RESUMEN
The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.
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Células Dendríticas , Receptores Tipo II del Factor de Necrosis Tumoral , Humanos , Diferenciación Celular , Linaje de la Célula , Factores Reguladores del Interferón/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Timo/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.
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Proteínas de Unión al ADN , Recombinasas , Humanos , ADN/genética , Reparación del ADN , Replicación del ADN , ADN Cruciforme , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinasas/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismoRESUMEN
Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.
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Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Dominios Proteicos , Pliegue de Proteína , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilasa/metabolismoRESUMEN
Phenotypic and metabolic heterogeneity within tumors is a major barrier to effective cancer therapy. How metabolism is implicated in specific phenotypes and whether lineage-restricted mechanisms control key metabolic vulnerabilities remain poorly understood. In melanoma, downregulation of the lineage addiction oncogene microphthalmia-associated transcription factor (MITF) is a hallmark of the proliferative-to-invasive phenotype switch, although how MITF promotes proliferation and suppresses invasion is poorly defined. Here, we show that MITF is a lineage-restricted activator of the key lipogenic enzyme stearoyl-CoA desaturase (SCD) and that SCD is required for MITFHigh melanoma cell proliferation. By contrast MITFLow cells are insensitive to SCD inhibition. Significantly, the MITF-SCD axis suppresses metastasis, inflammatory signaling, and an ATF4-mediated feedback loop that maintains de-differentiation. Our results reveal that MITF is a lineage-specific regulator of metabolic reprogramming, whereby fatty acid composition is a driver of melanoma phenotype switching, and highlight that cell phenotype dictates the response to drugs targeting lipid metabolism.
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Adaptación Fisiológica/fisiología , Ácidos Grasos/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación hacia Abajo/fisiología , Humanos , Ratones , Invasividad Neoplásica/patología , Fenotipo , Transducción de Señal/fisiologíaRESUMEN
In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.
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Proteínas Portadoras/genética , Inestabilidad Cromosómica/genética , Proteínas de Unión al ADN/genética , Mitosis/genética , Proteínas Nucleares/genética , Transactivadores/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN/genética , Fase G1/genética , Genoma Humano/genética , Inestabilidad Genómica/genética , Histonas , Humanos , Fosforilación , Transducción de Señal/genéticaRESUMEN
The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.
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Colon/citología , Colon/patología , Células Epiteliales/clasificación , Células Epiteliales/citología , Salud , Enfermedades Inflamatorias del Intestino/patología , Canales Iónicos/metabolismo , Animales , Biomarcadores/análisis , Colitis Ulcerosa/genética , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Células Epiteliales/microbiología , Células Epiteliales/patología , Predisposición Genética a la Enfermedad/genética , Células Caliciformes/citología , Células Caliciformes/metabolismo , Células Caliciformes/patología , Humanos , Concentración de Iones de Hidrógeno , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Péptidos Natriuréticos/metabolismo , Proteínas/metabolismo , Análisis de la Célula Individual , Células Madre/citología , Células Madre/metabolismo , Células Madre/patología , Uniones Estrechas/metabolismo , Transcripción Genética , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
Denisovans are members of a hominin group who are currently only known directly from fragmentary fossils, the genomes of which have been studied from a single site, Denisova Cave1-3 in Siberia. They are also known indirectly from their genetic legacy through gene flow into several low-altitude East Asian populations4,5 and high-altitude modern Tibetans6. The lack of morphologically informative Denisovan fossils hinders our ability to connect geographically and temporally dispersed fossil hominins from Asia and to understand in a coherent manner their relation to recent Asian populations. This includes understanding the genetic adaptation of humans to the high-altitude Tibetan Plateau7,8, which was inherited from the Denisovans. Here we report a Denisovan mandible, identified by ancient protein analysis9,10, found on the Tibetan Plateau in Baishiya Karst Cave, Xiahe, Gansu, China. We determine the mandible to be at least 160 thousand years old through U-series dating of an adhering carbonate matrix. The Xiahe specimen provides direct evidence of the Denisovans outside the Altai Mountains and its analysis unique insights into Denisovan mandibular and dental morphology. Our results indicate that archaic hominins occupied the Tibetan Plateau in the Middle Pleistocene epoch and successfully adapted to high-altitude hypoxic environments long before the regional arrival of modern Homo sapiens.
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Fósiles , Hominidae/anatomía & histología , Mandíbula/anatomía & histología , Altitud , Animales , Cuevas , Hominidae/clasificación , Migración Humana , Humanos , Filogenia , Tibet , Diente/anatomía & histologíaRESUMEN
Chronic inflammation underpins many human diseases. Morbidity and mortality associated with chronic inflammation are often mediated through metabolic dysfunction. Inflammatory and metabolic processes vary through circadian time, suggesting an important temporal crosstalk between these systems. Using an established mouse model of rheumatoid arthritis, we show that chronic inflammatory arthritis results in rhythmic joint inflammation and drives major changes in muscle and liver energy metabolism and rhythmic gene expression. Transcriptional and phosphoproteomic analyses revealed alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. Metabolomic analyses confirmed rhythmic metabolic rewiring with impaired ß-oxidation and lipid handling and revealed a pronounced shunt toward sphingolipid and ceramide accumulation. The arthritis-related production of ceramides was most pronounced during the day, which is the time of peak inflammation and increased reliance on fatty acid oxidation. Thus, our data demonstrate that localized joint inflammation drives a time-of-daydependent build-up of bioactive lipid species driven by rhythmic inflammation and altered EGFR-STAT signaling.
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Artritis , Relojes Circadianos , Ritmo Circadiano/fisiología , Metabolismo Energético , Humanos , Inflamación/metabolismoRESUMEN
While unbiased proteomics of human cerebrospinal fluid (CSF) has been used successfully to identify biomarkers of amyotrophic lateral sclerosis (ALS), high-abundance proteins mask the presence of lower abundance proteins that may have diagnostic and prognostic value. However, developments in mass spectrometry (MS) proteomic data acquisition methods offer improved protein depth. In this study, MS with library-free data-independent acquisition (DIA) was used to compare the CSF proteome of people with ALS (n = 40), healthy (n = 15) and disease (n = 8) controls. Quantified protein groups were subsequently correlated with clinical variables. Univariate analysis identified 7 proteins, all significantly upregulated in ALS versus healthy controls, and 9 with altered abundance in ALS versus disease controls (FDR < 0.1). Elevated chitotriosidase-1 (CHIT1) was common to both comparisons and was proportional to ALS disability progression rate (Pearson r = 0.41, FDR-adjusted p = 0.035) but not overall survival. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1; upregulated in ALS versus healthy controls) was proportional to disability progression rate (Pearson r = 0.53, FDR-adjusted p = 0.003) and survival (Kaplan Meier log-rank p = 0.013) but not independently in multivariate proportional hazards models. Weighted correlation network analysis was used to identify functionally relevant modules of proteins. One module, enriched for inflammatory functions, was associated with age at symptom onset (Pearson r = 0.58, FDR-adjusted p = 0.005) and survival (Hazard Ratio = 1.78, FDR = 0.065), and a second module, enriched for endoplasmic reticulum proteins, was negatively correlated with disability progression rate (r = -0.42, FDR-adjusted p = 0.109). DIA acquisition methodology therefore strengthened the biomarker candidacy of CHIT1 and UCHL1 in ALS, while additionally highlighted inflammatory and endoplasmic reticulum proteins as novel sources of prognostic biomarkers.
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Esclerosis Amiotrófica Lateral , Humanos , Proteómica/métodos , Biomarcadores/líquido cefalorraquídeo , Pronóstico , Espectrometría de MasasRESUMEN
53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.
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Reparación del ADN por Unión de Extremidades , ADN/química , ADN/metabolismo , Proteínas Mad2/metabolismo , Complejos Multiproteicos/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Cambio de Clase de Inmunoglobulina/genética , Proteínas Mad2/deficiencia , Proteínas Mad2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos/química , Mutación , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Recombinación V(D)J/genéticaRESUMEN
The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.
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Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/metabolismo , Succinatos/metabolismo , Alquilación , Animales , Carboxiliasas , Bovinos , Cisteína/química , Cisteína/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Retroalimentación Fisiológica , Femenino , Células HEK293 , Humanos , Hidroliasas/biosíntesis , Interferón beta/inmunología , Interferón beta/farmacología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteínas/metabolismo , Ratas , Ratas Wistar , Succinatos/químicaRESUMEN
The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.
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Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/química , Inestabilidad Genómica , Secuencia de Aminoácidos , Sitios de Unión , Reactivos de Enlaces Cruzados/química , ADN/genética , ADN/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Etopósido/química , Formaldehído/química , Expresión Génica , Humanos , Cinética , Mutación , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Síndrome , Rayos UltravioletaRESUMEN
Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing nonfunctional SP mutant proteins that affect SP's binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data therefore reveal a role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signaling functions seen in Dmelanogaster.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lípidos/química , Microesferas , Semen/química , Animales , Proteínas de Drosophila/genética , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Mutación/genética , Proteoma/metabolismo , Conducta Sexual Animal , Especificidad de la EspecieRESUMEN
Throughout pregnancy, some degree of insulin resistance is necessary to divert glucose towards the developing foetus. In gestational diabetes mellitus (GDM), insulin resistance is exacerbated in combination with insulin deficiency, causing new-onset maternal hyperglycaemia. The rapid reversal of insulin resistance following delivery strongly implicates the placenta in GDM pathogenesis. In this case-control study, we investigated the proteomic cargo of human syncytiotrophoblast-derived extracellular vesicles (STBEVs), which facilitate maternal-fetal signalling during pregnancy, in a UK-based cohort comprising patients with a gestational age of 38-40 weeks. Medium/large (m/l) and small (s) STBEVs were isolated from GDM (n = 4) and normal (n = 5) placentae using ex vivo dual-lobe perfusion and subjected to mass spectrometry. Bioinformatics were used to identify differentially carried proteins and mechanistic pathways. In m/lSTBEVs, 56 proteins were differently expressed while in sSTBEVs, no proteins reached statistical difference. Differences were also observed in the proteomic cargo between m/lSTBEVs and sSTBEVs, indicating that the two subtypes of STBEVs may have divergent modes of action and downstream effects. In silico functional enrichment analysis of differentially expressed proteins in m/lSTBEVs from GDM and normal pregnancy found positive regulation of cytoskeleton organisation as the most significantly enriched biological process. This work presents the first comparison of two populations of STBEVs' protein cargos (m/l and sSTBEVs) from GDM and normal pregnancy isolated using placenta perfusion. Further investigation of differentially expressed proteins may contribute to an understanding of GDM pathogenesis and the development of novel diagnostic and therapeutic tools.
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Diabetes Gestacional , Vesículas Extracelulares , Resistencia a la Insulina , Embarazo , Humanos , Femenino , Lactante , Placenta/metabolismo , Diabetes Gestacional/metabolismo , Resistencia a la Insulina/fisiología , Proteómica/métodos , Estudios de Casos y Controles , Vesículas Extracelulares/metabolismoRESUMEN
Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.
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Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Humanos , Encefalitis Japonesa/diagnóstico , Cromatografía Liquida/métodos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Proteoma/análisisRESUMEN
The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.
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Repetición de Anquirina , Oxigenasas de Función Mixta , Proteínas Represoras , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Catálisis , Humanos , Hidroxilación , Hipoxia , Oxigenasas de Función Mixta/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Nuclear actin participates in many essential cellular processes including gene transcription, chromatin remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin-9 and exportin-6, but how this transport is regulated remains unclear. Here, we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin-6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleocytoplasmic actin transport and the concomitant regulation of myocardin-related transcription factor A (MRTF-A), a co-activator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MRTF-A/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MRTF-A/SRF in tumour suppression.
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Actinas/metabolismo , Neoplasias de la Mama/genética , Neoplasias Hepáticas/genética , Membrana Nuclear/metabolismo , Factor de Respuesta Sérica/genética , Proteínas Supresoras de Tumor/metabolismo , Transporte Biológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Metilación de ADN , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Carioferinas/metabolismo , Neoplasias Hepáticas/metabolismo , Pronóstico , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Tumour necrosis factor (TNF) is a pleiotropic cytokine and master regulator of the immune system. It acts through two receptors resulting in often opposing biological effects, which may explain the lack of therapeutic potential obtained so far in multiple sclerosis (MS) with non-receptor-specific anti-TNF therapeutics. Under neuroinflammatory conditions, such as MS, TNF receptor-1 (TNFR1) is believed to mediate the pro-inflammatory activities associated with TNF, whereas TNF receptor-2 (TNFR2) may instead induce anti-inflammatory effects as well as promote remyelination and neuroprotection. In this study, we have investigated the therapeutic potential of blocking TNFR1 whilst simultaneously stimulating TNFR2 in a mouse model of MS. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG35-55) in humanized TNFR1 knock-in mice. These were treated with a human-specific TNFR1-selective antagonistic antibody (H398) and a mouse-specific TNFR2 agonist (EHD2-sc-mTNFR2), both in combination and individually. Histopathological analysis of spinal cords was performed to investigate demyelination and inflammatory infiltration, as well as axonal and neuronal degeneration. Retinas were examined for any protective effects on retinal ganglion cell (RGC) degeneration and neuroprotective signalling pathways analysed by Western blotting. RESULTS: TNFR modulation successfully ameliorated symptoms of EAE and reduced demyelination, inflammatory infiltration and axonal degeneration. Furthermore, the combinatorial approach of blocking TNFR1 and stimulating TNFR2 signalling increased RGC survival and promoted the phosphorylation of Akt and NF-κB, both known to mediate neuroprotection. CONCLUSION: These results further support the potential of regulating the balance of TNFR signalling, through the co-modulation of TNFR1 and TNFR2 activity, as a novel therapeutic approach in treating inflammatory demyelinating disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Humanos , Animales , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Inhibidores del Factor de Necrosis Tumoral , Encefalomielitis Autoinmune Experimental/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos/uso terapéuticoRESUMEN
TNF signaling is an essential regulator of cellular homeostasis. Through its two receptors TNFR1 and TNFR2, soluble versus membrane-bound TNF enable cell death or survival in a variety of cell types. TNF-TNFRs signaling orchestrates important biological functions such as inflammation, neuronal activity as well as tissue de- and regeneration. TNF-TNFRs signaling is a therapeutic target for neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer's disease (AD), but animal and clinical studies yielded conflicting findings. Here, we ask whether a sequential modulation of TNFR1 and TNFR2 signaling is beneficial in experimental autoimmune encephalomyelitis (EAE), an experimental mouse model that recapitulates inflammatory and demyelinating aspects of MS. To this end, human TNFR1 antagonist and TNFR2 agonist were administered peripherally at different stages of disease development in TNFR-humanized mice. We found that stimulating TNFR2 before onset of symptoms leads to improved response to anti-TNFR1 therapeutic treatment. This sequential treatment was more effective in decreasing paralysis symptoms and demyelination, when compared to single treatments. Interestingly, the frequency of the different immune cell subsets is unaffected by TNFR modulation. Nevertheless, treatment with only a TNFR1 antagonist increases T-cell infiltration in the central nervous system (CNS) and B-cell cuffing at the perivascular sites, whereas a TNFR2 agonist promotes Treg CNS accumulation. Our findings highlight the complicated nature of TNF signaling which requires a timely balance of selective activation and inhibition of TNFRs in order to exert therapeutic effects in the context of CNS autoimmunity.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Receptores Tipo II del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Animales , Humanos , Ratones , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Inflamación , Esclerosis Múltiple/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/agonistas , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Repeat expansions in the C9orf72 gene are a common cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, two devastating neurodegenerative disorders. One of the proposed mechanisms of GGGGCC repeat expansion is their translation into non-canonical dipeptide repeats, which can then accumulate as aggregates and contribute to these pathologies. There are five different dipeptide repeat proteins (polyGA, polyGR, polyPR, polyPA and polyGP), some of which are known to be neurotoxic. In the present study, we used BioID2 proximity labelling to identify the interactomes of all five dipeptide repeat proteins consisting of 125 repeats each. We identified 113 interacting partners for polyGR, 90 for polyGA, 106 for polyPR, 25 for polyPA and 27 for polyGP. Gene Ontology enrichment analysis of the proteomic data revealed that these target interaction partners are involved in a variety of functions, including protein translation, signal transduction pathways, protein catabolic processes, amide metabolic processes and RNA-binding. Using autopsy brain tissue from patients with C9orf72 expansion complemented with cell culture analysis, we evaluated the interactions between polyGA and valosin containing protein (VCP). Functional analysis of this interaction revealed sequestration of VCP with polyGA aggregates, altering levels of soluble valosin-containing protein. VCP also functions in autophagy processes, and consistent with this, we observed altered autophagy in cells expressing polyGA. We also observed altered co-localization of polyGA aggregates and p62 in cells depleted of VCP. All together, these data suggest that sequestration of VCP with polyGA aggregates contributes to the loss of VCP function, and consequently to alterations in autophagy processes in C9orf72 expansion disorders.