RESUMEN
The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.
Asunto(s)
Niacinamida , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Relación Estructura-Actividad , Compuestos de Piridinio/metabolismo , Compuestos de Piridinio/química , N-Glicosil Hidrolasas/metabolismo , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/química , Uridina/metabolismo , Uridina/análogos & derivados , Uridina/química , Especificidad por Sustrato , Humanos , Modelos MolecularesRESUMEN
Pathogenic parasites of the Trichomonas genus are causative agents of sexually transmitted diseases affecting millions of individuals worldwide and whose outcome may include stillbirths and enhanced cancer risks and susceptibility to HIV infection. Trichomonas vaginalis relies on imported purine and pyrimidine nucleosides and nucleobases for survival, since it lacks the enzymatic activities necessary for de novo biosynthesis. Here we show that T. vaginalis additionally lacks homologues of the bacterial or mammalian enzymes required for the synthesis of the nicotinamide ring, a crucial component in the redox cofactors NAD+ and NADP. Moreover, we show that a yet fully uncharacterized T. vaginalis protein homologous to bacterial and protozoan nucleoside hydrolases is active as a pyrimidine nucleosidase but shows the highest specificity toward the NAD+ metabolite nicotinamide riboside. Crystal structures of the trichomonal riboside hydrolase in different states reveals novel intermediates along the nucleoside hydrolase-catalyzed hydrolytic reaction, including an unexpected asymmetry in the homotetrameric assembly. The active site structure explains the broad specificity toward different ribosides and offers precise insights for the engineering of specific inhibitors that may simultaneously target different essential pathways in the parasite.
Asunto(s)
Hidrolasas , Parásitos , Trichomonas vaginalis , Animales , Hidrolasas/química , Hidrolasas/metabolismo , NAD/metabolismo , Niacinamida/metabolismo , Trichomonas vaginalis/enzimología , Cristalografía por Rayos X , Especificidad por Sustrato , Estructura Terciaria de Proteína , Modelos Moleculares , Unión ProteicaRESUMEN
Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Glicosilación , Antígenos/metabolismoRESUMEN
Although toxin may have some advantages compared to chemotherapeutic drugs in cancer therapy, e.g. a potent cytotoxic activity and a reduced risk of resistance, their successful application in the treatments to solid tumors still remains to be fully demonstrated. In this study, we genetically modified the structure of the plant-derived single-chain ribosome inactivating protein saporin (SAP) by fusing its N-terminus to the ACDCRGDCFCG peptide (RGD-4C), an αv-integrin ligand, and explored the anti-tumor activity of the resulting protein (called RGD-SAP) in vitro and in vivo, using a model of muscle invasive bladder cancer. We found that the RGD-4C targeting domain enhances the cytotoxic activity of SAP against various tumor cell lines, in a manner dependent on αv-integrin expression levels. In a subcutaneous syngeneic model of bladder cancer, RGD-SAP significantly reduced tumor growth in a dose-dependent manner. Furthermore, systemic administration of RGD-SAP in combination with mitomycin C, a chemotherapeutic drug currently used to treat patients with bladder cancer, increased the survival of mice bearing orthotopic bladder cancer with no evidence of systemic toxicity. Overall, the results suggest that RGD-SAP represents an efficient drug that could be exploited, either alone or in combination with the state-of-the-art therapies, for the treatment of bladder cancer and, potentially, of other solid tumors.
RESUMEN
Enzymes catalyzing the hydrolysis of the N-glycosidic bond in nucleosides and other ribosides (N-ribohydrolases, NHs) with diverse substrate specificities are found in all kingdoms of life. While the overall NH fold is highly conserved, limited substitutions and insertions can account for differences in substrate selection, catalytic efficiency, and distinct structural features. The NH structural module is also employed in monomeric proteins devoid of enzymatic activity with different physiological roles. The homo-oligomeric quaternary structure of active NHs parallels the different catalytic strategies used by each isozyme, while providing a buttressing effect to maintain the active site geometry and allow the conformational changes required for catalysis. The unique features of the NH catalytic strategy and structure make these proteins attractive targets for diverse therapeutic goals in different diseases.
Asunto(s)
Nucleósidos , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Especificidad por SustratoRESUMEN
Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, â¼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, â¼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.
Asunto(s)
Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Leucemia Linfocítica Crónica de Células B/genética , Receptores de Antígenos de Linfocitos B/genética , Cristalografía por Rayos X , Regulación Leucémica de la Expresión Génica , Reordenamiento Génico , Células HEK293 , Humanos , Modelos Moleculares , Dominios Proteicos , Receptores de Antígenos de Linfocitos B/química , Hipermutación Somática de InmunoglobulinaRESUMEN
The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify â¼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.
Asunto(s)
Cadenas lambda de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos B/inmunología , Estudios de Cohortes , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Predisposición Genética a la Enfermedad , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Mutación Puntual , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Línea Celular , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Supervivencia Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , Unión Proteica/efectos de los fármacos , Puromicina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The structural plasticity of G-protein coupled receptors (GPCRs) enables the long-range transmission of conformational changes induced by specific orthosteric site ligands and other pleiotropic factors. Here, we demonstrate that the ligand binding cavity in the sphingosine 1-phosphate receptor S1PR1, a class A GPCR, is in allosteric communication with both the ß-arrestin-binding C-terminal tail, and a receptor surface involved in oligomerization. We show that S1PR1 oligomers are required for full response to different agonists and ligand-specific association with arrestins, dictating the downstream signalling kinetics. We reveal that the active form of the immunomodulatory drug fingolimod, FTY720-P, selectively harnesses both these intramolecular networks to efficiently recruit ß-arrestins in a stable interaction with the receptor, promoting deep S1PR1 internalization and simultaneously abrogating ERK1/2 phosphorylation. Our results define a molecular basis for the efficacy of fingolimod for people with multiple sclerosis, and attest that GPCR signalling can be further fine-tuned by the oligomeric state.
Asunto(s)
Regulación Alostérica , Modelos Moleculares , Conformación Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Línea Celular , Membrana Celular/metabolismo , Clorhidrato de Fingolimod/química , Clorhidrato de Fingolimod/farmacología , Humanos , Cinética , Fosforilación , Proproteína Convertasas/química , Proproteína Convertasas/metabolismo , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Transducción de Señal , Relación Estructura-Actividad , beta-Arrestinas/química , beta-Arrestinas/metabolismoRESUMEN
Methionine adenosyltransferases catalyse the biosynthesis of S-adenosylmethionine, the primary methyl group donor in biochemical reactions, through the condensation of methionine and ATP. Here, we report the structural analysis of the Pyrococcus furiosus methionine adenosyltransferase (PfMAT) captured in the unliganded, substrate- and product-bound states. The conformational changes taking place during the enzymatic catalytic cycle are allosterically propagated by amino acid residues conserved in the archaeal orthologues to induce an asymmetric dimer structure. The distinct occupancy of the active sites within a PfMAT dimer is consistent with a half-site reactivity that is mediated by a product-induced negative cooperativity. The structures of intermediate states of PfMAT reported here suggest a distinct molecular mechanism for S-adenosylmethionine synthesis in Archaea, likely consequence of the evolutionary pressure to achieve protein stability under extreme conditions.
Asunto(s)
Metionina Adenosiltransferasa/metabolismo , Pyrococcus furiosus/enzimología , Sitios de Unión , Catálisis , Cristalografía por Rayos X/métodos , Metionina Adenosiltransferasa/química , Conformación Proteica , Pyrococcus furiosus/metabolismoRESUMEN
NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias/irrigación sanguínea , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Inhibidores de la Angiogénesis/química , Línea Celular Tumoral , Cristalografía por Rayos X , Células Endoteliales de la Vena Umbilical Humana , Humanos , Modelos Moleculares , Oligopéptidos/química , Proteínas Recombinantes de Fusión/química , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/químicaRESUMEN
Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.
Asunto(s)
Movimiento Celular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Línea Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Proteínas del Tejido Nervioso/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
Mutations in the NPHP1 gene, coding for human nephrocystin-1 (NPHP1), cause the autosomal recessive disease nephronophthisis, the most common cause of end-stage renal disease in children and adolescents. The function and structure of NPHP1 are still poorly characterized. NPHP1 presents a modular structure well in keeping with its role as an adaptor protein: it harbors an SH3 domain flanked by two glutamic acid-rich regions and a conserved C-terminal nephrocystin homology domain (NHD). Similar to other NPHP protein family members, its N-terminus contains a putative coiled-coil domain (NPHP1CC) that is supposed to play an important role in NPHP1 self-association and/or protein-protein interactions. Structural studies proving its structure and its oligomerization state are still lacking. Here we demonstrate that NPHP1CC is monomeric in solution and unexpectedly folds into an autonomous domain forming a three-stranded antiparallel coiled coil suitable for protein-protein interactions. Notably, we found that the NPHP1CC shares remarkable structural similarities with the three-stranded coiled coil of the BAG domain protein family, which is known to mediate the anti-apoptotic function of these proteins, suggesting a possible similar role for NPHP1CC. In agreement with this hypothesis, we show that in the context of the full-length protein the NPHP1CC is fundamental to regulate resistance to apoptotic stimuli.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas del Citoesqueleto/metabolismo , Secuencia de Aminoácidos , Animales , Perros , Humanos , Células de Riñón Canino Madin Darby , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Dominios Proteicos , Pliegue de Proteína , Alineación de SecuenciaRESUMEN
Improvements in the immunological, molecular, and genetic technologies such as next-generation sequencing have led to an exponential increase in the number of monogenic immune dysregulatory syndromes diagnosed, where type 1 diabetes (T1D) forms part of the autoimmune manifestations. Here, we reviewed the mutations in the signal transducer and activator of transcription (STAT) protein family, namely gain-of-function (GOF) mutations in STAT1 and STAT3 as well as STAT5b deficiency, that show strong association to T1D susceptibility. The equilibrium of T-helper 17 (Th17) and regulatory T cells (Tregs) is often found altered in patients affected by STAT GOF mutations. While the increased number of Th17 cells and the concomitant decrease in Treg cells may explain T1D in STAT3 GOF patients, the reduced number of Th17 cells found in those carrying STAT1 GOF mutations added a new level of complexity on the exact role of Th17 in the pathogenesis of T1D. Here, we describe the possible mechanisms through which STAT3 and STAT1 GOF mutations may perturb the fate and function of Th17 and Tregs and explore how this may lead to the development of T1D. We propose that the study of monogenic diseases, and in particular STAT mutations, may not only improve our understanding of the function of the human immune system but also shed light onto the pathogenic mechanisms of T1D and the genetic variants that confer predisposition to the disease.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Animales , Humanos , Mutación/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genéticaRESUMEN
ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/genética , Mutación de Línea Germinal , Síndromes de Inmunodeficiencia/genética , Linfocitos T/patología , Complejo 2-3 Proteico Relacionado con la Actina/química , Femenino , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/patología , Masculino , Modelos Moleculares , Linaje , Conformación Proteica , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/metabolismoRESUMEN
Low-level rifampin resistance associated with specific rpoB mutations (referred as "disputed") in Mycobacterium tuberculosis is easily missed by some phenotypic methods. To understand the mechanism by which some mutations are systematically missed by MGIT phenotypic testing, we performed an in silico analysis of their effect on the structural interaction between the RpoB protein and rifampin. We also characterized 24 representative clinical isolates by determining MICs on 7H10 agar and testing them by an extended MGIT protocol. We analyzed 2,097 line probe assays, and 156 (7.4%) cases showed a hybridization pattern referred to here as "no wild type + no mutation." Isolates harboring "disputed" mutations (L430P, D435Y, H445C/L/N/S, and L452P) tested susceptible in MGIT, with prevalence ranging from 15 to 57% (overall, 16 out of 55 isolates [29%]). Our in silico analysis did not highlight any difference between "disputed" and "undisputed" substitutions, indicating that all rpoB missense mutations affect the rifampin binding site. MIC testing showed that "undisputed" mutations are associated with higher MIC values (≥20 mg/liter) compared to "disputed" mutations (4 to >20 mg/liter). Whereas "undisputed" mutations didn't show any delay (Δ) in time to positivity of the test tube compared to the control tube on extended MGIT protocol, "disputed" mutations showed a mean Δ of 7.2 days (95% confidence interval [CI], 4.2 to 10.2 days; P < 0.05), providing evidence that mutations conferring low-level resistance are associated with a delay in growth on MGIT. Considering the proved relevance of L430P, D435Y, H445C/L/N, and L452P mutations in determining clinical resistance, genotypic drug susceptibility testing (DST) should be used to replace phenotypic results (MGIT) when such mutations are found.
Asunto(s)
Antibióticos Antituberculosos/farmacología , ARN Polimerasas Dirigidas por ADN/genética , Genotipo , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Rifampin/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Estudios Retrospectivos , Factores de Tiempo , Tuberculosis/microbiologíaRESUMEN
Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 (CECR1) gene, currently named ADA2. The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect.
Asunto(s)
Adenosina Desaminasa/deficiencia , Síndrome Linfoproliferativo Autoinmune/terapia , Trasplante de Células Madre Hematopoyéticas , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Acondicionamiento Pretrasplante , Donante no Emparentado , Adenosina Desaminasa/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Síndrome Linfoproliferativo Autoinmune/enzimología , Síndrome Linfoproliferativo Autoinmune/inmunología , Síndrome Linfoproliferativo Autoinmune/patología , Preescolar , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Humanos , Péptidos y Proteínas de Señalización Intercelular/inmunología , Neutropenia/enzimología , Neutropenia/inmunología , Neutropenia/patología , Neutropenia/terapia , Trasplante Homólogo , Receptor fas/inmunologíaRESUMEN
IL-27 and IL-35 are heterodimeric cytokines, members of the IL-12 family and considered to have immunomodulatory properties. Their role during neuroinflammation had been investigated using mutant mice devoid of either one of their subunits or lacking components of their receptors, yielding conflicting results. We sought to understand the therapeutic potential of IL-27 and IL-35 delivered by gene therapy in neuroinflammation. We constructed lentiviral vectors expressing IL-27 and IL-35 from a single polypeptide chain, and we validated in vitro their biological activity. We injected IL-27 and IL-35-expressing lentiviral vectors into the cerebrospinal fluid (CSF) of mice affected by experimental neuroinflammation (EAE), and performed clinical, neuropathological and immunological analyses. Both cytokines interfere with neuroinflammation, but only IL-27 significantly modulates disease development, both clinically and neuropathologically. IL-27 protects from autoimmune inflammation by inhibiting granulocyte macrophages colony-stimulating factor (GM-CSF) expression in CD4+ T cells and by inducing program death-ligand 1 (PD-L1) expression in both CNS-resident and CNS-infiltrating myeloid cells. We demonstrate here that IL-27 holds therapeutic potential during neuroinflammation and that IL-27 inhibits GM-CSF and induces pd-l1 mRNA in vivo.
Asunto(s)
Sistema Nervioso Central/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inflamación/metabolismo , Interleucina-27/metabolismo , Interleucinas/metabolismo , Leucocitos/metabolismo , Animales , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Líquido Cefalorraquídeo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR-BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies.