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1.
Blood ; 137(14): 1895-1904, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33036024

RESUMO

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% 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, ∼0.2% 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.


Assuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Cristalografia por Raios X , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Células HEK293 , Humanos , Modelos Moleculares , Domínios Proteicos , Receptores de Antígenos de Linfócitos B/química , Hipermutação Somática de Imunoglobulina
2.
Proc Natl Acad Sci U S A ; 117(8): 4320-4327, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32047037

RESUMO

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 ∼30% 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.


Assuntos
Cadeias lambda de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos B/imunologia , Estudos de Coortes , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Predisposição Genética para Doença , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Mutação Puntual , Receptores de Antígenos de Linfócitos B/genética
3.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

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.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
4.
J Struct Biol ; 210(1): 107462, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962159

RESUMO

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.


Assuntos
Metionina Adenosiltransferase/metabolismo , Pyrococcus furiosus/enzimologia , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Metionina Adenosiltransferase/química , Conformação Proteica , Pyrococcus furiosus/metabolismo
5.
Commun Biol ; 3(1): 27, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941999

RESUMO

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.


Assuntos
Regulação Alostérica , Modelos Moleculares , Conformação Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Linhagem Celular , Membrana Celular/metabolismo , Cloridrato de Fingolimode/química , Cloridrato de Fingolimode/farmacologia , Humanos , Cinética , Fosforilação , Pró-Proteína Convertases/química , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , beta-Arrestinas/química , beta-Arrestinas/metabolismo
6.
Sci Rep ; 9(1): 13530, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537859

RESUMO

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.


Assuntos
Movimento Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Proteínas do Tecido Nervoso/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia
7.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547231

RESUMO

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.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias/irrigação sanguínea , Oligopeptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Inibidores da Angiogênese/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Oligopeptídeos/química , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/química
8.
ACS Chem Biol ; 14(8): 1845-1854, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31345020

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Alinhamento de Sequência
9.
Diabetes ; 68(2): 258-265, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30665954

RESUMO

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.


Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Animais , Humanos , Mutação/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética
10.
Blood ; 132(22): 2362-2374, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

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.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Mutação em Linhagem Germinativa , Síndromes de Imunodeficiência/genética , Linfócitos T/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Feminino , Homozigoto , Humanos , Síndromes de Imunodeficiência/patologia , Masculino , Modelos Moleculares , Linhagem , Conformação Proteica , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/patologia , Linfócitos T/metabolismo
11.
J Clin Microbiol ; 56(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29540456

RESUMO

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.


Assuntos
Antibióticos Antituberculose/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Genótipo , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Rifampina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Fatores de Tempo , Tuberculose/microbiologia
12.
Front Immunol ; 9: 2767, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30692987

RESUMO

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.


Assuntos
Adenosina Desaminase/deficiência , Síndrome Linfoproliferativa Autoimune/terapia , Transplante de Células-Tronco Hematopoéticas , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Condicionamento Pré-Transplante , Doadores não Relacionados , Adenosina Desaminase/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Síndrome Linfoproliferativa Autoimune/enzimologia , Síndrome Linfoproliferativa Autoimune/imunologia , Síndrome Linfoproliferativa Autoimune/patologia , Pré-Escolar , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Neutropenia/enzimologia , Neutropenia/imunologia , Neutropenia/patologia , Neutropenia/terapia , Transplante Homólogo , Receptor fas/imunologia
13.
Sci Rep ; 7(1): 16547, 2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29185463

RESUMO

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.


Assuntos
Sistema Nervoso Central/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Inflamação/metabolismo , Interleucina-27/metabolismo , Interleucinas/metabolismo , Leucócitos/metabolismo , Animais , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Líquido Cefalorraquidiano/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Terapia Genética , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
14.
Nat Commun ; 8: 15746, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28598442

RESUMO

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.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/química , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Dimerização , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais
15.
Nucleic Acids Res ; 44(7): 3448-63, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26896805

RESUMO

Sotos syndrome is an overgrowth syndrome caused by mutations within the functional domains ofNSD1 gene coding for NSD1, a multidomain protein regulating chromatin structure and gene expression. In particular, PHDVC5HCHNSD1 tandem domain, composed by a classical (PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent transcriptional regulation and interacts with the C2HR domain of transcriptional repressor Nizp1 (C2HRNizp1)in vitro To get molecular insights into the mechanisms dictating the patho-physiological relevance of the PHD finger tandem domain, we solved its solution structure and provided a structural rationale for the effects of seven Sotos syndrome point-mutations. To investigate PHDVC5HCHNSD1 role as structural platform for multiple interactions, we characterized its binding to histone H3 peptides and to C2HRNizp1 by ITC and NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal tails, conversely we proved specific binding to C2HRNizp1 We solved C2HRNizp1 solution structure and generated a 3D model of the complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1 pathological mutations, thus impacting on the repression of growth-promoting genes, leading to overgrowth conditions.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Síndrome de Sotos/genética , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína
16.
Sci Rep ; 5: 15401, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26486184

RESUMO

Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.


Assuntos
Fatores de Iniciação em Eucariotos/genética , Técnicas In Vitro/métodos , Ribossomos/genética , Animais , Artemia/genética , Artemia/metabolismo , Sítios de Ligação , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Ligação Proteica/genética , Ribossomos/metabolismo
17.
mBio ; 5(5): e01819-14, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25336456

RESUMO

Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.


Assuntos
Amidoidrolases/genética , Amidoidrolases/metabolismo , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Variação Genética , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Humanos , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Análise de Sequência de DNA , Tuberculose/microbiologia
18.
J Cell Sci ; 127(Pt 19): 4260-9, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25097228

RESUMO

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Humanos , Chaperonas Moleculares/genética , Controle de Qualidade , Via Secretória
19.
Biochim Biophys Acta ; 1844(3): 656-62, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24473221

RESUMO

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480µM. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5'-OH group or with the 2'-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.


Assuntos
Escherichia coli/enzimologia , N-Glicosil Hidrolases/química , Catálise , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Peso Molecular , N-Glicosil Hidrolases/isolamento & purificação , Solventes/química , Especificidade por Substrato
20.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 8): 1553-66, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23897478

RESUMO

Sleeping sickness is a deadly disease that primarily affects sub-Saharan Africa and is caused by protozoan parasites of the Trypanosoma genus. Trypanosomes are purine auxotrophs and their uptake pathway has long been appreciated as an attractive target for drug design. Recently, one tight-binding competitive inhibitor of the trypanosomal purine-specific nucleoside hydrolase (IAGNH) showed remarkable trypanocidal activity in a murine model of infection. Here, the enzymatic characterization of T. brucei brucei IAGNH is presented, together with its high-resolution structures in the unliganded form and in complexes with different inhibitors, including the trypanocidal compound UAMC-00363. A description of the crucial contacts that account for the high-affinity inhibition of IAGNH by iminoribitol-based compounds is provided and the molecular mechanism underlying the conformational change necessary for enzymatic catalysis is identified. It is demonstrated for the first time that metalorganic complexes can compete for binding at the active site of nucleoside hydrolase enzymes, mimicking the positively charged transition state of the enzymatic reaction. Moreover, we show that divalent metal ions can act as noncompetitive IAGNH inhibitors, stabilizing a nonproductive conformation of the catalytic loop. These results open a path for rational improvement of the potency and the selectivity of existing compounds and suggest new scaffolds that may be used as blueprints for the design of novel antitrypanosomal compounds.


Assuntos
Inibidores Enzimáticos/química , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/química , Tripanossomicidas/química , Trypanosoma brucei brucei/enzimologia , Adenosina/análogos & derivados , Adenosina/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Isoenzimas , Cinética , Ligantes , Metais/química , Metais/farmacologia , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Conformação Proteica , Trypanosoma brucei brucei/genética
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