RESUMEN
Rab GTPase is critical for autophagy processes and is implicated in insect immunity against viruses. In this study, we aimed to investigate the role of FoRabs in the autophagic regulation of antiviral defense against tomato spotted wilt orthotospovirus (TSWV) in Frankliniella occidentalis. Transcriptome analysis revealed the downregulation of FoRabs in viruliferous nymph and adults of F. occidentalis in response to TSWV infection. Manipulation of autophagy levels with 3-MA and Rapa treatments resulted in a 5- to 15-fold increase and a 38-64% decrease in viral titers, respectively. Additionally, interference with FoRab10 in nymphs and FoRab29 in adults led to a 20-90% downregulation of autophagy-related genes, a decrease in ATG8-II (an autophagy marker protein), and an increase in the TSWV titers by 1.5- to 2.5-fold and 1.3- to 2.0-fold, respectively. In addition, the leaf disk and the living plant methods revealed increased transmission rates of 20.8-41.6 and 68.3-88.3%, respectively. In conclusion, FoRab10 and FoRab29 play a role in the autophagic regulation of the antiviral defense in F. occidentalis nymphs and adults against TSWV, respectively. These findings offer insights into the intricate immune mechanisms functional in F. occidentalis against TSWV, suggesting potential targeted strategies for F. occidentalis and TSWV management.
Asunto(s)
Autofagia , Resistencia a la Enfermedad , Proteínas de Insectos , Enfermedades de las Plantas , Thysanoptera , Tospovirus , Animales , Tospovirus/fisiología , Tospovirus/inmunología , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/genética , Thysanoptera/virología , Thysanoptera/inmunología , Thysanoptera/genética , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Proteínas de Insectos/metabolismo , Solanum lycopersicum/virología , Solanum lycopersicum/inmunología , Solanum lycopersicum/genética , Ninfa/inmunología , Ninfa/crecimiento & desarrollo , Ninfa/virología , Ninfa/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Background: Chitinase 3-like-1 (CHI3L1) is a secretion glycoprotein associated with the immunosuppressive tumor microenvironment (TME). The secretory mode of CHI3L1 makes it a promising target for cancer treatment. We have previously reported that Rab37 small GTPase mediates secretion of IL-6 in macrophages to promote cancer progression, whereas the roles of Rab37 in the intracellular trafficking and exocytosis of CHI3L1 are unclear. Methods: We examined the concentration of CHI3L1 in the culture medium of splenocytes and bone marrow derived macrophages (BMDMs) from wild-type or Rab37 knockout mice, and macrophage or T cell lines expressing wild type, active GTP-bound or inactive GDP-bound Rab37. Vesicle isolation, total internal reflection fluorescence microscopy, and real-time confocal microscopy were conducted. We developed polyclonal neutralizing-CHI3L1 antibodies (nCHI3L1 Abs) to validate the therapeutic efficacy in orthotopic lung, pancreas and colon cancer allograft models. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37 and CHI3L1, and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from cancer patients. Results: We demonstrate a novel secretion mode of CHI3L1 mediated by the small GTPase Rab37 in T cells and macrophages. Rab37 mediated CHI3L1 intracellular vesicle trafficking and exocytosis in a GTP-dependent manner, which is abolished in the splenocytes and BMDMs from Rab37 knockout mice and attenuated in macrophage or T cell lines expressing the inactive Rab37. The secreted CHI3L1 activated AKT, ß-catenin and NF-κB signal pathways in cancer cells and macrophages to foster a protumor TME characterized by activating M2 macrophages and increasing the population of regulatory T cells. Our developed nCHI3L1 Abs showed the dual properties of reducing tumor growth/metastases and eliciting an immunostimulatory TME in syngeneic orthotopic lung, pancreas and colon tumor models. Clinically, high plasma level or intratumoral expression of CHI3L1 correlated with poor survival in 161 lung cancer, 155 pancreatic cancer and 180 colon cancer patients. Conclusions: These results provide the first evidence that Rab37 mediates CHI3L1 secretion in immune cells and highlight nCHI3L1 Abs that can simultaneously target both cancer cells and tumor microenvironment.
Asunto(s)
Proteína 1 Similar a Quitinasa-3/inmunología , Inmunoterapia/métodos , Neoplasias , Proteínas de Unión al GTP rab/inmunología , Animales , Línea Celular Tumoral , Estudios de Cohortes , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/terapia , Microambiente TumoralRESUMEN
The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette-Guérin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo. We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.
Asunto(s)
Aparato de Golgi/inmunología , Macrófagos/inmunología , Infecciones por Mycobacterium , Factor de Necrosis Tumoral alfa/inmunología , Proteínas de Unión al GTP rab/inmunología , Animales , Ratones , Infecciones por Mycobacterium/inmunología , Mycobacterium bovisRESUMEN
The food-borne bacterial pathogen Salmonella Typhimurium uses a type III protein secretion system to deliver multiple proteins into host cells. These secreted effectors modulate the functions of host cells and activate specific signalling cascades that result in the production of pro-inflammatory cytokines and intestinal inflammation. Some of the Salmonella-encoded effectors counteract this inflammatory response and help to preserve host homeostasis. Here, we demonstrate that the Salmonella effector protein SopD, which is required for pathogenesis, functions to both activate and inhibit the inflammatory response by targeting the Rab8 GTPase, which is a negative regulator of inflammation. We show that SopD has GTPase-activating protein activity for Rab8 and, therefore, inhibits this GTPase and stimulates inflammation. We also show that SopD activates Rab8 by displacing it from its cognate guanosine dissociation inhibitor, resulting in the stimulation of a signalling cascade that suppresses inflammation. We solved the crystal structure of SopD in association with Rab8 to a resolution of 2.3 Å, which reveals a unique contact interface that underlies these complex interactions. These findings show the remarkable evolution of a bacterial effector protein to exert both agonistic and antagonistic activities towards the same host cellular target to modulate the inflammatory response.
Asunto(s)
Proteínas Bacterianas/inmunología , Interacciones Huésped-Patógeno , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Proteínas de Unión al GTP rab/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Humanos , Unión Proteica , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/química , Salmonella typhimurium/genética , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genéticaRESUMEN
The aorta and the large conductive arteries are immunoprivileged tissues and are protected against inflammatory attack. A breakdown of immunoprivilege leads to autoimmune vasculitis, such as giant cell arteritis, in which CD8+ Treg cells fail to contain CD4+ T cells and macrophages, resulting in the formation of tissue-destructive granulomatous lesions. Here, we report that the molecular defect of malfunctioning CD8+ Treg cells lies in aberrant NOTCH4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, NOTCH4 signaling restricted vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). Specifically, NOTCH4hiCD8+ Treg cells increased RAB5A and RAB11A expression and suppressed RAB7A, culminating in the accumulation of early and recycling endosomes and sequestering of NOX2 in an intracellular compartment. RAB7AloCD8+ Treg cells failed in the surface translocation and exosomal release of NOX2. NOTCH4hiRAB5AhiRAB7AloRAB11AhiCD8+ Treg cells left adaptive immunity unopposed, enabling a breakdown in tissue tolerance and aggressive vessel wall inflammation. Inhibiting NOTCH4 signaling corrected the defect and protected arteries from inflammatory insult. This study implicates NOTCH4-dependent transcriptional control of RAB proteins and intracellular vesicle trafficking in autoimmune disease and in vascular inflammation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Endosomas/inmunología , Receptor Notch4/inmunología , Linfocitos T Reguladores/inmunología , Vasculitis/inmunología , Anciano , Transporte Biológico Activo/inmunología , Linfocitos T CD8-positivos/patología , Endosomas/patología , Femenino , Humanos , Masculino , NADPH Oxidasa 2/inmunología , Linfocitos T Reguladores/patología , Vasculitis/patología , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión al GTP rab5/inmunología , Proteínas de Unión a GTP rab7RESUMEN
Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset important in the early response to bacterial and viral lung pathogens. MAIT cells recognize bacterial small molecule metabolites presented on the Class I-like molecule MR1. As with other Class I and Class II molecules, MR1 can likely sample ligands in the intracellular environment through multiple cellular pathways. Rab6, a small GTPase that regulates a number of endosomal trafficking pathways including retrograde transport to the trans-Golgi network (TGN), is involved in the presentation of ligands from Mycobacterium tuberculosis (Mtb) to MAIT cells. The Rab6-mediated trafficking pathway contains endosomal compartments that share features with the Mtb intracellular compartment. Using inducible expression of MR1, this study demonstrates that Rab6 regulates the recycling of MR1 molecules from the cell surface through endosomal trafficking compartments to the TGN. This Rab6-dependent pool of recycled MR1, which is available for reloading with ligands from bacterial pathogens like Mtb, may be important for early recognition of infected cells by MAIT cells in the lung.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Células T Invariantes Asociadas a Mucosa/inmunología , Proteínas de Unión al GTP rab/metabolismo , Adulto , Presentación de Antígeno , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Endosomas/inmunología , Endosomas/metabolismo , Silenciador del Gen , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunidad Innata , Técnicas In Vitro , Cinética , Ligandos , Antígenos de Histocompatibilidad Menor/genética , Células T Invariantes Asociadas a Mucosa/metabolismo , Células T Invariantes Asociadas a Mucosa/microbiología , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Regiones Promotoras Genéticas , Transporte de Proteínas , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Red trans-Golgi/inmunología , Red trans-Golgi/metabolismoRESUMEN
The guanosine triphosphatase (GTPase) Rab32 coordinates a cell-intrinsic host defense mechanism that restricts the replication of intravacuolar pathogens such as Salmonella Here, we show that this mechanism requires aconitate decarboxylase 1 (IRG1), which synthesizes itaconate, a metabolite with antimicrobial activity. We find that Rab32 interacts with IRG1 on Salmonella infection and facilitates the delivery of itaconate to the Salmonella-containing vacuole. Mice defective in IRG1 rescued the virulence defect of a S. enterica serovar Typhimurium mutant specifically defective in its ability to counter the Rab32 defense mechanism. These studies provide a link between a metabolite produced in the mitochondria after stimulation of innate immune receptors and a cell-autonomous defense mechanism that restricts the replication of an intracellular bacterial pathogen.
Asunto(s)
Hidroliasas/inmunología , Infecciones por Salmonella/inmunología , Salmonella enterica , Salmonella typhimurium , Proteínas de Unión al GTP rab/inmunología , Animales , Línea Celular , Interacciones Huésped-Patógeno , Humanos , Hidroliasas/metabolismo , Ratones , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Succinatos , Virulencia , Proteínas de Unión al GTP rab/metabolismoRESUMEN
White spot syndrome virus (WSSV) is the main pathogen of shrimp and has led to considerable economic losses to the shrimp industry around the world. However, so far there are still no effective strategies to address this problem. In this paper, the tissue distribution of Rab9 as well as its defense mechanism against WSSV in Japanese shrimp (Marsupenaeus japonicas) was investigated. The results revealed that Rab9 had a higher expression in hemocyte and gill while expression was lower in heart, muscle, intestine, liver, indicating Rab9 was involved in the innate immune process. The results showed that the Rab9 expression increased when shrimp was challenged with WSSV compared with that of control, while the silence of Rab9 led to the increase of WSSV copies. In order to explore the antiviral mechanism of Rab9, it was demonstrated that the expression level of Rab9 changed during autophagy process, which indicated that Rab9 is participated in the autophagy procedure of shrimp. The fact that autophagy decreased after Rab9 silenced, may also suggest that Rab9 protein could affect autophagy. In short, the results showed Rab9 played a key role in antivirus through regulating autophagy. The results not only enlarge the limited views about molecular mechanism of Rab in invertebrate, but also help to enrich the immunological content in marine invertebrate.
Asunto(s)
Proteínas de Artrópodos/inmunología , Autofagia , Infecciones por Virus ADN/inmunología , Penaeidae/inmunología , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1 , Proteínas de Unión al GTP rab/inmunología , Animales , Proteínas de Artrópodos/genética , Infecciones por Virus ADN/veterinaria , Proteínas de Unión al GTP rab/genéticaRESUMEN
Pathogenic variants in the gene encoding the small GTPase Ras analogue in Brain 39b (RAB39B) are associated with early-onset parkinsonism. In this study we investigated the expression and localization of RAB39B (RNA and protein) in mouse brain tissue to gain a better understanding of its normal physiological function(s) and role in disease.We developed novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, and performed real-time PCR and western blot analysis on whole brain lysates. To determine the spatial localization of Rab39b RNA and protein, we performed in-situ hybridization and immunohistochemistry on fresh frozen and fixed brain tissue. Our results show that RAB39B is localized throughout the cortex, hippocampus and substantia nigra of mice throughout postnatal life. We found high levels of RAB39B within MAP2 positive cortical and hippocampal neurons, and TH positive dopaminergic neurons in the substantia nigra pars compacta.Our studies support and extend current knowledge of the localization of RAB39B. We validate RAB39B as a neuron-enriched protein and demonstrate that it is present throughout the mouse cortex and hippocampus. Further, we observe high levels in the substantia nigra pars compacta, the brain region most affected in Parkinson's disease pathology. The distribution of Rab39b is consistent with human disease associations with parkinsonism and cognitive impairment. We also describe and validate novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, both of which are valuable tools for future studies of the molecular function of RAB39B.
Asunto(s)
Encéfalo/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Ratones Noqueados , Factores de Tiempo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunologíaRESUMEN
Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.
Asunto(s)
Infecciones por Alphavirus/veterinaria , Proteínas de Peces/genética , Salmo salar/genética , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP/genética , Proteínas de Unión al GTP rab5/genética , Alphavirus , Infecciones por Alphavirus/inmunología , Animales , Células Cultivadas , Endosomas/genética , Proteínas de Peces/inmunología , Expresión Génica , Regulación de la Expresión Génica , Riñón Cefálico/citología , Riñón Cefálico/inmunología , Leucocitos/inmunología , Lisosomas/genética , Salmo salar/inmunología , Homología de Secuencia , Proteínas de Unión al GTP rab/inmunología , Proteínas rab27 de Unión a GTP/inmunología , Proteínas de Unión al GTP rab5/inmunologíaRESUMEN
Upon activation by CD40 or TLR signaling, B lymphocytes activate NF-κB to induce activation-induced cytidine deaminase and, therefore, Ig class switch DNA recombination, as central to the maturation of the Ab and autoantibody responses. In this study, we show that NF-κB activation is boosted by colocalization of engaged immune receptors, such as CD40, with RAB7 small GTPase on mature endosomes, in addition to signals emanating from the receptors localized on the plasma membrane, in mouse B cells. In mature endosomes, RAB7 directly interacts with TRAF6 E3 ubiquitin ligase, which catalyzes K63 polyubiquitination for NF-κB activation. RAB7 overexpression in Cd19+/creRosa26fl-STOP-fl-Rab7 mouse B cells upregulates K63 polyubiquitination activity of TRAF6, enhances NF-κB activation and activation-induced cytidine deaminase induction, and boosts IgG Ab and autoantibody levels. This, together with the extensive intracellular localization of CD40 and the strong correlation of RAB7 expression with NF-κB activation in mouse lupus B cells, shows that RAB7 is an integral component of the B cell NF-κB activation machinery, likely through interaction with TRAF6 for the assembly of "intracellular membrane signalosomes."
Asunto(s)
Linfocitos B/inmunología , Endosomas/inmunología , Cambio de Clase de Inmunoglobulina , FN-kappa B/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Ubiquitinación/inmunología , Proteínas de Unión al GTP rab/inmunología , Animales , Antígenos CD19/genética , Antígenos CD19/inmunología , Linfocitos B/citología , Endosomas/genética , Ratones , Ratones Transgénicos , FN-kappa B/genética , Factor 6 Asociado a Receptor de TNF/genética , Ubiquitinación/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
The study of cancer has allowed researchers to describe some biological characteristics that tumor cells acquire during their development, known as the "hallmarks of cancer" but more research is needed to expand our knowledge about cancer biology and to generate new strategies of treatment. The role that RabGTPases might play in some hallmarks of cancer represents interesting areas of study since these proteins are frequently altered in cancer. However, their participation is not well known. Recently, Rab35was recognized as an oncogenic RabGTPase and and because of its association with different cellular functions, distinctly important in immune cells, a possible role of Rab35 in leukemia can be suggested. Nevertheless, the involvement of Rab35 in cancer remains poorly understood and its possible specific role in leukemia remains unknown. In this review, we analyze general aspects of the participation of RabGTPases in cancer, and especially, the plausible role of Rab35 in leukemia.
Asunto(s)
Leucemia/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Humanos , Leucemia/inmunología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunologíaRESUMEN
Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ε (IKKε) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKε substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKε activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNß. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKε in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic. SIGNIFICANCE: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.
Asunto(s)
Quinasa I-kappa B/metabolismo , Inmunidad Innata , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias de la Mama Triple Negativas/inmunología , Proteínas de Unión al GTP rab/metabolismo , Mama/inmunología , Mama/patología , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación/genética , Fosforilación/inmunología , Proteolisis , Serina/genética , Serina/metabolismo , Transducción de Señal/inmunología , Neoplasias de la Mama Triple Negativas/patología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7RESUMEN
The Rab family is the most significant subfamily of small GTP-binding proteins. These proteins have widespread intracellular localization and play an important role in many biological processes. Rab7 plays a crucial role in the innate immune system of crustaceans. In the present study, we cloned and characterized Rab7 from Chinese mitten crab (Eriocheir sinensis), designated EsRab7. The full-length of the EsRab7 cDNA sequence is 1,257 bp and contains a 618-bp open reading frame encoding a 205-amino acid polypeptide. Bioinformatics analysis showed that the Rab7 protein was highly conserved during evolution. Quantitative real-time PCR showed the highest tissue expression in muscle, followed by hepatopancreas. EsRab7 was significantly upregulated in hemocytes after stimulation by Gram-positive Staphylococcus aureus or Gram-negative Vibrio parahaemolyticus. Further studies showed that EsRab7 knockdown during bacterial stimulation resulted in decreased bacterial phagocytosis. In addition, EsRab7 regulated the expression of antimicrobial peptides via the Toll signaling pathway. Collectively, these results demonstrate that EsRab7 plays critical roles in antimicrobial function in the Chinese mitten crab.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Braquiuros/genética , Braquiuros/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Perfilación de la Expresión Génica , Fagocitosis/genética , Filogenia , Alineación de Secuencia , Proteínas de Unión al GTP rab/química , Proteínas de Unión a GTP rab7RESUMEN
Immune evasion is a critical survival mechanism for bacterial colonization of deeper tissues and may lead to life-threatening conditions such as endotoxaemia and sepsis. Understanding these immune evasion pathways would be an important step for the development of novel anti-microbial therapeutics. Here, we report a hitherto unknown mechanism by which Salmonella exploits an anti-inflammatory pathway in human immune cells to obtain survival advantage. We show that Salmonella enterica serovar Typhimurium strain 4/74 significantly (P < 0·05) increased expression of mRNA and surface protein of the type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in human monocytes. However, we also show that S. Typhimurium induced retrograde recycling of VPAC1 from early endosomes to Rab11a-containing sorting endosomes, associated with the Golgi apparatus, and anterograde trafficking via Rab3a and calmodulin 1. Expression of Rab3a and calmodulin 1 were significantly increased by S. Typhimurium infection and W-7 (calmodulin antagonist) decreased VPAC1 expression on the cell membrane while CALP-1 (calmodulin agonist) increased VPAC1 expression (P < 0·05). When infected monocytes were co-cultured with VIP, a significantly higher number of S. Typhimurium were recovered from these monocytes, compared with S. Typhimurium recovered from monocytes cultured only in cell media. We conclude that S. Typhimurium infection exploits host VPAC1/VIP to gain survival advantage in human monocytes.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Evasión Inmune , Monocitos , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Endosomas/inmunología , Endosomas/microbiología , Endosomas/patología , Humanos , Monocitos/inmunología , Monocitos/microbiología , Monocitos/patología , Infecciones por Salmonella/patología , Proteínas de Unión al GTP rab/inmunología , Proteína de Unión al GTP rab3A/inmunologíaRESUMEN
Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.
Asunto(s)
Burkholderia pseudomallei/inmunología , Melioidosis/inmunología , MicroARNs/inmunología , Fagosomas/inmunología , Proteínas de Unión al GTP rab/inmunología , Animales , Burkholderia pseudomallei/patogenicidad , Melioidosis/patología , Ratones , Viabilidad Microbiana/inmunología , Fagosomas/microbiología , Fagosomas/patología , Células RAW 264.7RESUMEN
Rab26 GTPase modulates the trafficking of cell surface receptors, such as G protein-coupled receptors including α2-adrenergic receptors in some cell types. However, the effect of Rab26 on ß2-adrenergic receptor (ß2-AR) trafficking or/and Toll-like receptor 4 (TLR4) expression in human pulmonary microvascular endothelial cells (HPMECs) is still unclear. Here, we investigated the role of Rab26 in regulating the expression of ß2-ARs and TLR4 in HPMECs and the effect of these receptors' imbalance on endothelial cell barrier function. The results showed that there was unbalance expression in these receptors, where ß2-AR expression was remarkably reduced, and TLR4 was increased on the cell membrane after lipopolysaccharide (LPS) treatment. Furthermore, we found that Rab26 overexpression not only upregulated ß2-ARs but also downregulated TLR4 expression on the cell membrane. Subsequently, the TLR4-related inflammatory response was greatly attenuated, and the hyperpermeability of HPMECs also was partially relived. Taken together, these data suggest that basal Rab26 maintains the balance between ß2-ARs and TLR4 on the cell surface, and it might be a potential therapeutic target for diseases involving endothelial barrier dysfunction.
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Células Endoteliales/metabolismo , Inflamación/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Citometría de Flujo , Humanos , Inflamación/inmunología , Microscopía Confocal , Microvasos/citología , Microvasos/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab/inmunologíaRESUMEN
Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.
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Ácidos Araquidónicos/farmacología , Endocannabinoides/farmacología , Glicéridos/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Lipopolisacáridos/toxicidad , Mastocitos/inmunología , Receptor Cannabinoide CB2/inmunología , Receptor Toll-Like 4/inmunología , Animales , Tolerancia Inmunológica/inmunología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/inmunología , Ratones , Ratones Noqueados , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptor Cannabinoide CB2/genética , Receptor Toll-Like 4/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7RESUMEN
Dendritic cells (DCs) trigger CD8â¯+â¯T cell responses after the internalization of exogenous antigens in a process called cross-presentation. Multiple intracellular transport events within the endocytic and secretory routes take place in order to accomplish this fundamental immunological process. The endomembrane system can be envisioned as a complex network of membrane domains coordinately working in the fusion of organelles, the budding of vesicles and tubules, and modifying the molecular composition of the limiting membranes. In this context of tightly regulated and dynamic endomembrane transport, small GTPases of the Rab family display a pivotal role by organizing membrane microdomains and defining specific identities to the different intracellular compartments. In this review, we synthesize and update the current knowledge about Rab22a, which has been involved in several immune functions. In this way, we analyze the intracellular localization of Rab22a and its important role in the endocytic recycling, including its relevance during MHC-I trafficking, antigen cross-presentation by DCs and the formation of T cell conjugates. We also describe how different pathogenic microorganisms hijack Rab22a functions to achieve efficient infection and intracellular survival strategies. Furthermore, we examine the oncogenic properties of Rab22a and how its expression determines the progression of many tumors. In summary, we highlight the role of Rab22a as a key effector of the intracellular trafficking that could be exploited in future therapies to modulate the immune system.
Asunto(s)
Proteínas de Unión al GTP rab/inmunología , Animales , Presentación de Antígeno/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Transporte de Proteínas/inmunologíaRESUMEN
BACKGROUND: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats. METHODS: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0. RESULTS: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-ß) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway. CONCLUSIONS: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.