RESUMEN
We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.
Asunto(s)
Señalización del Calcio , Reparación del ADN , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Mesotelioma , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Humanos , Reparación del ADN/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Mesotelioma/genética , Señalización del Calcio/genética , Femenino , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Fibroblastos/metabolismo , Amianto/toxicidad , Inestabilidad GenómicaRESUMEN
Carriers of heterozygous germline BAP1 mutations (BAP1+/-) are affected by the "BAP1 cancer syndrome." Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/- cells secrete increased amounts of HMGB1, and that BAP1+/- carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.
Asunto(s)
Amianto , Proteína HMGB1/química , Histona Desacetilasa 1/química , Proteínas Supresoras de Tumor/química , Ubiquitina Tiolesterasa/química , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis , Núcleo Celular/metabolismo , Femenino , Interacción Gen-Ambiente , Mutación de Línea Germinal , Proteína HMGB1/genética , Heterocigoto , Histona Desacetilasa 1/genética , Incidencia , Inflamación , Masculino , Mesotelioma/metabolismo , Ratones , Mutación , Pronóstico , Unión Proteica , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina/química , Ubiquitina Tiolesterasa/metabolismoRESUMEN
The Voltage-Gated Calcium Channel (VGCC) auxiliary subunit Cavα2δ-1 (CACNA2D1) is the target/receptor of gabapentinoids which are known therapeutics in epilepsy and neuropathic pain. Following damage to the peripheral sensory nervous system, Cavα2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of chronic neuropathic pain. Gabapentinoids, such as gabapentin and pregabalin, engage with Cavα2δ-1 via binding an arginine residue (R241) within an RRR motif located at the N-terminus of human Cavα2δ-1. A novel, next generation gabapentinoid, engineered not to penetrate the brain, was able to generate a strong analgesic response in Chronic Constriction Injury animal model of chronic neuropathic pain and showed binding specificity for Cavα2δ-1 versus the Cavα2δ-2 subunit. This novel non-brain penetrant gabapentinoid, binds to R241 and a novel binding site on Cavα2δ-1, which is located within the VGCC_α2 domain, identified as a lysine residue within an IKAK amino acid motif (K634). The overall whole cell current amplitudes were diminished by the compound, with these inhibitory effects being diminished in R241A mutant Cavα2δ-1 subunits. The functional effects occurred at lower concentrations than those needed for inhibition by gabapentin or pregabalin, which apparently bound the Cavα2δ-1 subunit only on the R241 and not on the K634 residue. Our work sets the stage for the identification and characterisation of novel compounds with therapeutic properties in neuropathic pain and possibly in other disorders and conditions which require engagement of the Cavα2δ-1 target.
Asunto(s)
Canales de Calcio Tipo L , Neuralgia , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Animales , Ligandos , Humanos , Masculino , Canales de Calcio/metabolismo , Canales de Calcio/genética , Gabapentina/farmacología , Ratas Sprague-Dawley , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ratas , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Analgésicos/farmacología , Modelos Animales de Enfermedad , Pregabalina/farmacologíaRESUMEN
NVA1309 is a non-brain penetrant next-generation gabapentinoid shown to bind Cavα2δ at R243 within a triple Arginine motif forming the binding site for gabapentin and pregabalin. In this study we have compared the effects of NVA1309 with Mirogabalin, a gabapentinoid drug with higher affinity for the voltage-gated calcium channel subunit Cavα2δ-1 than pregabalin which is approved for post-herpetic neuralgia in Japan, Korea and Taiwan. Both NVA1309 and mirogabalin inhibit Cav2.2 currents in vitro and decrease Cav2.2 plasma membrane expression with higher efficacy than pregabalin. Mutagenesis of the classical binding residue arginine R243 and the newly identified binding residue lysine K615 reverse the effect of mirogabalin on Cav2.2 current, but not that of NVA1309.
Asunto(s)
Gabapentina , Humanos , Gabapentina/farmacología , Animales , Unión Proteica , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Células HEK293 , Ácido gamma-Aminobutírico/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Pregabalina/farmacología , Canales de Calcio/metabolismo , Compuestos Bicíclicos con PuentesRESUMEN
Obesity is a classified epidemic, increasing the risk of secondary diseases such as diabetes, inflammation, cardiovascular disease, and cancer. The pleiotropic hormone leptin is the proposed link for the gut-brain axis controlling nutritional status and energy expenditure. Research into leptin signaling provides great promise toward discovering therapeutics for obesity and its related diseases targeting leptin and its cognate leptin receptor (LEP-R). The molecular basis underlying the human leptin receptor complex assembly remains obscure, due to the lack of structural information regarding the biologically active complex. In this work, we investigate the proposed receptor binding sites in human leptin utilizing designed antagonist proteins combined with AlphaFold predictions. Our results show that binding site I has a more intricate role in the active signaling complex than previously described. We hypothesize that the hydrophobic patch in this region engages a third receptor forming a higher-order complex, or a new LEP-R binding site inducing allosteric rearrangement.
Asunto(s)
Leptina , Receptores de Leptina , Humanos , Leptina/química , Receptores de Leptina/metabolismo , Obesidad/metabolismo , Transducción de Señal , Unión ProteicaRESUMEN
The NLRP3 inflammasome is a critical component of innate immunity that senses diverse pathogen- and host-derived molecules. However, its aberrant activation has been associated with the pathogenesis of multiple diseases, including cancer. In this study, we designed and synthesized a series of aryl sulfonamide derivatives (ASDs) to inhibit the NLRP3 inflammasome. Among these, compounds 6c, 7n, and 10 specifically inhibited NLRP3 activation at nanomolar concentrations without affecting the activation of the NLRC4 and AIM2 inflammasomes. Furthermore, we demonstrated that these compounds reduce interleukin-1ß (IL-1ß) production in vivo and attenuate melanoma tumor growth. Moreover, metabolic stability in liver microsomes of 6c, 7n, and 10 was studied along with plasma exposure in mice of the most interesting compound 6c. Therefore, we generated potent NLRP3 inflammasome inhibitors, which can be considered in future medicinal chemistry and pharmacological studies aimed at developing a new therapeutic approach for NLRP3 inflammasome-driven cancer.
Asunto(s)
Inflamasomas , Neoplasias , Ratones , Animales , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inmunidad Innata , Interleucina-1beta/metabolismo , Ratones Endogámicos C57BLRESUMEN
Uncontrolled inflammatory response arising from the tumor microenvironment (TME) significantly contributes to cancer progression, prompting an investigation and careful evaluation of counter-regulatory mechanisms. We identified a trimeric complex at the mitochondria-associated membranes (MAMs), in which the purinergic P2X7 receptor - NLRP3 inflammasome liaison is fine-tuned by the tumor suppressor PML. PML downregulation drives an exacerbated immune response due to a loss of P2X7R-NLRP3 restraint that boosts tumor growth. PML mislocalization from MAMs elicits an uncontrolled NLRP3 activation, and consequent cytokines blast fueling cancer and worsening the tumor prognosis in different human cancers. New mechanistic insights are provided for the PML-P2X7R-NLRP3 axis to govern the TME in human carcinogenesis, fostering new targeted therapeutic approaches.
Asunto(s)
Proteína con Dominio Pirina 3 de la Familia NLR , Proteína de la Leucemia Promielocítica , Receptores Purinérgicos P2X7 , Microambiente Tumoral , Humanos , Citocinas , Inflamasomas , Mitocondrias , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Receptores Purinérgicos P2X7/metabolismo , Proteína de la Leucemia Promielocítica/metabolismoRESUMEN
The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here, we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Humanos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2RESUMEN
IgE is a central molecule in allergic disease. We have isolated cDNAs coding for the heavy and light chains of a murine mAb specific to human IgE and expressed a recombinant single-chain variable fragment (ScFv) derived thereof in Escherichia coli. The purified recombinant ScFv has a molecular mass of 28 kDa as measured by mass spectrometry and shows a beta-sheet fold as determined by circular dichroism. In biosensor-based studies it was demonstrated that the ScFv rapidly and stably binds to human IgE with an affinity of K(D) of 1.52 x 10(-10) M, which is almost as high as the affinity of IgE for FcepsilonRI, and that the ScFv is able to recognize FcepsilonRI-bound IgE and to prevent IgE binding to FcepsilonRI. The ScFv reacts specifically with IgE but not with other isotypes, allows the measurement of allergen-specific IgE in serum samples, and specifically targets cells that contain FcepsilonRI- or FcepsilonRII-bound IgE or that secrete IgE. Using negative-stain electron microscopy we demonstrated the formation of bimolecular complexes consisting of two ScFv molecules and one IgE and trimolecular complexes consisting of IgE, FcepsilonRI, and ScFv in which only one ScFv is able to bind to IgE. Accordingly, we found that the ScFv does not cross-link basophil-bound IgE and hence does not induce histamine release or activation of basophils as demonstrated by FACS analysis of CD203c expression and by histamine release experiments. In vivo skin testing confirmed the lack of allergenic activity of the ScFv. The recombinant ScFv may represent a universal tool for the IgE-targeted treatment of allergies.
Asunto(s)
Anafilaxia/inmunología , Inmunoglobulina E/biosíntesis , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Alérgenos/inmunología , Secuencia de Aminoácidos , Anafilaxia/genética , Anafilaxia/metabolismo , Animales , Secuencia de Bases , Basófilos/inmunología , Dicroismo Circular , Humanos , Inmunoglobulina E/genética , Inmunoglobulina E/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is therefore paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.
RESUMEN
The activation of dendritic cells is marked by changes both on their cell surfaces and in their functions. We define EWI-2/CD316 as an early activation marker of dendritic cells upregulated by Toll-like receptor ligands clearly before CD86 and CD83. By expression cloning, human heat shock protein A8 (HSPA8), a member of the hsp70 family, was identified as the ligand for EWI-2. Soluble EWI-2 bound both to cells expressing HSPA8 and also to immobilized HSPA8 protein. Although heat shock proteins are evolutionarily well conserved, other members of this class, including human hsp60 and mycobacterial hsp65, did not bind to EWI-2. The ligation of EWI-2 enhanced the CCL21/SLC-dependent migration of activated mature dendritic cells but attenuated their antigen-specific stimulatory capacities. Important functions of recently activated dendritic cells are thus critically modulated by the newly discovered HSPA8-EWI-2 interaction.
Asunto(s)
Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas de la Membrana/metabolismo , Antígenos CD/genética , Línea Celular , Proliferación Celular , Células Dendríticas/citología , Humanos , Cinética , Ligandos , Proteínas de la Membrana/genética , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Receptores Toll-Like/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Cross-linking of mast cell-bound IgE releases proinflammatory mediators, cytokines, and proteolytic enzymes and is a key event in allergic inflammation. OBJECTIVE: We sought to study the effect of proteases released on effector cell activation on receptor-bound IgE and their possible role in the regulation of allergic inflammation. METHODS: Using molar ratios of purified recombinant tryptase and human IgE, we studied whether tryptase can cleave IgE. Similar experiments were performed with mast cell lysates in the presence or absence of protease inhibitors. IgE cleavage products were detected in supernatants of allergen cross-linked, cultivated mast cells and in tissue fluids collected from patients' skin after IgE-mediated degranulation. The effects of protamine, an inhibitor of heparin-dependent proteases on IgE-mediated allergic in vivo skin inflammation in human subjects were studied. RESULTS: We show that beta-tryptase, a major protease released during mast cell activation, cleaves IgE. IgE degradation products were detected in tryptase-containing tissue fluids collected from sites of allergic inflammation. The biologic significance of this mechanism is demonstrated by in vivo experiments showing that protease inhibition enhances allergic skin inflammation. CONCLUSION: We suggest that IgE cleavage by effector cell proteases is a natural mechanism for controlling allergic inflammation.
Asunto(s)
Hipersensibilidad/inmunología , Inmunoglobulina E/metabolismo , Inflamación/inmunología , Mastocitos/enzimología , Triptasas/metabolismo , Alérgenos/efectos adversos , Alérgenos/metabolismo , Femenino , Humanos , Hipersensibilidad/etiología , Hipersensibilidad Inmediata/etiología , Hipersensibilidad Inmediata/inmunología , Mastocitos/inmunología , Receptores de IgE/metabolismo , Piel/inmunologíaRESUMEN
Aggregation of the alpha-chain of the high affinity IgE receptor (Fc epsilon RI alpha) on mast cells or basophils after cross-linking of receptor-bound IgE by its antigen or an anti-IgE antibody results in cell activation and release of inflammatory mediators. Omalizumab (Xolair), Novartis Pharmaceuticals; Genentech Inc.) is a recombinant humanized anti-IgE mAb developed for the treatment of severe allergic asthma. It complexes with free serum IgE, which prevents its binding to Fc epsilon RI and thereby interrupts the allergic cascade. Administration of an inhibitory anti-Fc epsilon RI alpha mAb may represent an alternative strategy to neutralize IgE-mediated receptor activation. In the present report, for the first time, we have performed direct side of side comparison between the inhibitory anti-Fc epsilon RI alpha mAb designated 15/1 and Omalizumab for their effects on human cord blood-derived mast cells. We provide the first evidence that both 15/1 mAb and Omalizumab efficiently inhibit Fc epsilon RI-mediated human mast cell responses in vitro (degranulation, activation, release of IL-8 and IL-13, phosphorylation of Akt) and that mAb 15/1 is a non-anaphylactogenic antibody, which compared to Omalizumab, displays markedly higher inhibitory potency in the presence of high IgE levels.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/farmacología , Sangre Fetal/citología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Receptores de IgE/inmunología , Anafilaxia/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Citocinas/metabolismo , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Omalizumab , Proteína Oncogénica v-akt/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
According to the network theory antibodies may act as antigens thus generating anti-idiotypic antibodies that can function as regulators of immune responses. Designed ankyrin repeat proteins (DARPins) are a new class of binding proteins and may serve as an alternative to antibodies. Selections from large DARPin libraries against the variable regions of a murine monoclonal anti-human IgE antibody, termed BSW17, yielded two highly specific anti-idiotypic DARPins both with high affinity. Their binding characteristics were comparable with these of a previously selected anti-idiotypic antibody. In vitro cell assays showed that the anti-idiotypic DARPins were able to inhibit the binding of BSW17 to cell-bound IgE and prevented BSW17 functional activity. These experiments demonstrate the possibility to isolate anti-idiotypic DARPins recognizing idiotypic determinants analogous to antibodies. In the future these DARPins may be further analyzed for their potential as putative vaccine candidates.