RESUMEN
Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.
Asunto(s)
Flavivirus , Interferones , Replicación Viral , Apolipoproteínas L/genética , Apolipoproteínas L/metabolismo , Flavivirus/fisiología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Interferones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , SARS-CoV-2/fisiología , Virus Zika/fisiologíaRESUMEN
Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.
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Apolipoproteínas L/metabolismo , Membrana Celular/metabolismo , Citosol/microbiología , Bacterias Gramnegativas/fisiología , Interferón gamma/inmunología , Apolipoproteínas L/química , Apolipoproteínas L/genética , Membrana Externa Bacteriana/metabolismo , Bacteriólisis , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular , Células Cultivadas , Detergentes/metabolismo , Proteínas de Unión al GTP/metabolismo , Edición Génica , Bacterias Gramnegativas/inmunología , Bacterias Gramnegativas/patogenicidad , Bacterias Gramnegativas/ultraestructura , Humanos , Inmunidad Innata , Lipoproteínas/química , Viabilidad Microbiana , Antígenos O/metabolismo , Dominios Proteicos , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Salmonella typhimurium/ultraestructura , SolubilidadRESUMEN
Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.
Asunto(s)
Apolipoproteína L1/química , Apolipoproteínas L/química , Dominios Proteicos , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Apolipoproteínas L/genética , Apolipoproteínas L/metabolismo , HumanosRESUMEN
BACKGROUND: Recent studies have shown that exosomes (Exos) derived from stem cells can be used as paracrine factors to regenerate cells and tissues via shuttling miRNAs. Exos derived from human umbilical cord derived mesenchymal stem cells (UCMSCs) have been found to alleviate mifepristone-induced endometrial stromal cell (ESC) injury in vitro. Information on the functions and mechanisms of Exos from UCMSC-induced endometrial repair is limited and requires more study. METHODS: UCMSC-Exos were isolated and identified by Transmission Electron Microscopy, Nanoparticle Tracking Analysis software, and western blot assays. The damaged-ESC model and the UCMSC co-culture system were established, while GW4869, a noncompetitive neutral sphingomyelinase (N-SMase) inhibitor, was used to investigate the effects of UCMSC-Exos on mifepristone-induced ESC injury. Cell apoptosis of damaged ESCs treated with UCMSCs was detected using the TUNEL assay and flow cytometry analysis. Then, miRNA microarrays were performed to detect differentially expressed miRNA profiles in both UCMSCs and ESCs after co-culturing. A subset of upregulated miRNAs was validated by qRT-PCR, and miRNA mimics/inhibitor were used to investigate the functions of miR-7162-3p. The miRNA-mRNA interactions were predicted by Targetscan software, while the miRNA binding sites were predicted by miRcode software. Moreover, dual-luciferase reporter, western blot assays and qPCR were conducted to identify the regulatory mechanisms between miR-7162-3p and APOL6. RESULTS: UCMSCs attenuated mifepristone-induced endometrial stromal cell apoptosis by Exos, while three miRNAs (miR-6831-5p, miR-4669, and miR-7162-3p) were both upregulated in UCMSCs and ESCs after co-culture, and were candidate effectors of UCMSC-Exos-mediated endometrial repair. We showed that miR-7162-3p was shuttled by Exos from UCMSCs and regulated the expression of APOL6 by targeting its 3'-UTR in ESCs. CONCLUSIONS: These results showed UCMSC-Exos protected ESCs from mifepristone-induced apoptosis and played an active role in repairing the damaged ESCs by in vitro shuttling of miR-7162-3p. The miR-7162-3p-overexpressed UCMSC-Exos may therefore be used in cell-free therapy of endometrial injury.
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Apolipoproteínas L/genética , Endometrio/patología , Exosomas/metabolismo , Células Madre Mesenquimatosas/citología , MicroARNs/genética , Células del Estroma/patología , Cordón Umbilical/citología , Femenino , Humanos , MicroARNs/metabolismo , Células del Estroma/metabolismoRESUMEN
The discovery that apolipoprotein L1 (APOL1) is the trypanolytic factor of human serum raised interest about the function of APOLs, especially following the unexpected finding that in addition to their protective action against sleeping sickness, APOL1 C-terminal variants also cause kidney disease. Based on the analysis of the structure and trypanolytic activity of APOL1, it was proposed that APOLs could function as ion channels of intracellular membranes and be involved in mechanisms triggering programmed cell death. In this review, the recent finding that APOL1 and APOL3 inversely control the synthesis of phosphatidylinositol-4-phosphate (PI(4)P) by the Golgi PI(4)-kinase IIIB (PI4KB) is commented. APOL3 promotes Ca2+ -dependent activation of PI4KB, but due to their increased interaction with APOL3, APOL1 C-terminal variants can inactivate APOL3, leading to reduction of Golgi PI(4)P synthesis. The impact of APOLs on several pathological processes that depend on Golgi PI(4)P levels is discussed. I propose that through their effect on PI4KB activity, APOLs control not only actomyosin activities related to vesicular trafficking, but also the generation and elongation of autophagosomes induced by inflammation.
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Apolipoproteína L1/genética , Apolipoproteínas L/genética , Trastorno Autístico/genética , Neoplasias/genética , Insuficiencia Renal/genética , Esquizofrenia/genética , Virosis/genética , Actomiosina/genética , Actomiosina/metabolismo , Animales , Apolipoproteína L1/metabolismo , Apolipoproteínas L/metabolismo , Trastorno Autístico/metabolismo , Trastorno Autístico/patología , Autofagosomas/metabolismo , Calcio/metabolismo , Regulación de la Expresión Génica , Aparato de Golgi/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Esquizofrenia/metabolismo , Esquizofrenia/patología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/parasitología , Virosis/metabolismo , Virosis/patologíaRESUMEN
Humans homozygous or hemizygous for variants predicted to cause a loss of function (LoF) of the corresponding protein do not necessarily present with overt clinical phenotypes. We report here 190 autosomal genes with 207 predicted LoF variants, for which the frequency of homozygous individuals exceeds 1% in at least one human population from five major ancestry groups. No such genes were identified on the X and Y chromosomes. Manual curation revealed that 28 variants (15%) had been misannotated as LoF. Of the 179 remaining variants in 166 genes, only 11 alleles in 11 genes had previously been confirmed experimentally to be LoF. The set of 166 dispensable genes was enriched in olfactory receptor genes (41 genes). The 41 dispensable olfactory receptor genes displayed a relaxation of selective constraints similar to that observed for other olfactory receptor genes. The 125 dispensable nonolfactory receptor genes also displayed a relaxation of selective constraints consistent with greater redundancy. Sixty-two of these 125 genes were found to be dispensable in at least three human populations, suggesting possible evolution toward pseudogenes. Of the 179 LoF variants, 68 could be tested for two neutrality statistics, and 8 displayed robust signals of positive selection. These latter variants included a known FUT2 variant that confers resistance to intestinal viruses, and an APOL3 variant involved in resistance to parasitic infections. Overall, the identification of 166 genes for which a sizeable proportion of humans are homozygous for predicted LoF alleles reveals both redundancies and advantages of such deficiencies for human survival.
Asunto(s)
Genética Humana , Mutación con Pérdida de Función , Alelos , Apolipoproteínas L/genética , Fucosiltransferasas/genética , Variación Genética , Homocigoto , Humanos , Proteínas/genética , Cromosomas Sexuales/genética , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
To investigate the potential role of alcohol use disorder (AUD) in aging processes, we employed Levine's epigenetic clock (DNAm PhenoAge) to estimate DNA methylation age in 331 individuals with AUD and 201 healthy controls (HC). We evaluated the effects of heavy, chronic alcohol consumption on epigenetic age acceleration (EAA) using clinical biomarkers, including liver function test enzymes (LFTs) and clinical measures. To characterize potential underlying genetic variation contributing to EAA in AUD, we performed genome-wide association studies (GWAS) on EAA, including pathway analyses. We followed up on relevant top findings with in silico expression quantitative trait loci (eQTL) analyses for biological function using the BRAINEAC database. There was a 2.22-year age acceleration in AUD compared to controls after adjusting for gender and blood cell composition (p = 1.85 × 10-5). This association remained significant after adjusting for race, body mass index, and smoking status (1.38 years, p = 0.02). Secondary analyses showed more pronounced EAA in individuals with more severe AUD-associated phenotypes, including elevated gamma-glutamyl transferase (GGT) and alanine aminotransferase (ALT), and higher number of heavy drinking days (all ps < 0.05). The genome-wide meta-analysis of EAA in AUD revealed a significant single nucleotide polymorphism (SNP), rs916264 (p = 5.43 × 10-8), in apolipoprotein L2 (APOL2) at the genome-wide level. The minor allele A of rs916264 was associated with EAA and with increased mRNA expression in hippocampus (p = 0.0015). Our data demonstrate EAA in AUD and suggest that disease severity further accelerates epigenetic aging. EAA was associated with genetic variation in APOL2, suggesting potential novel biological mechanisms for age acceleration in AUD.
Asunto(s)
Envejecimiento/genética , Alcoholismo/genética , Apolipoproteínas L/genética , Epigénesis Genética/genética , Variación Genética/genética , Estudio de Asociación del Genoma Completo/métodos , Adulto , Envejecimiento/sangre , Alcoholismo/sangre , Alcoholismo/diagnóstico , Estudios de Cohortes , Metilación de ADN/genética , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Copy number variations (CNVs) have been identified as another important structural variation of genome. In recent years, a large amount of CNVRs have been identified in humans and animals. However, association and dosage effects studies of CNVs are very limited. Apolipoprotein L3 (APOL3) gene plays a central role in modulating gene transcription and is located within a CNVR that encompasses quantitative trait locis (QTLs) for economic traits like meat quality. Herein, we analyzed the CNV polymorphism of APOL3 in 421 individuals from five distinct cattle breeds, and then correlated their genotypes with growth traits. Association analysis revealed that the APOL3 CNV was significantly associated with hip height and cannon circumference of Xianan (XN) cattle (P < .01), and visibly associated with body slanting length and hucklebone width of Pinan (PN) cattle (P < .05). Overall, the data provide evidence for the functional role of APOL3 CNV and a basis for future applications in cattle breeding.
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Apolipoproteínas L/genética , Tamaño Corporal/genética , Bovinos/genética , Variaciones en el Número de Copia de ADN/genética , Animales , Cruzamiento , Bovinos/crecimiento & desarrollo , Sitios de Carácter CuantitativoRESUMEN
MicroRNAs (miRNAs) are small, non-coding RNAs that have been proposed to control or fine-tune complex genetic pathways by post-transcriptional regulation of target genes. It was proved that numerous miRNAs have influence on the biology of adipocytes as well as on the function of adipose tissues. This study shows that miR-10b-5p expression was decreased in mice, rats, and human under obesity. In addition, the obtained results indicated that the expression level of miR-10b-5p was increased in 3T3-L1 pre-adipocytes without manifesting a significant role in 3T3-L1 cells proliferation. On the other hand, the downregulation of miR-10b-5p by the inhibitor played a role in 3T3-L1 cells differentiation and adipogenesis. Our results strongly suggest that Apol6 was the target gene of miR-10b-5p. The inhibition of miR-10b-5p promoted the differentiation of 3T3-L1 cells and adipogenesis by upregulating the Apol6 expression. Then, the upregulated Apol6 acted as an oncogene in certain obesity-related cancers. These results indicate that miR-10b-5p may have a therapeutic significance for obesity and obesity-related cancers.
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Apolipoproteínas L/metabolismo , Biomarcadores de Tumor/metabolismo , Diferenciación Celular , MicroARNs/genética , Neoplasias/patología , Obesidad/patología , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis , Animales , Apolipoproteínas L/genética , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/etiología , Neoplasias/metabolismo , Obesidad/complicaciones , Obesidad/genética , Pronóstico , Ratas , Tasa de SupervivenciaRESUMEN
BACKGROUND AND AIMS: Endothelial cells are main actors in vascular homeostasis as they regulate vascular pressure and permeability as well as hemostasis and inflammation. Disturbed stimuli delivered to and by endothelial cells correlate with the so-called endothelial dysfunction and disrupt this homeostasis. As constituents of the inner layer of blood vessels, endothelial cells are also involved in angiogenesis. Apolipoprotein Ls (APOL) comprise a family of newly discovered apolipoproteins with yet poorly understood function, and are suggested to be involved in inflammatory processes and cell death mechanisms. Here we investigate the role of APOLs in endothelial cells stimulated with factors known to be involved in atherogenesis and their possible contribution to endothelial dysfunction with an emphasis on inflammation driven-angiogenesis in vitro. METHODS: Using the CRISPR/Cas9 technique, we analyzed the effect of APOL3 gene knock out in HMEC-1 endothelial cells on cell migration, tubulogenesis, endothelial permeability, intracellular signal transduction as assessed by kinase phosphorylation, and angiogenesis gene expression (measured by qRT-PCR). RESULTS: Our results indicate that among the family, APOL3 was the only member induced by myeloperoxidase, oxidized LDL, VEGF and FGF treatments. APOL3 invalidation increased endothelial permeability, reduced wound repair and tubule formation in vitro, the latter only in MPO and VEGF-induced conditions. Accordingly, some pro-angiogenic signaling pathways (ERK1/2 and FAK but not Akt) and some pro-angiogenic genes were partially inhibited in APOL3 knock out cells. CONCLUSIONS: These findings suggest the involvement of APOL3 in angiogenesis in vitro and as a modulator of MAPK and FAK signaling in endothelial cells.
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Apolipoproteínas L/metabolismo , Células Endoteliales/enzimología , Quinasa 1 de Adhesión Focal/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Inductores de la Angiogénesis/farmacología , Apolipoproteínas L/genética , Aterosclerosis/enzimología , Aterosclerosis/patología , Permeabilidad Capilar , Movimiento Celular , Proliferación Celular , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Humanos , Inflamación/enzimología , Inflamación/patología , Mediadores de Inflamación/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Transducción de SeñalRESUMEN
Background: Inheritance of apolipoprotein L1 gene (APOL1) renal-risk variants in a recessive pattern strongly associates with non-diabetic end-stage kidney disease (ESKD). Further evidence supports risk modifiers in APOL1-associated nephropathy; some studies demonstrate that heterozygotes possess excess risk for ESKD or show earlier age at ESKD, relative to those with zero risk alleles. Nearby loci are also associated with ESKD in non-African Americans. Methods: We assessed the role of the APOL3 null allele rs11089781 on risk of non-diabetic ESKD. Four cohorts containing 2781 ESKD cases and 2474 controls were analyzed. Results: Stratifying by APOL1 risk genotype (recessive) and adjusting for African ancestry identified a significant additive association between rs11089781 and ESKD in each stratum and in a meta-analysis [meta-analysis P = 0.0070; odds ratio (OR) = 1.29]; ORs were consistent across APOL1 risk strata. The biological significance of this association is supported by the finding that the APOL3 gene is co-regulated with APOL1, and that APOL3 protein was able to bind to APOL1 protein. Conclusions: Taken together, the genetic and biological data support the concept that other APOL proteins besides APOL1 may also influence the risk of non-diabetic ESKD.
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Apolipoproteínas L/genética , Predisposición Genética a la Enfermedad , Glomerulonefritis/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Fallo Renal Crónico/genética , Polimorfismo de Nucleótido Simple , Estudios de Casos y Controles , Genotipo , Humanos , Metaanálisis como Asunto , PronósticoRESUMEN
The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1-4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei, but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense, T.b. gambiense and the animal pathogens Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi. This trypanolytic potential was partially shared by rAPOL1 from Papio papio (rPpAPOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental rPpAPOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs.
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Apolipoproteína L1/farmacología , Apolipoproteínas L/farmacología , Trypanosoma/efectos de los fármacos , Animales , Apolipoproteína L1/genética , Apolipoproteínas L/genética , Concentración de Iones de Hidrógeno , Ratones , Papio papio , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/farmacología , Trypanosoma/fisiología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacos , Trypanosoma congolense/efectos de los fármacos , Trypanosoma vivax/efectos de los fármacos , Tripanosomiasis Africana/parasitologíaRESUMEN
Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention.