RESUMO
Fatty acid elongase ELOVL5 is part of a protein family of multipass transmembrane proteins that reside in the endoplasmic reticulum where they regulate long-chain fatty acid elongation. A missense variant (c.689G>T p.Gly230Val) in ELOVL5 causes Spinocerebellar Ataxia subtype 38 (SCA38), a neurodegenerative disorder characterized by autosomal dominant inheritance, cerebellar Purkinje cell demise and adult-onset ataxia. Having previously showed aberrant accumulation of p.G230V in the Golgi complex, here we further investigated the pathogenic mechanisms triggered by p.G230V, integrating functional studies with bioinformatic analyses of protein sequence and structure. Biochemical analysis showed that p.G230V enzymatic activity was normal. In contrast, SCA38-derived fibroblasts showed reduced expression of ELOVL5, Golgi complex enlargement and increased proteasomal degradation with respect to controls. By heterologous overexpression, p.G230V was significantly more active than wild-type ELOVL5 in triggering the unfolded protein response and in decreasing viability in mouse cortical neurons. By homology modelling, we generated native and p.G230V protein structures whose superposition revealed a shift in Loop 6 in p.G230V that altered a highly conserved intramolecular disulphide bond. The conformation of this bond, connecting Loop 2 and Loop 6, appears to be elongase-specific. Alteration of this intramolecular interaction was also observed when comparing wild-type ELOVL4 and the p.W246G variant which causes SCA34. We demonstrate by sequence and structure analyses that ELOVL5 p.G230V and ELOVL4 p.W246G are position-equivalent missense variants. We conclude that SCA38 is a conformational disease and propose combined loss of function by mislocalization and gain of toxic function by ER/Golgi stress as early events in SCA38 pathogenesis.
Assuntos
Ataxias Espinocerebelares , Animais , Camundongos , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Ataxia , Elongases de Ácidos Graxos/genética , Sequência de Aminoácidos , MutaçãoRESUMO
Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Ataxias Espinocerebelares/congênito , Animais , Feminino , Técnicas de Introdução de Genes , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Células de Purkinje/fisiologia , Células de Purkinje/ultraestrutura , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologiaRESUMO
Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (â¼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.
Assuntos
Elementos Facilitadores Genéticos , Lamina Tipo B/genética , Doença de Pelizaeus-Merzbacher/genética , Deleção de Sequência , Animais , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Lamina Tipo B/metabolismo , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Dados de Sequência Molecular , LinhagemRESUMO
Analysis of genomic DNA methylation by generating epigenetic signature profiles (episignatures) is increasingly being implemented in genetic diagnosis. Here we report our experience using episignature analysis to resolve both uncomplicated and complex cases of neurodevelopmental disorders (NDDs). We analyzed 97 NDDs divided into (1) a validation cohort of 59 patients with likely pathogenic/pathogenic variants characterized by a known episignature and (2) a test cohort of 38 patients harboring variants of unknown significance or unidentified variants. The expected episignature was obtained in most cases with likely pathogenic/pathogenic variants (53/59 [90%]), a revealing exception being the overlapping profile of two SMARCB1 pathogenic variants with ARID1A/B:c.6200, confirmed by the overlapping clinical features. In the test cohort, five cases showed the expected episignature, including (1) novel pathogenic variants in ARID1B and BRWD3; (2) a deletion in ATRX causing MRXFH1 X-linked mental retardation; and (3) confirmed the clinical diagnosis of Cornelia de Lange (CdL) syndrome in mutation-negative CdL patients. Episignatures analysis of the in BAF complex components revealed novel functional protein interactions and common episignatures affecting homologous residues in highly conserved paralogous proteins (SMARCA2 M856V and SMARCA4 M866V). Finally, we also found sex-dependent episignatures in X-linked disorders. Implementation of episignature profiling is still in its early days, but with increasing utilization comes increasing awareness of the capacity of this methodology to help resolve the complex challenges of genetic diagnoses.
Assuntos
Metilação de DNA , Transtornos do Neurodesenvolvimento , Humanos , Metilação de DNA/genética , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/diagnóstico , Masculino , Feminino , Fatores de Transcrição/genética , Criança , Epigênese Genética , Pré-Escolar , Proteínas de Ligação a DNA/genética , Mutação , AdolescenteRESUMO
Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.
Assuntos
Genes Ligados ao Cromossomo X , Inativação do Cromossomo X , Feminino , Humanos , Masculino , Mães , Alelos , Cromossomos , Cromossomos Humanos X/genética , Proteínas de Neoplasias/genéticaRESUMO
CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of ß(1) and ß(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Microdomínios da Membrana/metabolismo , Monócitos/metabolismo , Transdução de Sinais/fisiologia , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase/genética , Anticorpos Bloqueadores/farmacologia , Antígenos CD/genética , Antígenos CD18/genética , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Microdomínios da Membrana/genética , Monócitos/citologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , ADP-Ribosil Ciclase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antimetabólitos Antineoplásicos/toxicidade , Apoptose , Células Cultivadas , Citarabina/toxicidade , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Células THP-1 , Tiofenos/farmacologiaRESUMO
CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.
Assuntos
ADP-Ribosil Ciclase 1/genética , Transformação Celular Neoplásica/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Glicoproteínas de Membrana/genética , Polimorfismo Genético , Células Cultivadas , Estudos de Coortes , Frequência do Gene , Marcadores Genéticos , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Interleucina-2/farmacologia , Itália/epidemiologia , Leucemia Linfocítica Crônica de Células B/epidemiologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Linfoma Difuso de Grandes Células B/epidemiologia , Prognóstico , Fatores de Risco , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Nicotinamide adenine dinucleotide (NAD(+)), a precursor of molecules involved in cell regulatory processes, is released in extra-cellular compartments after stress or inflammation.This study investigates the expression in the human cornea of CD38 and CD157, two NAD(+)-consuming ectoenzymes and surface receptors. The analysis in corneal epithelial and stromal cells was performed by means of multiple approaches, which included immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and confocal microscopy. The presence of enzymatically active NAD(+)-consumers in intact corneal cells was analyzed by high performance liquid chromatography (HPLC)-based assays. The results obtained show that CD38 and CD157 are expressed constitutively by corneal cells: CD38 appears as a 45-kDa monomer, while CD157 is a 42- to 45-kDa doublet. The molecules are enzymatically active, with features reminiscent of those observed in human leukocytes. CD38 is expressed by cells of the suprabasal limbal epithelium, whereas it is not detectable in central corneal epithelium and stroma. CD157 is expressed by basal limbal clusters, a p63(+)/cytokeratin 19(+) cell subset reported to contain corneal stem cells, and by stromal cells. The results of the work indicates that the human cornea is equipped with molecular tools capable of consuming extracellular NAD(+), and that CD157 is a potential marker of corneal limbal cells in the stem cell niche. The presence and characteristics of these ectoenzymes may be exploited to design drugs for wound repair or for applications in tissue transplantation.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Limbo da Córnea , Nicho de Células-Tronco , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase 1/genética , Antígenos CD/genética , Biomarcadores/metabolismo , Córnea/anatomia & histologia , Córnea/metabolismo , Proteínas Ligadas por GPI , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/enzimologia , NAD/metabolismoRESUMO
The leukocyte ectonucleotidases are a recently defined family included in the last Human Leukocyte Differentiation Antigens Workshop, giving prominence to these membrane proteins whose catalytic activity is expressed outside the cell. Among the most important substrates of the leukocyte ectonucleotidases are extracellular ATP and NAD+ whose transient increases are not immunologically silent but rather perceived as danger signals by the host. Among the host responses to the release of ATP, NAD+ and related small molecules is their breakdown on behalf of a panel of leukocyte ectonucleotidases - CD38, CD39, CD73, CD157, CD203a and CD203c -, whose activities are concatenated to form two nucleotide-catabolizing channels defined as the canonical and non-canonical adenosinergic pathways. Here, after briefly reviewing the structure and function of the proteins involved in these pathwys, we focus on the genes encoding the ectoenzymes of these adenosinergic pathways. The chromosomal localizations of the enzyme-encoding genes yield a first level of information concerning their origins by duplication and modes of regulation. Further information was obtained from phylogenetic analyses that show ectoenzyme orthologs are conserved in major tetrapod species whereas examination of synteny conservation revealed that the chromosomal regions harboring the ADP-ribosyl cyclases on human chromosome 4 and the ENTPDase CD39 on chromosome 10 show striking similarities in gene content consistent with their being paralogous chromosomal regions derived from a vertebrate whole genome duplication. Thus the connections between some of the leukocyte ectoenzymes run deeper than previously imagined.
Assuntos
Antígenos CD/classificação , Antígenos CD/genética , Leucócitos/enzimologia , Filogenia , Adenosina/metabolismo , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Evolução Molecular , Regulação da Expressão Gênica/imunologia , Humanos , Leucócitos/metabolismo , Nucleotidases/genética , Domínios Proteicos , Transdução de SinaisRESUMO
: Human CD157/BST-1 and CD38 are dual receptor-enzymes derived by gene duplication that belong to the ADP ribosyl cyclase gene family. First identified over 30 years ago as Mo5 myeloid differentiation antigen and 10 years later as Bone Marrow Stromal Cell Antigen 1 (BST-1), CD157 proved not to be restricted to the myeloid compartment and to have a diversified functional repertoire ranging from immunity to cancer and metabolism. Despite being a NAD+-metabolizing ectoenzyme anchored to the cell surface through a glycosylphosphatidylinositol moiety, the functional significance of human CD157 as an enzyme remains unclear, while its receptor role emerged from its discovery and has been clearly delineated with the identification of its high affinity binding to fibronectin. The aim of this review is to provide an overview of the immunoregulatory functions of human CD157/BST-1 in physiological and pathological conditions. We then focus on CD157 expression in hematological tumors highlighting its emerging role in the interaction between acute myeloid leukemia and extracellular matrix proteins and its potential utility for monoclonal antibody targeted therapy in this disease.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase/química , Imunidade Adaptativa , Antígenos CD/química , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Suscetibilidade a Doenças , Ativação Enzimática , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunidade Inata , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Terapia de Alvo Molecular , Células Mieloides/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Distribuição TecidualRESUMO
BACKGROUND: CD157/Bst1 glycoprotein is expressed in >85% of malignant pleural mesotheliomas and is a marker of enhanced tumor aggressiveness. RESULTS: In vitro, mesothelial cells (malignant and non-malignant) released CD157 in soluble form or as an exosomal protein. In vivo, sCD157 is released and can be measured in pleural effusions by ELISA. Significantly higher levels of effusion sCD157 were detected in patients with malignant pleural mesothelioma than in patients with non-mesothelioma tumors or with non-malignant conditions. In our patient cohort, the area under the receiver-operating characteristic curve for sCD157 that discriminated malignant pleural mesothelioma from all other causes of pleural effusion was 0.685, cut-off (determined by the Youden Index) = 23.66 ng/ml (62.3% sensitivity; 73.93% specificity). Using a cut-off that yielded 95.58% specificity, measurement of sCD157 in cytology-negative effusions increased sensitivity of malignant pleural mesothelioma diagnosis from 34.42% to 49.18%. CONCLUSIONS: Evaluation of soluble CD157 in pleural effusions provides a diagnostic aid in malignant mesothelioma. METHODS: Soluble CD157 (sCD157) was detected biochemically in culture supernatants of malignant and non-malignant mesothelial cells, and in pleural effusions from various pathological conditions. An ELISA system was established to measure the concentration of sCD157 in fluids, and extended to analyze sCD157 in pleural effusions from a cohort of 295 patients.
RESUMO
CD157/Bst1 is a dual-function receptor and ß-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclase family. Expressed in human peripheral blood neutrophils and monocytes, CD157 interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signalling in inflammation. CD157 also regulates cell migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. One form of CD157 is known to date: the canonical sequence of 318 aa from a 9-exon transcript encoded by BST1 on human chromosome 4. Here we describe a second BST1 transcript, consisting of 10 exons, in human neutrophils. This transcript includes an unreported exon, exon 1b, located between exons 1 and 2 of BST1. Inclusion of exon 1b in frame yields CD157-002, a novel proteoform of 333 aa: exclusion of exon 1b by alternative splicing generates canonical CD157, the dominant proteoform in neutrophils and other tissues analysed here. In comparative functional analyses, both proteoforms were indistinguishable in cell surface localization, specific mAb binding, and behaviour in cell adhesion and migration. However, NAD glycohydrolase activity was detected in canonical CD157 alone. Comparative phylogenetics indicate that exon 1b is a genomic innovation acquired during primate evolution, pointing to the importance of alternative splicing for CD157 function.
Assuntos
ADP-Ribosil Ciclase/genética , Processamento Alternativo/genética , Antígenos CD/genética , Éxons/genética , Primatas/genética , ADP-Ribosil Ciclase/metabolismo , Animais , Antígenos CD/metabolismo , Sequência de Bases , Adesão Celular , Sequência Conservada/genética , Evolução Molecular , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HeLa , Humanos , Neutrófilos/metabolismo , Filogenia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Especificidade da Espécie , Células THP-1RESUMO
Demyelinating polyneuropathiy associated with IgM paraproteinemia and high titers of anti-MAG IgM antibodies (MAG-PN) is considered different from chronic inflammatory demyelinating polyneuropathy, particularly because of the poorer response to treatment of MAG-PN patients. Therefore, anti-MAG anitbodies may have relevant prognostic value. Available anti-MAG antibody assays require central nervous system myelin proteins from autopsied human brains. This study investigated the feasibility of detecting anti-MAG antibody by immunofluorescence and flow cytometry using a panel of human neuroblastoma cell lines as targets. We report here on the evaluation of the LA-N-1 cell line as an appropriate substrate for the detection of anti-MAG antibody by indirect immunoflourescence.
Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Glicoproteína Associada a Mielina/imunologia , Neuroblastoma/metabolismo , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anti-Idiotípicos/imunologia , Ligação Competitiva/imunologia , Antígenos CD57/imunologia , Antígenos CD57/metabolismo , Linhagem Celular Tumoral , Demografia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , Immunoblotting/métodos , Masculino , Pessoa de Meia-Idade , Glicoproteína Associada a Mielina/metabolismo , Neuroblastoma/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/sangueRESUMO
CD38 is a progression marker in HIV-1 infection, it displays lateral association with CD4, and down-modulates gp120/CD4 binding. The aim of this study was to elucidate the mechanism behind the interplay between CD4, CD38, and HIV-1. We used mouse cell transfectants expressing human CD4 and either CD38 or other CD4-associated molecules to show that CD38 specifically inhibits gp120/CD4 binding. Human cell transfectants expressing truncated forms of CD38 and bioinformatic analysis were used to map the anti-HIV activity and show that it is concentrated in the membrane-proximal region. This region displayed significant sequence-similarity with the V3 loop of the HIV-1 gp120 glycoprotein. In line with this similarity, synthetic soluble peptides derived from this region reproduced the anti-HIV effects of full-length CD38 and inhibited HIV-1 and HIV-2 primary isolates from different subtypes and with different coreceptor use. A multiple-branched peptide construct presenting part of the sequence of the V3-like region potently and selectively inhibited HIV-1 replication in the nanomolar range. Conversely, a deletion in the V3-like region abrogated the anti-HIV-1 activity of CD38 and its lateral association with CD4. These findings may provide new insights into the early events of HIV-1 fusion and strategies to intervene.
Assuntos
ADP-Ribosil Ciclase/química , Antígenos CD/química , Proteína gp120 do Envelope de HIV/química , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase 1 , Motivos de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD4/fisiologia , Linhagem Celular , Regulação para Baixo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Humanos , Fusão de Membrana , Glicoproteínas de Membrana , Camundongos , Modelos Biológicos , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Receptores Virais/fisiologia , Homologia de Sequência de Aminoácidos , Transfecção , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.
Assuntos
ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/genética , Antígenos CD/química , Antígenos CD/genética , Mapeamento de Epitopos/métodos , Macaca fascicularis/genética , Filogenia , ADP-Ribosil Ciclase/biossíntese , ADP-Ribosil Ciclase/imunologia , ADP-Ribosil Ciclase 1 , Animais , Anticorpos Monoclonais/biossíntese , Antígenos CD/biossíntese , Antígenos CD/imunologia , Linfócitos B/química , Linfócitos B/metabolismo , Células COS/química , Células COS/metabolismo , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular/métodos , Reações Cruzadas/genética , DNA Complementar/genética , Dissulfetos/química , Dissulfetos/metabolismo , Ditiotreitol/farmacologia , Epitopos/genética , Epitopos/metabolismo , Regulação da Expressão Gênica/genética , Genoma , Genoma Humano , Humanos , Glicoproteínas de Membrana , Camundongos , Peso Molecular , Células NIH 3T3/química , Células NIH 3T3/metabolismo , Especificidade da EspécieRESUMO
The major ADP-ribosylating enzyme families are the focus of this special issue of Frontiers in Bioscience . However, there is room for another family of enzymes with the capacity to utilize nicotinamide adenine dinucleotide (NAD): the ADP-ribosyl cyclases (ARCs). These unique enzymes catalyse the cyclization of NAD to cyclic ADP ribose (cADPR), a widely distributed second messenger. However, the ARCs are versatile enzymes that can manipulate NAD, NAD phosphate (NADP) and other substrates to generate various bioactive molecules including nicotinic acid adenine dinucleotide diphosphate (NAADP) and ADP ribose (ADPR). This review will focus on the group of well-characterized invertebrate and vertebrate ARCs whose common gene structure allows us to trace their origin to the ancestor of bilaterian animals. Behind a facade of gene and protein homology lies a family with a disparate functional repertoire dictated by the animal model and the physical trait under investigation. Here we present a phylogenetic view of the ARCs to better understand the evolution of function in this family.
Assuntos
ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase/genética , Antígenos CD/genética , Evolução Molecular , Filogenia , ADP-Ribosil Ciclase/classificação , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/classificação , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antígenos CD/classificação , Antígenos CD/metabolismo , Proteínas Ligadas por GPI/classificação , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , NAD/metabolismo , Especificidade da EspécieRESUMO
CD157 is a member of the ADP-ribosyl cyclase gene family that is involved in the metabolism of NAD. CD157 behaves both as an ectoenzyme and as a receptor. Though CD157 is anchored to the membrane by a glycosylphosphatidylinositol moiety, which makes it unsuitable to transduce signals on its own, it exploits its localization in selected membrane microdomains and its proclivity to interact with integrins to accomplish receptor functions. Initially characterized as a stromal and myeloid antigen involved in the control of leukocyte adhesion, migration and diapedesis, CD157 was subsequently found to have a far wider distribution. In particular, CD157 was found to be expressed by epithelial ovarian cancer cells where it is involved in interactions among tumor cells, extracellular matrix proteins and mesothelium. The overall picture inferred from experimental and clinical observations is that CD157 is a critical player both in leukocyte trafficking and in ovarian cancer invasion and metastasis formation. In this review, we will discuss the biological mechanisms underpinning the role of CD157 in the control of leukocyte migration and ovarian cancer dissemination.
Assuntos
ADP-Ribosil Ciclase/imunologia , Antígenos CD/imunologia , Leucócitos/patologia , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Feminino , Proteínas Ligadas por GPI/imunologia , Humanos , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Ovarianas/imunologiaRESUMO
Malignant mesothelioma is a deadly tumor whose diagnosis and treatment remain very challenging. There is an urgent need to advance our understanding of mesothelioma biology and to identify new molecular markers for improving management of patients. CD157 is a membrane glycoprotein linked to ovarian cancer progression and mesenchymal differentiation. The common embryonic origin of ovarian epithelial cells and mesothelial cells and the evident similarities between ovarian and mesothelial cancer prompted us to investigate the biological role and clinical significance of CD157 in malignant pleural mesothelioma (MPM). CD157 mRNA and protein were detected in four of nine MPM cell lines of diverse histotype and in 85.2% of MPM surgical tissue samples (32/37 epithelioid; 37/44 biphasic). CD157 expression correlated with clinical aggressiveness in biphasic MPM. Indeed, high CD157 was a negative prognostic factor and an independent predictor of poor survival for patients with biphasic MPM by multivariate survival analysis (HR = 2.433, 95% CI 1.120-5.284; p = 0.025). In mesothelioma cell lines, CD157 gain (in CD157-negative cells) or knockdown (in CD157-positive cells) affected cell growth, migration, invasion and tumorigenicity, most notably in biphasic MPM cell lines. In these cells, CD157 expression was associated with increased activation of the mTOR signaling pathway, resulting in decreased platinum sensitivity. Moreover, a trend towards reduced survival was observed in patients with biphasic MPM receiving postoperative platinum-based chemotherapy. These findings indicate that CD157 is implicated in multiple aspects of MPM progression and suggest that CD157 expression could be used to stratify patients into different prognostic groups or to select patients that might benefit from particular chemotherapeutic approach.
Assuntos
ADP-Ribosil Ciclase/biossíntese , Antígenos CD/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , ADP-Ribosil Ciclase/análise , Antígenos CD/análise , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/biossíntese , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/diagnóstico , Mesotelioma/patologia , Mesotelioma Maligno , Pessoa de Meia-Idade , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/patologia , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Resultado do TratamentoRESUMO
CD38 (also known as T10) was identified in the late 1970s in the course of pioneering work carried out at the Dana-Farber Cancer Center (Boston, MA) that focused on the identification of surface molecules involved in antigen recognition. CD38 was initially found on thymocytes and T lymphocytes, but today we know that the molecule is found throughout the immune system, although its expression levels vary. Because of this, CD38 was considered an "activation marker," a term still popular in routine flow cytometry. This review summarizes the findings obtained from different approaches, which led to CD38 being re-defined as a multifunctional molecule. CD38 and its homologue CD157 (BST-1), contiguous gene duplicates on human chromosome 4 (4p15), are part of a gene family encoding products that modulate the social life of cells by means of bidirectional signals. Both CD38 and CD157 play dual roles as receptors and ectoenzymes, endowed with complex activities related to signaling and cell homeostasis. The structure-function analysis presented here is intended to give clinical scientists and flow cytometrists a background knowledge of these molecules. The link between CD38/CD157 and human diseases will be explored here in the context of chronic lymphocytic leukemia, myeloma and ovarian carcinoma, although other disease associations are also known. Thus CD38 and CD157 have evolved from simple leukocyte activation markers to multifunctional molecules involved in health and disease. Future tasks will be to explore their potential as targets for in vivo therapeutic interventions and as regulators of the immune response.