RESUMEN

Striking morphological transformations characterize the invasion of a red blood cell by the malaria parasite. Shortly after the infection, parasite-induced membranes appear in the cytosol of the affected host erythrocyte. One intensely investigated membrane type, commonly called Maurer's clefts, has a slit-like morphology and can be arranged in the form of extended three-dimensional membrane stacks or networks. Here we report the three-dimensional reconstruction of a second membrane type, giant or extended membrane rings/loops, that have only occasionally been described on single ultrathin sections, however that have never been systematically examined so far. Serial ultrathin sectioning of P. falciparum-infected red blood cells, subsequent three-dimensional reconstructions, and in addition examination of Giemsa-stained blood films revealed that intraerythrocytic membrane rings/loops are not isolated structures but are locally in contact with the parasite. They consist either of the parasitophorous vacuolar membrane alone or contain the parasitophorous vacuolar membrane including the plasma membrane of the parasite and small amounts of parasite cytoplasm. We demonstrate that membrane rings/loops represent surface extensions of the parasite that maybe involved in ring stage parasite formation and Maurer's cleft generation at least in a subset of infected red blood cells.

Asunto(s)

Citosol , Eritrocitos , Plasmodium falciparum , Eritrocitos/parasitología , Plasmodium falciparum/fisiología , Citosol/parasitología , Citosol/química , Humanos , Membrana Eritrocítica/parasitología , Membrana Eritrocítica/ultraestructura , Malaria Falciparum/parasitología , Imagenología Tridimensional , Membrana Celular/parasitología

RESUMEN

Neuroligin-2 (Nlgn2) is a key synaptic adhesion protein at virtually all GABAergic synapses, which recruits GABAARs by promoting assembly of the postsynaptic gephyrin scaffold. Intriguingly, loss of Nlgn2 differentially affects subsets of GABAergic synapses, indicating that synapse-specific interactors and redundancies define its function, but the nature of these interactions remain poorly understood. Here we investigated how Nlgn2 function in hippocampal area CA1 is modulated by two proposed interaction partners, MDGA1 and MDGA2. We show that loss of MDGA1 expression, but not heterozygous deletion of MDGA2, ameliorates the abnormal cytosolic gephyrin aggregation, the reduction in inhibitory synaptic transmission and the exacerbated anxiety-related behaviour characterizing Nlgn2 knockout (KO) mice. Additionally, combined Nlgn2 and MDGA1 deletion causes an exacerbated layer-specific loss of gephyrin puncta. Given that both Nlgn2 and the MDGA1 have been correlated with many psychiatric disorders, our data support the notion that cytosolic gephyrin aggregation may represent an interesting target for novel therapeutic strategies.

Asunto(s)

Proteínas Portadoras , Moléculas de Adhesión Celular Neuronal , Proteínas de la Membrana , Ratones Noqueados , Receptores de GABA-A , Sinapsis , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Sinapsis/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Citosol/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Transmisión Sináptica , Ratones Endogámicos C57BL , Región CA1 Hipocampal/metabolismo

RESUMEN

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.

Asunto(s)

Señalización del Calcio , Calcio , Fosfolipasa C beta , Receptores Acoplados a Proteínas G , Humanos , Fosfolipasa C beta/metabolismo , Fosfolipasa C beta/genética , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Sistemas CRISPR-Cas , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , AMP Cíclico/metabolismo , Animales , Edición Génica , Citosol/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética

RESUMEN

GRK2 and arrestin3, key players in the functional regulation of G protein-coupled receptors (GPCRs), are ubiquitinated by Mdm2, a nuclear protein. The agonist-induced increase in arrestin3 ubiquitination occurs in the nucleus, underscoring the crucial role of its nuclear translocation in this process. The ubiquitination of arrestin3 occurs in the nucleus, highlighting the pivotal role of its nuclear translocation in this process. In contrast, GRK2 cannot translocate into the nucleus; thus, facilitation of the cytosolic translocation of nuclear Mdm2 is required to ubiquitinate GRK2 in the cytosol. Among the explored cellular components and processes, arrestin, Gßγ, clathrin, and receptor phosphorylation were found to be required for the nuclear import of arrestin3, the ubiquitination of arrestin3 in the nucleus, nuclear export of Mdm2, and the ubiquitination of GRK2 in the cytosol. In conclusion, our findings demonstrate that agonist-induced ubiquitination of arrestin3 in the nucleus is interconnected with cytosolic GRK2 ubiquitination.

Asunto(s)

Transporte Activo de Núcleo Celular , Núcleo Celular , Citosol , Quinasa 2 del Receptor Acoplado a Proteína-G , Proteínas Proto-Oncogénicas c-mdm2 , Ubiquitinación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Humanos , Citosol/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Núcleo Celular/metabolismo , Fosforilación , Arrestinas/metabolismo , Células HEK293 , Animales

RESUMEN

Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

Asunto(s)

Gutatión-S-Transferasa pi , Lipoilación , Palmitatos , Humanos , Gutatión-S-Transferasa pi/metabolismo , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/química , Células MCF-7 , Palmitatos/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Cisteína/metabolismo , Procesamiento Proteico-Postraduccional , Ácido Palmítico/metabolismo

RESUMEN

Endoplasmic reticulum quality control is crucial for maintaining cellular homeostasis and adapting to stress conditions. Although several ER-phagy receptors have been identified, the collaboration between cytosolic and ER-resident factors in ER fragmentation and ER-phagy regulation remains unclear. Here, we perform a phenotype-based gain-of-function screen and identify a cytosolic protein, FKBPL, functioning as an ER-phagy regulator. Overexpression of FKBPL triggers ER fragmentation and ER-phagy. FKBPL has multiple protein binding domains, can self-associate and might act as a scaffold connecting CKAP4 and LC3/GABARAPs. CKAP4 serves as a bridge between FKBPL and ER-phagy cargo. ER-phagy-inducing conditions increase FKBPL-CKAP4 interaction followed by FKBPL oligomerization at the ER, leading to ER-phagy. In addition, FKBPL-CKAP4 deficiency leads to Golgi disassembly and lysosome impairment, and an increase in ER-derived secretory vesicles and enhances cytosolic protein secretion via microvesicle shedding. Taken together, FKBPL with the aid of CKAP4 induces ER fragmentation and ER-phagy, and FKBPL-CKAP4 deficiency facilitates protein secretion.

Asunto(s)

Citosol , Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Humanos , Citosol/metabolismo , Animales , Células HEK293 , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Ratones , Células HeLa , Unión Proteica , Estrés del Retículo Endoplásmico

RESUMEN

The escape of mitochondrial double-stranded dsRNA (mt-dsRNA) into the cytosol has been recently linked to a number of inflammatory diseases. Here, we report that the release of mt-dsRNA into the cytosol is a general feature of senescent cells and a critical driver of their inflammatory secretome, known as senescence-associated secretory phenotype (SASP). Inhibition of the mitochondrial RNA polymerase, the dsRNA sensors RIGI and MDA5, or the master inflammatory signaling protein MAVS, all result in reduced expression of the SASP, while broadly preserving other hallmarks of senescence. Moreover, senescent cells are hypersensitized to mt-dsRNA-driven inflammation due to their reduced levels of PNPT1 and ADAR1, two proteins critical for mitigating the accumulation of mt-dsRNA and the inflammatory potency of dsRNA, respectively. We find that mitofusin MFN1, but not MFN2, is important for the activation of the mt-dsRNA/MAVS/SASP axis and, accordingly, genetic or pharmacologic MFN1 inhibition attenuates the SASP. Finally, we report that senescent cells within fibrotic and aged tissues present dsRNA foci, and inhibition of mitochondrial RNA polymerase reduces systemic inflammation associated to senescence. In conclusion, we uncover the mt-dsRNA/MAVS/MFN1 axis as a key driver of the SASP and we identify novel therapeutic strategies for senescence-associated diseases.

Asunto(s)

Senescencia Celular , Citosol , Inflamación , Mitocondrias , ARN Bicatenario , ARN Bicatenario/metabolismo , Humanos , Citosol/metabolismo , Mitocondrias/metabolismo , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Animales , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Fenotipo Secretor Asociado a la Senescencia , Helicasa Inducida por Interferón IFIH1/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Transducción de Señal

RESUMEN

Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.

Asunto(s)

Retículo Endoplásmico , Neuronas , Transporte de Proteínas , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Animales , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/toxicidad , Toxinas Botulínicas Tipo A/genética , Ratas , Aparato de Golgi/metabolismo , Línea Celular , Citosol/metabolismo

RESUMEN

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.

Asunto(s)

Citosol , Estrés del Retículo Endoplásmico , Interferón beta , Proteínas de la Membrana , Nucleotidiltransferasas , Respuesta de Proteína Desplegada , Humanos , Animales , Ratones , Nucleotidiltransferasas/metabolismo , Citosol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Interferón beta/metabolismo , ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismo , Endorribonucleasas/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Tapsigargina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional , ADN Mitocondrial/metabolismo

RESUMEN

Mitochondrial membranes define distinct structural and functional compartments. Cristae of the inner mitochondrial membrane (IMM) function as independent bioenergetic units that undergo rapid and transient remodelling, but the significance of this compartmentalized organization is unknown1. Using super-resolution microscopy, here we show that cytosolic IMM vesicles, devoid of outer mitochondrial membrane or mitochondrial matrix, are formed during resting state. These vesicles derived from the IMM (VDIMs) are formed by IMM herniation through pores formed by voltage-dependent anion channel 1 in the outer mitochondrial membrane. Live-cell imaging showed that lysosomes in proximity to mitochondria engulfed the herniating IMM and, aided by the endosomal sorting complex required for transport machinery, led to the formation of VDIMs in a microautophagy-like process, sparing the remainder of the organelle. VDIM formation was enhanced in mitochondria undergoing oxidative stress, suggesting their potential role in maintenance of mitochondrial function. Furthermore, the formation of VDIMs required calcium release by the reactive oxygen species-activated, lysosomal calcium channel, transient receptor potential mucolipin 1, showing an interorganelle communication pathway for maintenance of mitochondrial homeostasis. Thus, IMM compartmentalization could allow for the selective removal of damaged IMM sections via VDIMs, which should protect mitochondria from localized injury. Our findings show a new pathway of intramitochondrial quality control.

Asunto(s)

Calcio , Lisosomas , Mitocondrias , Membranas Mitocondriales , Estrés Oxidativo , Lisosomas/metabolismo , Humanos , Membranas Mitocondriales/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Animales , Canales de Potencial de Receptor Transitorio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratones , Autofagia , Homeostasis , Citosol/metabolismo

RESUMEN

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Asunto(s)

Linfocitos T CD8-positivos , Citosol , Activación de Linfocitos , Mitocondrias , Biosíntesis de Proteínas , Receptores de Antígenos de Linfocitos T , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Activación de Linfocitos/inmunología , Citosol/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Mitocondrias/metabolismo , Mitocondrias/inmunología , Citoesqueleto/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Ribosomas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

In vitro reconstitution studies enable the controllable and stepwise investigation of complicated biochemical processes. In yeast and mammals, in vitro reconstitution of COPII vesicles marked a pivotal point in characterizing the endoplasmic reticulum-to-Golgi anterograde trafficking route and revealed how vesicles mediate the selective and reliable transportation among topologically equivalent compartments. By providing the necessary physiological conditions in a cell-free environment, it enables the dissection of essential components required for the vesicle formation. To enrich and purify the small amount in vivo membrane-bounded compartments, it simplifies the evaluation of vesicle regulation by distinct external stimuli or upstream signals. Here, we describe the preparation of plant microsomes and cytosol for the reconstitution of plant COPII vesicles. Purified vesicles can be used for further biochemical or microscopical analyses.

Asunto(s)

Vesículas Cubiertas por Proteínas de Revestimiento , Microsomas , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Microsomas/metabolismo , Retículo Endoplásmico/metabolismo , Citosol/metabolismo , Aparato de Golgi/metabolismo , Plantas/metabolismo

RESUMEN

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Asunto(s)

Chaperonina 10 , Chaperonina 60 , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteínas de Escherichia coli , Escherichia coli , Sitios de Unión , Chaperonina 10/metabolismo , Chaperonina 10/química , Chaperonina 10/ultraestructura , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/ultraestructura , Citosol/química , Citosol/metabolismo , Citosol/ultraestructura , Escherichia coli/química , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Reproducibilidad de los Resultados , Especificidad por Sustrato , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura

RESUMEN

Production of the amyloidogenic prion protein, PrPSc, which forms infectious protein aggregates, or prions, is a key pathogenic event in prion diseases. Functional prion-like protein aggregations, such as the mitochondrial adaptor protein MAVS and the inflammasome component protein ASC, have been identified to play a protective role in viral infections in mammalian cells. In this study, to investigate if PrPSc could play a functional role against external stimuli, we infected prion-infected cells with a neurotropic influenza A virus strain, IAV/WSN. We found that prion-infected cells were highly resistant to IAV/WSN infection. In these cells, NF-κB nuclear translocation was disturbed; therefore, mitochondrial superoxide dismutase (mtSOD) expression was suppressed, and mitochondrial reactive oxygen species (mtROS) was increased. The elevated mtROS subsequently activated NLRP3 inflammasomes, leading to the suppression of IAV/WSN-induced necroptosis. We also found that prion-infected cells accumulated a portion of PrP molecules in the cytosol, and that the N-terminal potential nuclear translocation signal of PrP impeded NF-κB nuclear translocation. These results suggest that PrPSc might play a functional role in protection against viral infections by stimulating the NLRP3 inflammasome-dependent antivirus mechanism through the cytosolic PrP-mediated disturbance of NF-κB nuclear translocation, which leads to suppression of mtSOD expression and consequently upregulation of the NLRP3 inflammasome activator mtROS. IMPORTANCE: Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections.

Asunto(s)

Citosol , Inflamasomas , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno , Animales , Citosol/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratones , Humanos , Mitocondrias/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Línea Celular , Superóxido Dismutasa/metabolismo , Priones/metabolismo , Proteínas Priónicas/metabolismo , Necroptosis

RESUMEN

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture. IMPORTANCE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.

Asunto(s)

Proteínas Bacterianas , Citosol , Interferón Tipo I , Macrófagos , Mycobacterium marinum , Serpinas , Animales , Femenino , Ratones , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Muerte Celular , Citosol/microbiología , Citosol/metabolismo , Retroalimentación Fisiológica , Interacciones Huésped-Patógeno , Interferón Tipo I/metabolismo , Macrófagos/microbiología , Ratones Endogámicos C57BL , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/patogenicidad , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Serpinas/metabolismo , Serpinas/genética , Transducción de Señal , Sistemas de Secreción Tipo VII/metabolismo , Sistemas de Secreción Tipo VII/genética

RESUMEN

Crassulacean acid metabolism (CAM) leaves are characterized by nocturnal acidification and diurnal deacidification processes related with the timed actions of phosphoenolpyruvate carboxylase and Rubisco, respectively. How CAM leaves manage cytosolic proton homeostasis, particularly when facing massive diurnal proton effluxes from the vacuole, remains unclear. A 12-phase flux balance analysis (FBA) model was constructed for a mature malic enzyme-type CAM mesophyll cell in order to predict diel kinetics of intracellular proton fluxes. The charge- and proton-balanced FBA model identified the mitochondrial phosphate carrier (PiC, Pi/H+ symport), which provides Pi to the matrix to sustain ATP biosynthesis, as a major consumer of cytosolic protons during daytime (> 50%). The delivery of Pi to the mitochondrion, co-transported with protons, is required for oxidative phosphorylation and allows sufficient ATP to be synthesized to meet the high energy demand during CAM Phase III. Additionally, the model predicts that mitochondrial pyruvate originating from decarboxylation of malate is exclusively exported to the cytosol, probably via a pyruvate channel mechanism, to fuel gluconeogenesis. In this biochemical cycle, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acts as another important cytosolic proton consumer. Overall, our findings emphasize the importance of mitochondria in CAM and uncover a hitherto unappreciated role in metabolic proton homeostasis.

Asunto(s)

Metabolismo Ácido de las Crasuláceas , Homeostasis , Mitocondrias , Modelos Biológicos , Fosfatos , Hojas de la Planta , Protones , Ácido Pirúvico , Hojas de la Planta/metabolismo , Mitocondrias/metabolismo , Ácido Pirúvico/metabolismo , Fosfatos/metabolismo , Ritmo Circadiano , Citosol/metabolismo , Malatos/metabolismo , Adenosina Trifosfato/metabolismo

RESUMEN

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

Asunto(s)

ATP Citrato (pro-S)-Liasa , Acetatos , Acetilcoenzima A , Linfocitos T CD8-positivos , Cromatina , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Animales , Cromatina/metabolismo , Acetilcoenzima A/metabolismo , ATP Citrato (pro-S)-Liasa/metabolismo , ATP Citrato (pro-S)-Liasa/genética , Ratones , Acetatos/metabolismo , Acetato CoA Ligasa/metabolismo , Acetato CoA Ligasa/genética , Acetilación , Ratones Noqueados , Citosol/metabolismo , Histonas/metabolismo

RESUMEN

Mitochondrial functions can be regulated by membrane contact sites with the endoplasmic reticulum (ER). These mitochondria-ER contact sites (MERCs) are functionally heterogeneous and maintained by various tethers. Here, we found that REEP5, an ER tubule-shaping protein, interacts with Mitofusins 1/2 to mediate mitochondrial distribution throughout the cytosol by a new transport mechanism, mitochondrial "hitchhiking" with tubular ER on microtubules. REEP5 depletion led to reduced tethering and increased perinuclear localization of mitochondria. Conversely, increasing REEP5 expression facilitated mitochondrial distribution throughout the cytoplasm. Rapamycin-induced irreversible REEP5-MFN1/2 interaction led to mitochondrial hyperfusion, implying that the dynamic release of mitochondria from tethering is necessary for normal mitochondrial distribution and dynamics. Functionally, disruption of MFN2-REEP5 interaction dynamics by forced dimerization or silencing REEP5 modulated the production of mitochondrial reactive oxygen species (ROS). Overall, our results indicate that dynamic REEP5-MFN1/2 interaction mediates cytosolic distribution and connectivity of the mitochondrial network by "hitchhiking" and this process regulates mitochondrial ROS, which is vital for multiple physiological functions.

Asunto(s)

Retículo Endoplásmico , GTP Fosfohidrolasas , Mitocondrias , Especies Reactivas de Oxígeno , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Humanos , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Especies Reactivas de Oxígeno/metabolismo , Células HeLa , Microtúbulos/metabolismo , Células HEK293 , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Unión Proteica , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Citosol/metabolismo , Dinámicas Mitocondriales

RESUMEN

Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here, we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains.

Asunto(s)

Citosol , Mitocondrias , Membranas Mitocondriales , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Citosol/metabolismo , Membranas Mitocondriales/metabolismo , Humanos , Tomografía con Microscopio Electrónico , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Células HeLa , Animales

RESUMEN

Mitochondrial RNA (mtRNA) in the cytosol can trigger the innate immune sensor MDA5, and autoinflammatory disease due to type I IFN. Here, we show that a dominant negative mutation in the gene encoding the mitochondrial exonuclease REXO2 may cause interferonopathy by triggering the MDA5 pathway. A patient characterized by this heterozygous de novo mutation (p.T132A) presented with persistent skin rash featuring hyperkeratosis, parakeratosis and acanthosis, with infiltration of lymphocytes and eosinophils around small blood vessels. In addition, circulating IgE levels and inflammatory cytokines, including IFNα, are found consistently elevated. Transcriptional analysis highlights a type I IFN gene signature in PBMC. Mechanistically, REXO2 (T132A) lacks the ability to cleave RNA and inhibits the activity of wild-type REXO2. This leads to an accumulation of mitochondrial dsRNA in the cytosol, which is recognized by MDA5, leading to the associated type I IFN gene signature. These results demonstrate that in the absence of appropriate regulation by REXO2, aberrant cellular nucleic acids may accumulate and continuously trigger innate sensors, resulting in an inborn error of immunity.

Asunto(s)

Heterocigoto , Interferón Tipo I , Helicasa Inducida por Interferón IFIH1 , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferón Tipo I/metabolismo , Interferón Tipo I/genética , Mutación , Masculino , Mitocondrias/metabolismo , Mitocondrias/genética , Femenino , Inmunidad Innata/genética , Exonucleasas/metabolismo , Exonucleasas/genética , Células HEK293 , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Citosol/metabolismo , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Genes Dominantes