humanos , Presión Osmótica , Fosforilación , Potasio/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Sodio/metabolismoRESUMEN
Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/genética , Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Canales Iónicos/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
TRPV4 cation channel activation by cytochrome P450-mediated derivatives of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), constitute a major mechanisms of endothelium-derived vasodilatation. Besides, TRPV4 mechano/osmosensitivity depends on phospholipase A2 (PLA2) activation and subsequent production of AA and EETs. However, the lack of evidence for a direct interaction of EETs with TRPV4 together with claims of EET-independent mechanical activation of TRPV4 has cast doubts on the validity of this mechanism. We now report: 1) The identification of an EET-binding pocket that specifically mediates TRPV4 activation by 5',6'-EET, AA and hypotonic cell swelling, thereby suggesting that all these stimuli shared a common structural target within the TRPV4 channel; and 2) A structural insight into the gating of TRPV4 by a natural agonist (5',6'-EET) in which K535 plays a crucial role, as mutant TRPV4-K535A losses binding of and gating by EET, without affecting GSK1016790A, 4α-phorbol 12,13-didecanoate and heat mediated channel activation. Together, our data demonstrates that the mechano- and osmotransducing messenger EET gates TRPV4 by a direct action on a site formed by residues from the S2-S3 linker, S4 and S4-S5 linker.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Canales Catiónicos TRPV/química , Ácido 8,11,14-Eicosatrienoico/química , Ácido 8,11,14-Eicosatrienoico/farmacología , Sustitución de Aminoácidos , Sitios de Unión , Células HEK293 , Células HeLa , Humanos , Activación del Canal Iónico , Simulación del Acoplamiento Molecular , Unión Proteica , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.
Asunto(s)
Asma/genética , Cromosomas Humanos Par 17/química , Proteínas del Huevo/inmunología , Activación de Linfocitos/efectos de los fármacos , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/inmunología , Fitohemaglutininas/farmacología , Alelos , Asma/inmunología , Asma/patología , Calcio/inmunología , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Cromosomas Humanos Par 17/inmunología , Proteínas del Huevo/genética , Expresión Génica , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Pulmón/inmunología , Pulmón/patología , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Cultivo Primario de Células , Riesgo , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/patología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/patologíaRESUMEN
As an organism is exposed to pathogens during very early development, specific defense mechanisms must take effect. In this study, we used a germ-free zebrafish embryo model to show that osmotic stress regulates the activation of immunity and host protection in newly hatched embryos. Mechanistically, skin keratinocytes were responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms. This occurred through a transient potential receptor vanilloid 4/Ca(2+)/TGF-ß-activated kinase 1/NF-κB signaling pathway. Surprisingly, the genes encoding antimicrobial effectors, which do not have the potential to cause tissue damage, are constitutively expressed during development, independently of both commensal microbes and osmotic stress. Our results reveal that osmotic stress is associated with the induction of developmental immunity in the absence of tissue damage and point out to the embryo skin as the first organ with full capacities to mount an innate immune response.
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Inmunidad Innata/inmunología , Queratinocitos/inmunología , Piel/embriología , Canales Catiónicos TRPV/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/embriología , Pez Cebra/inmunología , Animales , Embrión no Mamífero/inmunología , Técnica del Anticuerpo Fluorescente , Presión Osmótica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/inmunología , Transcriptoma , TransfecciónRESUMEN
Functional transient receptor potential (TRP) channels result from the assembly of four subunits. Here, we show an interaction between the pre-S1, TRP, and the ankyrin repeat domain (ARD)-S1 linker domains of TRPV1 and TRPV4 that is essential for proper channel assembly. Neutralization of TRPV4 pre-S1 K462 resulted in protein retention in the ER, defective glycosylation and trafficking, and unresponsiveness to TRPV4-activating stimuli. Similar results were obtained with the equivalent mutation in TRPV1 pre-S1. Molecular dynamics simulations revealed that TRPV4-K462 generated an alternating hydrogen network with E745 (TRP box) and D425 (pre-S1 linker), and that K462Q mutation affected subunit folding. Consistently, single TRPV4-E745A or TRPV4-D425A mutations moderately affected TRPV4 biogenesis while double TRPV4-D425A/E745A mutation resumed the TRPV4-K462Q phenotype. Thus, the interaction between pre-S1, TRP, and linker domains is mandatory to generate a structural conformation that allows the contacts between adjacent subunits to promote correct assembly and trafficking to the plasma membrane.
Asunto(s)
Subunidades de Proteína/química , Canales Catiónicos TRPV/química , Secuencia de Aminoácidos , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Enlace de Hidrógeno , Potenciales de la Membrana/fisiología , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Alineación de Secuencia , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.
Asunto(s)
Ceramidas/metabolismo , Orosomucoide/metabolismo , Serina C-Palmitoiltransferasa/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Orosomucoide/genética , Palmitatos/metabolismo , Isoformas de Proteínas/metabolismo , Serina C-Palmitoiltransferasa/genética , Esfingolípidos/metabolismoRESUMEN
Alzheimer's disease (AD) is characterized by the oxidative stress generated from amyloid ß-peptide (Aß) aggregates. It produces protein nitrotyrosination, after the reaction with nitric oxide to form peroxynitrite, being triosephosphate isomerase (TPI) one of the most affected proteins. TPI is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Methylglyoxal (MG) is a by-product of TPI activity whose production is triggered when TPI is nitrotyrosinated. MG is harmful to cells because it glycates proteins. Here we found protein glycation when human neuroblastoma cells were treated with Aß. Moreover glycation was also observed when neuroblastoma cells overexpressed mutated TPI where Tyr165 or Tyr209, the two tyrosines close to the catalytic center, were changed by Phe in order to mimic the effect of nitrotyrosination. The pathological relevance of these findings was studied by challenging cells with Aß oligomers and MG. A significant decrease in mitochondrial transmembrane potential, one of the first apoptotic events, was obtained. Therefore, increasing concentrations of MG were assayed searching for MG effect in neuronal apoptosis. We found a decrease of the protective Bcl2 and an increase of the proapoptotic caspase-3 and Bax levels. Our results suggest that MG is triggering apoptosis in neurons and it would play a key role in AD neurodegeneration.
Asunto(s)
Caspasa 3/metabolismo , Potencial de la Membrana Mitocondrial , Neuronas/metabolismo , Piruvaldehído/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Glicosilación , Humanos , Mutación , Neuronas/efectos de los fármacos , Neuronas/patología , Fragmentos de Péptidos/toxicidad , Piruvaldehído/farmacología , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Amyloid-ß peptide (Aß) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aß42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aß42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aß action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aß oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Apoptosis/fisiología , Neuronas/fisiología , Fragmentos de Péptidos/metabolismo , Piruvaldehído/metabolismo , Triosa-Fosfato Isomerasa/metabolismo , Anciano , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Apoptosis/genética , Encéfalo/fisiopatología , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Simulación por Computador , Femenino , Humanos , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Modelos Moleculares , Presenilina-1/genética , Triosa-Fosfato Isomerasa/genéticaRESUMEN
Calcium signaling in the brain is fundamental to the learning and memory process and there is evidence to suggest that its dysfunction is involved in the pathological pathways underlying Alzheimer's disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca(2+)-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca(2+) permeability and is associated with an earlier age-at-onset of AD. To investigate the role of CALHM1 variants in early-onset AD (EOAD), we sequenced all CALHM1 coding regions in three independent series comprising 284 EOAD patients and 326 controls. Two missense mutations in patients (p.G330D and p.R154H) and one (p.A213T) in a control individual were identified. Calcium imaging analyses revealed that while the mutation found in a control (p.A213T) behaved as wild-type CALHM1 (CALHM1-WT), a complete abolishment of the Ca(2+) influx was associated with the mutations found in EOAD patients (p.G330D and p.R154H). Notably, the previously reported p. P86L mutation was associated with an intermediate Ca(2+) influx between the CALHM1-WT and the p.G330D and p.R154H mutations. Since neither expression of wild-type nor mutant CALHM1 affected amyloid ß-peptide (Aß) production or Aß-mediated cellular toxicity, we conclude that rare genetic variants in CALHM1 lead to Ca(2+) dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Calcio/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Polimorfismo Genético , Adulto , Edad de Inicio , Anciano , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Señalización del Calcio , Estudios de Casos y Controles , Análisis Mutacional de ADN , Femenino , Homeostasis/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Alineación de SecuenciaRESUMEN
Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP2), although the structural rearrangements occurring on PIP2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence (121)KRWRK(125), which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP2 facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5'-6'-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP2-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP2 mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4-PIP2 interacting site and conditions that deplete PIP2 or restrict access of TRPV4 to PIP2--in the case of PACSIN3--change tail conformation and negatively affect channel activation by hypotonicity and heat.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales Catiónicos TRPV/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Análisis de Varianza , Calcio/metabolismo , Células Cultivadas , Clonación Molecular , Citoplasma/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Técnicas de Placa-Clamp , Forboles/metabolismo , Estructura Terciaria de ProteínaRESUMEN
T lymphocytes rely on a Ca(2+) signal known as store-operated calcium entry (SOCE) for their activation. This Ca(2+) signal is generated by activation of a T-cell receptor, depletion of endoplasmic reticulum (ER) Ca(2+) stores and activation of Ca(2+) release-activated Ca(2+) currents (I(CRAC)). Here, we report that the ER protein orosomucoid like 3 (ORMDL3), the product of the ORMDL3 gene associated with several autoimmune and/or inflammatory diseases, negatively modulates I(CRAC), SOCE, nuclear factor of activated T cells nuclear translocation and interleukin-2 production. ORMDL3 inhibits the Ca(2+) influx mechanism at the outer mitochondrial membrane, resulting in a Ca(2+)-dependent inhibition of I(CRAC) and reduced SOCE. The effect of ORMDL3 could be mimicked by interventions that decreased mitochondrial Ca(2+) influx and reverted by buffering of cytosolic Ca(2+) or activation of mitochondrial Ca(2+) influx. In conclusion, ORMDL3 modifies key steps in the process of T-lymphocyte activation, providing a functional link between the genetic associations of the ORMDL3 gene with autoimmune and/or inflammatory diseases.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Interleucina-2/metabolismo , Transporte Iónico , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Orosomucoide/metabolismo , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Alterations of protein folding or Ca(2+) levels within the endoplasmic reticulum (ER) result in the unfolded-protein response (UPR), a process considered as an endogenous inducer of inflammation. Thereby, understanding how genetic factors modify UPR is particularly relevant in chronic inflammatory diseases such as asthma. Here we identified that ORMDL3, the only genetic risk factor recently associated to asthma in a genome wide study, alters ER-mediated Ca(2+) homeostasis and facilitates the UPR. Heterologous expression of human ER-resident transmembrane ORMDL3 protein increased resting cytosolic Ca(2+) levels and reduced ER-mediated Ca(2+) signaling, an effect reverted by co-expression with the sarco-endoplasmic reticulum Ca(2+) pump (SERCA). Increased ORMDL3 expression also promoted stronger activation of UPR transducing molecules and target genes while siRNA-mediated knock-down of endogenous ORMDL3 potentiated ER Ca(2+) release and attenuated the UPR. In conclusion, our findings are consistent with a model in which ORMDL3 binds and inhibits SERCA resulting in a reduced ER Ca(2+) concentration and increased UPR. Thus, we provide a first insight into the molecular mechanism explaining the association of ORMDL3 with proinflammatory diseases.
Asunto(s)
Asma/genética , Asma/patología , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Proteínas de la Membrana/genética , Estrés Fisiológico , Asma/metabolismo , Silenciador del Gen , Homeostasis , Humanos , Células Jurkat , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Unión Proteica , Transporte de Proteínas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Integrative genomic and gene-expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene-expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated 20 homozygous deletions at 7 chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms. Notably, the pattern of genetic and epigenetic inactivation differed among B-NHL subtypes. Thus, the P53-inducible PIG7/LITAF was silenced by homozygous deletion in primary mediastinal B-cell lymphoma and by promoter hypermethylation in germinal center lymphoma, the proapoptotic BIM gene presented homozygous deletion in mantle cell lymphoma and promoter hypermethylation in Burkitt lymphoma, the proapoptotic BH3-only NOXA was mutated and preferentially silenced in diffuse large B-cell lymphoma, and INK4c/P18 was silenced by biallelic mutation in mantle-cell lymphoma. Our microarray strategy has identified novel candidate tumor suppressor genes inactivated by genetic and epigenetic mechanisms that substantially vary among the B-NHL subtypes.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas Portadoras/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Genes Supresores de Tumor , Proteínas de Homeodominio/genética , Linfoma de Células B/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas/genética , Eliminación de Secuencia , Factores de Transcripción/genética , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/genética , Proteína 11 Similar a Bcl2 , Biopsia , Línea Celular Tumoral , Mapeo Cromosómico , Cromosomas Humanos/genética , Cromosomas Humanos/ultraestructura , Metilación de ADN , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Epigénesis Genética , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Homocigoto , Humanos , Linfoma de Células B/clasificación , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Mutación Puntual , Regiones Promotoras Genéticas/genética , Proteínas de Unión al ARN , Nexinas de ClasificaciónRESUMEN
Deletions of chromosome 8p are a recurrent event in B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of a tumor suppressor gene. We have characterized these deletions using comparative genomic hybridization to microarrays, fluorescence in situ hybridization (FISH) mapping, DNA sequencing, and functional studies. A minimal deleted region (MDR) of 600 kb was defined in chromosome 8p21.3, with one mantle cell lymphoma cell line (Z138) exhibiting monoallelic deletion of 650 kb. The MDR extended from bacterial artificial chromosome (BAC) clones RP11-382J24 and RP11-109B10 and included the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene loci. Sequence analysis of the individual expressed genes within the MDR and DNA sequencing of the entire MDR in Z138 did not reveal any mutation. Gene expression analysis and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) showed down-regulation of TRAIL-R1 and TRAIL-R2 receptor genes as a consistent event in B-NHL with 8p21.3 loss. Epigenetic inactivation was excluded via promoter methylation analysis. In vitro studies showed that TRAIL-induced apoptosis was dependent on TRAIL-R1 and/or -R2 dosage in most tumors. Resistance to apoptosis of cell lines with 8p21.3 deletion was reversed by restoration of TRAIL-R1 or TRAIL-R2 expression by gene transfection. Our data suggest that TRAIL-R1 and TRAIL-R2 act as dosage-dependent tumor suppressor genes whose monoallelic deletion can impair TRAIL-induced apoptosis in B-cell lymphoma.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 8/genética , Genes Supresores de Tumor/fisiología , Linfoma de Células B/genética , Receptores del Factor de Necrosis Tumoral/genética , Proteínas Supresoras de Tumor/genética , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/metabolismo , Mutación/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
To identify recurrent genomic changes in mantle cell lymphoma (MCL), we used high-resolution comparative genomic hybridization (CGH) to bacterial artificial chromosome (BAC) microarrays in 68 patients and 9 MCL-derived cell lines. Array CGH defined an MCL genomic signature distinct from other B-cell lymphomas, including deletions of 1p21 and 11q22.3-ATM gene with coincident 10p12-BMI1 gene amplification and 10p14 deletion, along with a previously unidentified loss within 9q21-q22. Specific genomic alterations were associated with different subgroups of disease. Notably, 11 patients with leukemic MCL showed a different genomic profile than nodal cases, including 8p21.3 deletion at tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene cluster (55% versus 19%; P = .01) and gain of 8q24.1 at MYC locus (46% versus 14%; P = .015). Additionally, leukemic MCL exhibited frequent IGVH mutation (64% versus 21%; P = .009) with preferential VH4-39 use (36% versus 4%; P = .005) and followed a more indolent clinical course. Blastoid variants, increased number of genomic gains, and deletions of P16/INK4a and TP53 genes correlated with poorer outcomes, while 1p21 loss was associated with prolonged survival (P = .02). In multivariate analysis, deletion of 9q21-q22 was the strongest predictor for inferior survival (hazard ratio [HR], 6; confidence interval [CI], 2.3 to 15.7). Our study highlights the genomic profile as a predictor for clinical outcome and suggests that "genome scanning" of chromosomes 1p21, 9q21-q22, 9p21.3-P16/INK4a, and 17p13.1-TP53 may be clinically useful in MCL.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Leucemia/genética , Linfoma de Células del Manto/genética , Anciano , Anciano de 80 o más Años , Cromosomas Artificiales Bacterianos , Femenino , Genómica/métodos , Genotipo , Humanos , Leucemia/mortalidad , Leucemia/patología , Linfoma de Células del Manto/mortalidad , Linfoma de Células del Manto/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Eliminación de Secuencia , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Genomic aberrations in a series of paired biopsy samples from patients who presented initially with follicle center lymphoma (FCL) and subsequently transformed to diffuse large B-cell lymphoma (DLBCL) were measured by array comparative genomic hybridization (CGH). The consequences of these aberrations on gene expression were determined by comparison with expression analysis on these specimens using cDNA microarrays. A heterogeneous pattern of acquired genomic abnormalities was observed upon transformation, some of which were recurrent in small subsets of patients. Some of the genomic aberration acquired upon transformation, such as gain/amplification of 1q21-q24, 2p16 (REL/BCL11A gene loci), 3q27-q29 (including the BCL6 locus), 7q11.2-q22.1, 12pter-q12, 18q21 (including the BCL2 locus) and Xq, and deletion of 6q22-q24, 13q14-q21 and 17p13 (P53 locus) have been previously implicated in the FCL/DLBCL pathogenesis. In addition, novel genomic imbalances not previously reported in association with FCL transformation, such as overrepresentation of 4p12-pter, 5p12-p15, 6p12.3-p21, 9p23, 9q13-q31, 16q, 17q21, and loss of 1p36.3, 4q21-q23, 5q21-q23, 9q31-qter, 11q24-q25, and 15q23, were identified. We observed a differential expression profile of many genes within regions of gain and deletion upon transformation, including novel target genes associated with FCL transformation. However, other genes did not show deregulated expression despite their location within these areas. In summary, the combination of array CGH and expression analysis provides a more comprehensive picture of the transformation of FCL to DLBCL. This process is associated with the acquisition of a variable spectrum of genomic imbalances affecting recurrent chromosomal areas that harbor overexpressed or underexpressed genes targeted upon transformation.