RESUMEN
The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.
Asunto(s)
Betacoronavirus/fisiología , Betacoronavirus/ultraestructura , Glicoproteína de la Espiga del Coronavirus/fisiología , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/patogenicidad , COVID-19 , Células Cultivadas , Infecciones por Coronavirus/virología , Femenino , Variación Genética , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Conformación Proteica , Procesamiento Proteico-Postraduccional , Receptores de Coronavirus , Receptores Virales/metabolismo , SARS-CoV-2 , Especificidad de la EspecieRESUMEN
Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.
Asunto(s)
Citocinas/sangre , VIH-1/inmunología , VIH-1/patogenicidad , Inmunidad Innata , Células Asesinas Naturales/inmunología , Linfocitos/inmunología , Factor 1 de Transcripción de Linfocitos T/inmunología , Regulación de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Homeostasis/inmunología , Humanos , Memoria Inmunológica , Técnicas In Vitro , Inflamación/genética , Inflamación/inmunología , Inflamación/virología , Factor 1 de Transcripción de Linfocitos T/genética , Vía de Señalización Wnt/inmunologíaRESUMEN
Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV-1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL-2, and IL-15, and inhibited by TGFB1, a cytokine increased in people living with HIV-1. In HIV-1 infection, the percentage of AREG+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL-6. NK-cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV-1 viremic people, and anti-inflammatory gene MYDGF was increased in an NK-cell subset from HIV-1-infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV-1 correlated inversely with ILC percentage and CD4+ T-cell counts. CD4+ T cells and their production of IL-2 prevented the loss of NK-cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Ratones , Animales , Inmunidad Innata , Linfocitos/metabolismo , VIH-1/metabolismo , Interleucina-2/metabolismo , Cromatina , Células Asesinas Naturales , Citocinas , Infecciones por VIH/genéticaRESUMEN
The maintenance of a proton gradient across the thylakoid membrane is an integral aspect of photosynthesis that is mainly established by the splitting of water molecules in photosystem II and plastoquinol oxidation at the cytochrome complex, and it is necessary for the generation of ATP in the last step of photophosphorylation. Although environmental stresses, such as high temperatures, are known to disrupt this fundamental process, only a few studies have explored the molecular mechanisms underlying proton gradient regulation during stress. The present study identified a heat-sensitive mutant that displays aberrant photosynthesis at high temperatures. This mutation was mapped to AtFtsH11, which encodes an ATP-dependent AAA-family metalloprotease. We showed that AtFtsH11 localizes to the chloroplast inner envelope membrane and is capable of degrading the ATP synthase assembly factor BFA3 under heat stress. We posit that this function limits the amount of ATP synthase integrated into the thylakoid membrane to regulate proton efflux from the lumen to the stroma. Our data also suggest that AtFtsH11 is critical in stabilizing photosystem II and cytochrome complexes at high temperatures, and additional studies can further elucidate the specific molecular functions of this critical regulator of photosynthetic thermotolerance.
Asunto(s)
Arabidopsis , ATPasas de Translocación de Protón de Cloroplastos , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Fotosíntesis/fisiología , Respuesta al Choque Térmico , Adenosina Trifosfato/metabolismo , Citocromos/metabolismoRESUMEN
Intestinal innate lymphoid cells (ILCs) contribute to the protective immunity and homeostasis of the gut, and the microbiota are critically involved in shaping ILC function. However, the role of the gut microbiota in regulating ILC development and maintenance still remains elusive. Here, we identified opposing effects on ILCs by two Helicobacter species, Helicobacter apodemus and Helicobacter typhlonius, isolated from immunocompromised mice. We demonstrated that the introduction of both Helicobacter species activated ILCs and induced gut inflammation; however, these Helicobacter species negatively regulated RORγt+ group 3 ILCs (ILC3s), especially T-bet+ ILC3s, and diminished their proliferative capacity. Thus, these findings underscore a previously unknown dichotomous regulation of ILC3s by Helicobacter species, and may serve as a model for further investigations to elucidate the host-microbe interactions that critically sustain the maintenance of intestinal ILC3s.
Asunto(s)
Colitis/inmunología , Infecciones por Enterobacteriaceae/inmunología , Microbioma Gastrointestinal/inmunología , Helicobacter/inmunología , Mucosa Intestinal/inmunología , Linfocitos/inmunología , Animales , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Colitis/inducido químicamente , Colitis/microbiología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/microbiología , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Interacciones Microbiota-Huesped/inmunología , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Inmunidad Mucosa , Mucosa Intestinal/citología , Mucosa Intestinal/microbiología , Linfocitos/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Proteínas de Dominio T Box/inmunología , Proteínas de Dominio T Box/metabolismoRESUMEN
High light intensity as one of the stresses could lead to generation of large amounts of reactive oxygen species (ROS) in plants, resulting in severe plant growth retardation. The photorespiration metabolism plays an important role in producing and removing a variety of ROS, maintaining the dynamic balance of the redox reaction, and preventing photoinhibition. Arabidopsis hydroxypyruvate reductase 1 (HPR1) is a primary metabolic enzyme in the photorespiration cycle. However, the role of HPR1 in plants response to high light is not clear. Here, we found that the expression of HPR1 could be induced by high light intensity. The growth and photosynthetic capacity of hpr1 mutants are seriously affected under high light intensity. The absence of HPR1 suppresses the rates of photorepair of Photosystem II (PSII), aggravates the production of ROS, and accelerates photorespiration rates. Moreover, the activity of ROS scavenging enzymes in the hpr1 mutants is significantly higher. These results indicate that HPR1 is involved in plant response to high light intensity and is essential for maintaining the dynamic balance of ROS and photorespiration.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hidroxipiruvato Reductasa/metabolismo , Luz , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: This study aims to investigate the mechanism of proliferation potential-related protein (PP-RP) in influencing the proliferation, migration, and apoptosis of esophageal cancer cells. METHODS: Quantitative real-time PCR and western blotting were performed to analyze the expression of PP-RP gene, p53, and interleukin (IL)-17 in human normal tissues and tumor tissues, as well as the expression of p53 and IL-17 in Eca109 and TE3 cells. The esophageal cancer cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell apoptosis was detected by flow cytometry, and cell migration was detected by transwell migration. RESULTS: PP-RP expressed highly in tumor tissue and Eca109 and TE3 cells, PP-RP overexpression inhibited the expression of p53 and promoted the expression of IL-17 in Eca109 and TE3 cells. PP-RP overexpression increased the expression of F-actin, promoted cell proliferation, and migration and suppressed cell apoptosis. Cell proliferation ability and cell migration ability were significantly strengthened while apoptosis was suppressed by PP-RP + pyruvate carboxylase deoxyribonucleic acid (PCDNA)-p53 group and PP-RP + IL-17 siRNA group in TE3 cells. CONCLUSION: Our data suggest that PP-RP promotes esophageal cancer cell proliferation and migration, and suppresses apoptosis by mediating the expression of p53 and IL-17.
Asunto(s)
Apoptosis/genética , Proliferación Celular/genética , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica/genética , Interleucina-17/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Femenino , Humanos , Interleucina-17/metabolismo , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Studies examining the association between alcohol intake and the risk of pancreatic cancer have given inconsistent results. The purpose of this study was to summarize and examine the evidence regarding the association between alcohol intake and pancreatic cancer risk based on results from prospective cohort studies. METHODS: We searched electronic databases consisting of PubMed, Ovid, Embase, and the Cochrane Library identifying studies published up to Aug 2015. Only prospective studies that reported effect estimates with 95% confidence intervals (CIs) for the risk of pancreatic cancer, examining different alcohol intake categories compared with a low alcohol intake category were included. Results of individual studies were pooled using a random-effects model. RESULTS: We included 19 prospective studies (21 cohorts) reporting data from 4,211,129 individuals. Low-to-moderate alcohol intake had little or no effect on the risk of pancreatic cancer. High alcohol intake was associated with an increased risk of pancreatic cancer (risk ratio [RR], 1.15; 95% CI: 1.06-1.25). Pooled analysis also showed that high liquor intake was associated with an increased risk of pancreatic cancer (RR, 1.43; 95% CI: 1.17-1.74). Subgroup analyses suggested that high alcohol intake was associated with an increased risk of pancreatic cancer in North America, when the duration of follow-up was greater than 10 years, in studies scored as high quality, and in studies with adjustments for smoking status, body mass index, diabetes mellitus, and energy intake.. CONCLUSIONS: Low-to-moderate alcohol intake was not significantly associated with the risk of pancreatic cancer, whereas high alcohol intake was associated with an increased risk of pancreatic cancer. Furthermore, liquor intake in particular was associated with an increased risk of pancreatic cancer.
Asunto(s)
Consumo de Bebidas Alcohólicas/epidemiología , Consumo de Bebidas Alcohólicas/patología , Neoplasias Pancreáticas/epidemiología , Neoplasias Pancreáticas/patología , Consumo de Bebidas Alcohólicas/efectos adversos , Alcoholes/toxicidad , Índice de Masa Corporal , Diabetes Mellitus/epidemiología , Diabetes Mellitus/patología , Metabolismo Energético , Femenino , Humanos , Masculino , América del Norte , Neoplasias Pancreáticas/inducido químicamente , Factores de RiesgoRESUMEN
The casein kinase 1 (CK1) plays an important role in various biological processes by phosphorylating its target proteins. In this study, we demonstrate that CK1γ1 inhibits RNA virus-mediated activation of retinoic acid-inducible gene I (RIG-I) signaling by affecting the stability of NF-κB subunit p65. First, we found that ectopic expression of CK1γ1 inhibits RIG-I pathway-mediated activation of IFN-ß, whereas knockdown of CK1γ1 potentiates the activation of IFN-ß and NF-κB induced by Sendai virus (SeV). We then revealed that CK1γ1 interacts with p65 and specifically enhances its phosphorylation at Ser(536) induced by SeV. By using an in vitro kinase assay, we confirmed that CK1γ1 can phosphorylate p65 at Ser(536). We also showed that the kinase dead mutants CK1γ1K73A and CK1γ1N169A did not inhibit SeV-induced activation of IFN-ß and NF-κB, suggesting that the kinase activity of CK1γ1 is critical for its inhibitory effect on RIG-I signaling. Additionally, we found that CK1γ1 also has the similar effect on TLR signaling. Further analysis indicated that CK1γ1 phosphorylates p65 and consequently promotes its degradation by ubiquitin E3 ligases CUL2 and COMMD1. These results revealed a novel negative regulatory manner of CK1γ1 on innate immune signaling.
Asunto(s)
Quinasa de la Caseína I/metabolismo , ARN Helicasas DEAD-box/metabolismo , Receptores Toll-Like/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa de la Caseína I/genética , Línea Celular , Proteínas Cullin/metabolismo , Proteína 58 DEAD Box , Activación Enzimática , Células HEK293 , Humanos , Interferón beta/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Receptores Inmunológicos , Virus Sendai/metabolismo , Transducción de SeñalRESUMEN
Glycosylphosphatidylinositols (GPI) are complex glycolipids that are covalently linked to the C terminus of proteins as a post-translational modification and tether proteins to the plasma membrane. One of the most striking features of GPI-anchored proteins (APs) is their enrichment in lipid rafts. The biosynthesis of GPI and its attachment to proteins occur in the endoplasmic reticulum. In the Golgi, GPI-APs are subjected to fatty acid remodeling, which replaces an unsaturated fatty acid at the sn-2 position of the phosphatidylinositol moiety with a saturated fatty acid. We previously reported that fatty acid remodeling is critical for the enrichment of GPI-APs in lipid rafts. To investigate the biological significance of GPI-AP enrichment in lipid rafts, we generated a PGAP3 knock-out mouse (PGAP3(-/-)) in which fatty acid remodeling of GPI-APs does not occur. We report here that a significant number of aged PGAP3(-/-) mice developed autoimmune-like symptoms, such as increased anti-DNA antibodies, spontaneous germinal center formation, and enlarged renal glomeruli with deposition of immune complexes and matrix expansion. A possible cause for this was the impaired engulfment of apoptotic cells by resident peritoneal macrophages in PGAP3(-/-) mice. Mice with conditional targeting of PGAP3 in either B or T cells did not develop such autoimmune-like symptoms. In addition, PGAP3(-/-) mice exhibited the tendency of Th2 polarization. These data demonstrate that PGAP3-dependent fatty acid remodeling of GPI-APs has a significant role in the control of autoimmunity, possibly by the regulation of apoptotic cell clearance and Th1/Th2 balance.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Glicosilfosfatidilinositoles/inmunología , Enfermedades del Complejo Inmune/inmunología , Microdominios de Membrana/inmunología , Proteínas de la Membrana/inmunología , Animales , Anticuerpos Antinucleares/genética , Anticuerpos Antinucleares/inmunología , Anticuerpos Antinucleares/metabolismo , Apoptosis/genética , Apoptosis/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/inmunología , Hidrolasas de Éster Carboxílico/metabolismo , Células Cultivadas , Mesangio Glomerular/inmunología , Mesangio Glomerular/metabolismo , Mesangio Glomerular/patología , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Enfermedades del Complejo Inmune/genética , Enfermedades del Complejo Inmune/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Microdominios de Membrana/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/patología , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/patologíaRESUMEN
Ndfip1 functions as both a recruiter and an activator of multiple HECT domain E3 ubiquitin ligases of the Nedd4 family. In this study, we demonstrate that Ndfip1 is involved in the ubiquitin-mediated degradation of mitochondrial antiviral signaling (MAVS), which is a key adaptor protein in RIG-I-like receptor-mediated immune signaling. We found that overexpression of Ndfip1 severely impaired MAVS and Sendai virus-mediated activation of IFN-stimulated response element, NF-κB, IFN-ß promoter, and polyinosinic-polycytidylic acid or influenza virus RNA-stimulated IRF-3 phosphorylation, as well as the transcription of IFN-ß. This functional interaction was confirmed by knockdown of Ndfip1, which facilitated MAVS-mediated downstream signaling and elevated MAVS protein levels. Further analysis indicated that Ndfip1 enhances both self-ubiquitination of HECT domain-containing E3 ubiquitin ligase Smurf1 and its interaction with MAVS, and eventually promotes MAVS degradation. In addition, the activation of IFN-ß by MAVS, influenza virus RNA, polyinosinic-polycytidylic acid, and Sendai virus was enhanced in Ndfip1 knockdown cells. These results reveal that Ndfip1 is a potent inhibitor of MAVS-mediated antiviral response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Portadoras/inmunología , ARN Helicasas DEAD-box/inmunología , Proteínas de la Membrana/inmunología , ARN Viral/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Proteínas Adaptadoras Transductoras de Señales/agonistas , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón beta/genética , Interferón beta/inmunología , Interferón gamma/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , FN-kappa B/genética , FN-kappa B/inmunología , Poli I-C/farmacología , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteolisis/efectos de los fármacos , ARN Viral/genética , ARN Viral/farmacología , Receptores Inmunológicos , Virus Sendai/genética , Virus Sendai/inmunología , Transducción de Señal/efectos de los fármacos , Ubiquitina/genética , Ubiquitina/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Glycosylphosphatidylinositol (GPI) enriches GPI-anchored proteins (GPI-AP) in lipid rafts by intimate interaction of its lipid moiety with sphingolipids and cholesterol. In addition to such lipid-lipid interactions, it has been reported that GPI may interact with protein moiety linked to GPI and affect protein conformations because GPI delipidation reduced immunoreactivities of protein. Here, we report that GPI-APs that have not undergone fatty acid remodeling exhibit reduced immunoreactivities in Western blotting, similar to delipidated proteins, compared with normal remodeled GPI-APs. In contrast, immunostaining in flow cytometry and immunoprecipitation did not show significant differences between remodeled and unremodeled GPI-APs. Moreover, detection with premixed primary/secondary antibody complexes or Fab fragments eliminated this difference in Western blotting. These results indicate that normally remodeled GPI enhanced oligomerization of GPI-APs and that inefficient oligomerization of unremodeled GPI-APs was responsible for reduced immunoreactivities. Moreover, the reduction in immunoreactivities of delipidated GPI-APs was most likely caused by the same effect. Finally, by chemical cross-linking of surface proteins in living cells and cell killing assay using a pore-forming bacterial toxin, we showed that enhanced oligomerization by GPI-remodeling occurs under a physiological membrane environment. Thus, this study clarifies the significance of GPI fatty acid remodeling in oligomerization of GPI-APs and provides useful information for technical studies of these cell components.
Asunto(s)
Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Animales , Inmunoprecipitación , Microdominios de Membrana , Ratones , Ratones Noqueados , Multimerización de ProteínaRESUMEN
The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-ß promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Transducción de Señal/fisiología , Tetraspaninas/metabolismo , Ubiquitinación/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica/fisiología , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores Inmunológicos , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Tetraspaninas/genéticaRESUMEN
Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), such as RIG-I, melanoma differentiation-associated gene 5 (MDA5), and virus-induced signaling adaptor (VISA), are intracellular molecules that sense diverse viral RNAs and trigger immune responses. In this study, we demonstrate that the ankyrin repeat protein ankrd17 interacts with RIG-I, MDA5, and VISA and upregulates RLR-mediated immune signaling. Overexpression of ankrd17 enhances RLR-mediated activation of IRF-3 and NF-κB and upregulates the transcription of IFN-ß. It also promotes RLR signaling in response to poly (I:C), influenza virus RNA, and Sendai virus. Consistently, knockdown of ankrd17 impairs RLR signaling. Furthermore, we demonstrate that ankrd17 enhances the interaction of RIG-I and MDA5 with VISA; the ankyrin repeat domain of ankrd17 is required for its interaction with RIG-I as well as for its function in regulating the RLR pathway. Taken together, our results indicate that ankrd17 is a positive regulator of the RLR signaling pathway.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/inmunología , Repetición de Anquirina/inmunología , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Factor 3 Regulador del Interferón/inmunología , Helicasa Inducida por Interferón IFIH1 , Interferón beta/biosíntesis , Interferón beta/inmunología , FN-kappa B/metabolismo , Orthomyxoviridae/inmunología , Poli I-C/inmunología , ARN Viral/inmunología , Proteínas de Unión al ARN , Receptores Inmunológicos , Virus Sendai/inmunología , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
The cellular innate immune response is essential for recognizing and defending against viral infection. Retinoic acid-inducible gene-I (RIG-I) and virus-induced signaling adaptor (VISA) mediated immune signalling is critically involved in RNA-virus-induced innate immune responses. Here we demonstrate that the complement C1qA interacts with different RIG-I pathway components and enhances RIG-I-VISA-mediated signalling pathway as well as TBK1-mediated activation of interferon-ß (IFN-ß) promoter. Our data show that over-expression of C1qA up-regulates RIG-I-mediated activation of IFN-stimulated responsive element (ISRE) and nuclear factor-κB reporters and IFN-ß transcription, but not IFN regulatory factor-3-mediated and inhibitor of κB kinase-mediated activation of ISRE and nuclear factor-κB promoter. In addition, C1qA can counteract the function of the C1q receptor gC1qR in RIG-I-mediated signalling. Our results reveal the important role of complement C1qA in the innate immune response.
Asunto(s)
Complemento C1q/inmunología , ARN Helicasas DEAD-box/inmunología , Transducción de Señal , Animales , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Perros , Humanos , Inmunidad Innata , Interferón beta/inmunología , Interferón beta/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Receptores InmunológicosRESUMEN
Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.
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Linfocitos T CD4-Positivos/inmunología , Hidrolasas de Éster Carboxílico/genética , Encefalomielitis Autoinmune Experimental/inmunología , Ácidos Grasos/inmunología , Glicosilfosfatidilinositoles/inmunología , Activación de Linfocitos/genética , Microdominios de Membrana/inmunología , Antígenos Thy-1/inmunología , Animales , Antígenos/inmunología , Femenino , Masculino , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat-containing protein (NLR) family play important roles in pathogen recognition and inflammation. However, we previously reported that human PYNOD/NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in reconstitution experiments using HEK293 cells. In this study, we investigated the molecular mechanism of PYNOD's anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1-mediated IL-1beta processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1-mediated IL-1beta secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF-kappaB activation and apoptosis and to reduce ASC's ability to promote caspase-1-mediated IL-1beta production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1beta processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1-mediated IL-1beta processing without inhibiting caspase-4 (mouse caspase-11)-mediated caspase-1 processing. Furthermore, PYNOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo.
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Antiinflamatorios no Esteroideos , Proteínas Portadoras/fisiología , Mediadores de Inflamación/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/genética , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Inhibidores de Caspasas , Caspasas/fisiología , Caspasas Iniciadoras , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Citocinas/biosíntesis , Proteínas del Citoesqueleto/sangre , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Precursores Enzimáticos/antagonistas & inhibidores , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/sangre , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Choque Séptico/inmunología , Choque Séptico/prevención & control , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.
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Background: Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on abundance of specific lymphoid subsets explain these correlations. Methods: Multiple regression was used to determine the relationship between abundance of specific blood lymphoid cell types, age, sex, requirement for hospitalization, duration of hospitalization, and elevation of blood markers of systemic inflammation, in adults hospitalized for severe COVID-19 (n = 40), treated for COVID-19 as outpatients (n = 51), and in uninfected controls (n = 86), as well as in children with COVID-19 (n = 19), recovering from COVID-19 (n = 14), MIS-C (n = 11), recovering from MIS-C (n = 7), and pediatric controls (n = 17). Results: This observational study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan - T cell subsets decrease less than 2-fold - and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis. Among controls, the percentage of ILCs that produced amphiregulin was higher in females than in males, and people hospitalized with COVID-19 had a lower percentage of ILCs that produced amphiregulin than did controls. Conclusions: These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance contributes to increased COVID-19 severity with age and in males. Funding: This work was supported in part by the Massachusetts Consortium for Pathogen Readiness and NIH grants R37AI147868, R01AI148784, F30HD100110, 5K08HL143183.
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COVID-19 , Linfopenia , Anfirregulina , COVID-19/complicaciones , Niño , Femenino , Humanos , Inmunidad Innata , Inflamación , Masculino , SARS-CoV-2 , Síndrome de Respuesta Inflamatoria Sistémica , Subgrupos de Linfocitos TRESUMEN
Recently emerged deep learning methods have achieved great success in single image rain streaks removal. However, existing methods ignore an essential factor in the rain streaks generation mechanism, i.e., the motion blur leading to the line pattern appearances. Thus, they generally produce overderaining or underderaining results. In this article, inspired by the generation mechanism, we propose a novel rain streaks removal framework using a kernel-guided convolutional neural network (KGCNN), achieving state-of-the-art performance with a simple network architecture. More precisely, our framework consists of three steps. First, we learn the motion blur kernel by a plain neural network, termed parameter network, from the detail layer of a rainy patch. Then, we stretch the learned motion blur kernel into a degradation map with the same spatial size as the rainy patch. Finally, we use the stretched degradation map together with the detail patches to train a deraining network with a typical ResNet architecture, which produces the rain streaks with the guidance of the learned motion blur kernel. Experiments conducted on extensive synthetic and real data demonstrate the effectiveness of the proposed KGCNN, in terms of rain streaks removal and image detail preservation.