RESUMEN
The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Proteínas del Choque Térmico HSP110/antagonistas & inhibidores , Macrófagos/metabolismo , Fragmentos de Péptidos/administración & dosificación , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Biblioteca de Péptidos , Tomografía de Emisión de Positrones , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterised by myofibroblast proliferation and abnormal extracellular matrix accumulation in the lungs. Transforming growth factor (TGF)-ß1 initiates key profibrotic signalling involving the SMAD pathway and the small heat shock protein B5 (HSPB5). Tripartite motif-containing 33 (TRIM33) has been reported to negatively regulate TGF-ß/SMAD signalling, but its role in fibrogenesis remains unknown. The objective of this study was to elucidate the role of TRIM33 in IPF. METHODS: TRIM33 expression was assessed in the lungs of IPF patients and rodent fibrosis models. Bone marrow-derived macrophages (BMDM), primary lung fibroblasts and 3D lung tissue slices were isolated from Trim33-floxed mice and cultured with TGF-ß1 or bleomycin (BLM). Trim33 expression was then suppressed by adenovirus Cre recombinase (AdCre). Pulmonary fibrosis was evaluated in haematopoietic-specific Trim33 knockout mice and in Trim33-floxed mice that received AdCre and BLM intratracheally. RESULTS: TRIM33 was overexpressed in alveolar macrophages and fibroblasts in IPF patients and rodent fibrotic lungs. Trim33 inhibition in BMDM increased TGF-ß1 secretion upon BLM treatment. Haematopoietic-specific Trim33 knockout sensitised mice to BLM-induced fibrosis. In primary lung fibroblasts and 3D lung tissue slices, Trim33 deficiency increased expression of genes downstream of TGF-ß1. In mice, AdCre-Trim33 inhibition worsened BLM-induced fibrosis. In vitro, HSPB5 was able to bind directly to TRIM33, thereby diminishing its protein level and TRIM33/SMAD4 interaction. CONCLUSION: Our results demonstrate a key role of TRIM33 as a negative regulator of lung fibrosis. Since TRIM33 directly associates with HSPB5, which impairs its activity, inhibitors of TRIM33/HSPB5 interaction may be of interest in the treatment of IPF.
Asunto(s)
Fibrosis Pulmonar Idiopática , Factor de Crecimiento Transformador beta1 , Animales , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Fibroblastos , Humanos , Pulmón , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Factores de TranscripciónRESUMEN
ß-thalassemia major (ß-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ß-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ß-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ß-TM.
Asunto(s)
Carioferinas , Receptores Citoplasmáticos y Nucleares , Talasemia beta , Diferenciación Celular , Eritroblastos , Eritropoyesis , Humanos , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/genética , Talasemia beta/tratamiento farmacológico , Talasemia beta/genética , Proteína Exportina 1RESUMEN
ß-Thalassaemia major (ß-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing ß-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human ß-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of ß-TM erythroblasts, which may provide a rationale for new targeted therapies of ß-TM.
Asunto(s)
Eritroblastos/metabolismo , Eritropoyesis , Proteínas HSP70 de Choque Térmico/metabolismo , Globinas alfa/metabolismo , Talasemia beta/sangre , Talasemia beta/metabolismo , Apoptosis , Médula Ósea/metabolismo , Caspasa 3/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular/genética , Células Cultivadas , Citoplasma/metabolismo , Activación Enzimática , Eritroblastos/citología , Eritroblastos/patología , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Humanos , Cinética , Terapia Molecular Dirigida , Unión Proteica , Replegamiento Proteico , Talasemia beta/patologíaRESUMEN
BACKGROUND & AIMS: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracilbased chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. METHODS: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage IIIII colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17). RESULTS: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage IIIII cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.0120.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009). CONCLUSIONS: About 25% of patients with stages IIIII colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Secuencia de Bases , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas del Choque Térmico HSP110/genética , Inestabilidad de Microsatélites , Eliminación de Secuencia , Anciano , Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral , Quimioterapia Adyuvante , Colectomía , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/cirugía , Femenino , Fluorouracilo/administración & dosificación , Estudios de Seguimiento , Proteínas del Choque Térmico HSP110/química , Proteínas del Choque Térmico HSP110/metabolismo , Humanos , Intrones , Leucovorina/administración & dosificación , Masculino , Modelos Moleculares , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Estudios Retrospectivos , Resonancia por Plasmón de Superficie , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Radioimmunotherapy (RIT) is a cancer treatment that combines radiation therapy with tumor-directed monoclonal antibodies (Abs). Although RIT had been introduced for the treatment of CD20 positive non-Hodgkin lymphoma decades ago, it never found a broad clinical application. In recent years, researchers have developed theranostic agents based on Ab fragments or small Ab mimetics such as peptides, affibodies or single-chain Abs with improved tumor-targeting capacities. Theranostics combine diagnostic and therapeutic capabilities into a single pharmaceutical agent; this dual application can be easily achieved after conjugation to radionuclides. The past decade has seen a trend to increased specificity, fastened pharmacokinetics, and personalized medicine. In this review, we discuss the different strategies introduced for the noninvasive detection and treatment of hematological malignancies by radiopharmaceuticals. We also discuss the future applications of these radiotheranostic agents.
Asunto(s)
Neoplasias Hematológicas , Linfoma no Hodgkin , Neoplasias , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Linfoma no Hodgkin/diagnóstico por imagen , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/radioterapia , Neoplasias/tratamiento farmacológico , RadioinmunoterapiaRESUMEN
A water-soluble fluorescent aza-BODIPY platform (Wazaby) was prepared and functionalized by a polyazamacrocycle agent and a bioconjugable arm. The resulting fluorescent derivative was characterized and bioconjugated onto a trastuzumab monoclonal antibody as a vector. After bioconjugation, the imaging agent appeared to be stable in serum (>72 h at 37 °C) and specifically labeled HER-2-positive breast tumors slices. The bioconjugate was radiolabeled with [111In] indium and studied in vivo. The developed monomolecular multimodal imaging probe (MOMIP) is water-soluble and chemically and photochemically stable, emits in the near infrared (NIR) region (734 nm in aqueous media), and displays a good quantum yield of fluorescence (around 15%). Single-photon emission-computed tomography and fluorescence imaging have been performed in nude mice bearing HER2-overexpressing HCC1954 human breast cancer xenografts and have evidenced the good tumor targeting of the [111In] In bimodal agent. Finally, the proof of concept of using it as a new tool for fluorescence-guided surgery has been shown.
Asunto(s)
Compuestos de Boro/química , Neoplasias de la Mama/diagnóstico por imagen , Desarrollo de Medicamentos , Colorantes Fluorescentes/química , Imagen Óptica , Tomografía Computarizada de Emisión de Fotón Único , Animales , Anticuerpos Monoclonales/química , Compuestos de Boro/síntesis química , Relación Dosis-Respuesta a Droga , Femenino , Colorantes Fluorescentes/síntesis química , Células Hep G2 , Humanos , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Ratones , Ratones Desnudos , Estructura Molecular , Solubilidad , Relación Estructura-Actividad , Agua/químicaRESUMEN
BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.
Asunto(s)
Chaperonina 60/farmacología , Colon/efectos de los fármacos , Inflamación/prevención & control , Lactobacillus/metabolismo , Animales , Chaperonina 60/uso terapéutico , Colon/fisiopatología , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Limosilactobacillus reuteri/efectos de los fármacos , Limosilactobacillus reuteri/metabolismo , Ratones Endogámicos BALB C , Estadísticas no ParamétricasRESUMEN
BACKGROUND: Cancer is the second leading cause of death globally. Early detection and disease management lead to a better survival rate. Consequently, discovery of novel methods in cancer early diagnosis is a field of active research. Minimally invasive liquid biopsies are generating growing interest. Circulating tumour cells (CTCs) have been identified in patients' blood; nevertheless, these cells are rare and heterogeneous. Exosomes are extracellular nanovesicles released into the extracellular environment via the endosomal vesicle pathway and found in different body fluids. Exosomes deliver bioactive cargo such as proteins, mRNA and miRNA to recipient cells in the tumour environment. We have recently shown that heat shock protein 70 (HSP70) is detected in the membrane of tumour-derived exosomes, in contrast to normal cells. One single cancer cell can release thousands of HSP70-exosomes, facilitating detection. The aim of the pilot study ExoDiag is to determine whether it is possible to detect and quantify HSP70-exosomes in blood in patients with solid cancers. METHODS: Bicentric pilot study that will include 60 adult patients with metastatic and non-metastatic solid tumours and 20 healthy volunteers. Exosomes will be isolated from blood and urine samples, and HSP70 concentration will be determined. Patients will be followed for 1 year. The study is sponsored by Georges-François Leclerc Centre and is currently ongoing. DISCUSSION: We expect to demonstrate that HSP70-exosomes could be a powerful tool to diagnose cancer and to guide clinicians in therapeutic decision-making, improving patient's care. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02662621. Registered 20 January 2016, https://clinicaltrials.gov/ct2/show/study/NCT02662621?term=NCT02662621&rank=1.
RESUMEN
Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas del Choque Térmico HSP110/antagonistas & inhibidores , Animales , Antineoplásicos/toxicidad , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Cristalografía por Rayos X , Proteínas del Choque Térmico HSP110/química , Proteínas del Choque Térmico HSP110/metabolismo , Humanos , Ratones , Modelos Moleculares , Factor de Transcripción STAT3/metabolismoRESUMEN
A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.
Asunto(s)
Núcleo Celular/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Reparación del ADN por Unión de Extremidades/genética , Proteínas del Choque Térmico HSP110/genética , Mutágenos/farmacología , Translocación Genética/genética , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Proteínas de Unión al ADN/genética , Células HCT116 , Humanos , Autoantígeno Ku/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Oxaliplatino/farmacología , Translocación Genética/efectos de los fármacosRESUMEN
Correction to:Cell Death & Disease8, e2816 (2017); https://doi.org/10.1038/cddis.2017.222 ; published online 25 May 2017.
RESUMEN
The E2F transcription factor 1 is subtly regulated along the cell cycle progression and in response to DNA damage by post-translational modifications. Here, we demonstrated that the E3-ubiquitin ligase cellular inhibitor of apoptosis 1 (cIAP1) increases E2F1 K63-poly-ubiquitination on the lysine residue 161/164 cluster, which is associated with the transcriptional factor stability and activity. Mutation of these lysine residues completely abrogates the binding of E2F1 to CCNE, TP73 and APAF1 promoters, thus inhibiting transcriptional activation of these genes and E2F1-mediated cell proliferation control. Importantly, E2F1 stabilization in response to etoposide-induced DNA damage or during the S phase of cell cycle, as revealed by cyclin A silencing, is associated with K63-poly-ubiquitinylation of E2F1 on lysine 161/164 residues and involves cIAP1. Our results reveal an additional level of regulation of the stability and the activity of E2F1 by a non-degradative K63-poly-ubiquitination and uncover a novel function for the E3-ubiquitin ligase cIAP1.
Asunto(s)
Daño del ADN , Factor de Transcripción E2F1/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Fase S , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Arginina/metabolismo , Humanos , Metilación , Ratones , Estabilidad Proteica , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.
Asunto(s)
Apoptosis/efectos de los fármacos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/toxicidad , Secuencia de Aminoácidos , Animales , Línea Celular , Citomegalovirus/metabolismo , Glicosilación , Células HCT116 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Nanopartículas/química , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/deficiencia , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Alineación de Secuencia , Tunicamicina/toxicidad , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749-954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3-deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease.
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Complemento C3/metabolismo , Enfermedad Injerto contra Huésped/sangre , Glicoproteínas de Membrana/sangre , Chaperonas Moleculares/sangre , Adolescente , Adulto , Animales , Activación de Complemento , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Exosomes, via heat shock protein 70 (HSP70) expressed in their membrane, are able to interact with the toll-like receptor 2 (TLR2) on myeloid-derived suppressive cells (MDSCs), thereby activating them. METHODS: We analyzed exosomes from mouse (C57Bl/6) and breast, lung, and ovarian cancer patient samples and cultured cancer cells with different approaches, including nanoparticle tracking analysis, biolayer interferometry, FACS, and electron microscopy. Data were analyzed with the Student's t and Mann-Whitney tests. All statistical tests were two-sided. RESULTS: We showed that the A8 peptide aptamer binds to the extracellular domain of membrane HSP70 and used the aptamer to capture HSP70 exosomes from cancer patient samples. The number of HSP70 exosomes was higher in cancer patients than in healthy donors (mean, ng/mL ± SD = 3.5 ± 1.7 vs 0.17 ± 0.11, respectively, P = .004). Accordingly, all cancer cell lines examined abundantly released HSP70 exosomes, whereas "normal" cells did not. HSP70 had higher affinity for A8 than for TLR2; thus, A8 blocked HSP70/TLR2 association and the ability of tumor-derived exosomes to activate MDSCs. Treatment of tumor-bearing C57Bl/6 mice with A8 induced a decrease in the number of MDSCs in the spleen and inhibited tumor progression (n = 6 mice per group). Chemotherapeutic agents such as cisplatin or 5FU increase the amount of HSP70 exosomes, favoring the activation of MDSCs and hampering the development of an antitumor immune response. In contrast, this MDSC activation was not observed if cisplatin or 5FU was combined with A8. As a result, the antitumor effect of the drugs was strongly potentiated. CONCLUSIONS: A8 might be useful for quantifying tumor-derived exosomes and for cancer therapy through MDSC inhibition.
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Aptámeros de Péptidos/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias del Colon/inmunología , Exosomas/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias Pulmonares/inmunología , Células Mieloides/inmunología , Neoplasias Ováricas/inmunología , Receptor Toll-Like 2/metabolismo , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/tratamiento farmacológico , Exosomas/efectos de los fármacos , Femenino , Humanos , Interferometría/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Neoplasias Ováricas/tratamiento farmacológico , BazoRESUMEN
BACKGROUND AND PURPOSE: Leptin, an adipokine synthesized by the placenta during pregnancy, has been proposed for the management of preterm labour (PTL), as it is able to prevent in vitro uterine contractility and remodelling associated with labour onset. Another common feature of labour onset is the phenotypic switch of myometrial smooth muscle cells from a proliferative to a hypertrophic state. As proliferative effects have been demonstrated for leptin in other tissues, we aimed to investigate its ability to induce myometrial proliferation and thus to maintain uterine quiescence. EXPERIMENTAL APPROACH: We stimulated human primary myometrial smooth muscle cells with leptin in the presence or absence of receptor antagonists or signalling pathway inhibitors. KEY RESULTS: Leptin induced myometrial cell proliferation in a biphasic manner. At 6.25 ng · mL(-1), leptin-induced proliferation was mediated by the leptin receptor and required the early activation of ERK1/2. At a concentration above 25 ng · mL(-1), leptin induced direct non-specific stimulation of the IL-6 receptor, leading to NF-κB activation, and exerted anti-proliferative effects. However, at 50 ng · mL(-1), leptin re-induces proliferation via IL-6 receptor stimulation that requires STAT3 and delayed ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS: These data bring new insights into leptin signalling-induced myometrial proliferation and its interrelationship with the IL-6/IL-6 receptor axis. In the light of our previous work, the present study emphasizes the potential value of leptin in the pharmacological management of PTL and it also strengthens the hypothesis that leptin might be a contributory factor in the parturition-related disorders observed in obese women.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Leptina/farmacología , Miometrio/efectos de los fármacos , Receptores de Leptina/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Interleucina-6/metabolismo , Leptina/administración & dosificación , Leptina/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miometrio/citología , FN-kappa B/metabolismo , Embarazo , Receptores de Leptina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.