RESUMO
Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXß (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.
Assuntos
Inibidores Enzimáticos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , 2,6-Dicloroindofenol/química , 2,6-Dicloroindofenol/farmacologia , Coenzimas/química , Coenzimas/metabolismo , Simulação por Computador , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HL-60 , Humanos , Niacinamida/química , Niacinamida/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
Bioenergetic requirements of hematopoietic stem cells and pluripotent stem cells (PSCs) vary with lineage fate, and cellular adaptations rely largely on substrate (glucose/glutamine) availability and mitochondrial function to balance tricarboxylic acid (TCA)-derived anabolic and redox-regulated antioxidant functions. Heme synthesis and degradation converge in a linear pathway that utilizes TCA cycle-derived carbon in cataplerotic reactions of tetrapyrrole biosynthesis, terminated by NAD(P)H-dependent biliverdin reductases (IXα, BLVRA and IXß, BLVRB) that lead to bilirubin generation and cellular antioxidant functions. We now demonstrate that PSCs with targeted deletion of BLVRB display physiologically defective antioxidant activity and cellular viability, associated with a glutamine-restricted defect in TCA entry that was computationally predicted using gene/metabolite topological network analysis and subsequently validated by bioenergetic and isotopomeric studies. Defective BLVRB-regulated glutamine utilization was accompanied by exaggerated glycolytic accumulation of the rate-limiting hexokinase reaction product glucose-6-phosphate. BLVRB-deficient embryoid body formation (a critical size parameter of early lineage fate potential) demonstrated enhanced sensitivity to the pentose phosphate pathway (PPP) inhibitor 6-aminonicotinamide with no differences in the glycolytic pathway inhibitor 2-deoxyglucose. These collective data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism and suggest that early stages of lineage fate potential require glutamine anaplerotic functions and an intact PPP, which are, in part, regulated by BLVRB activity. In principle, BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization with consequences for cancer and hematopoietic metabolism.
Assuntos
Células-Tronco Embrionárias/metabolismo , Glutamina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/fisiologia , Células Cultivadas , Metabolismo Energético/genética , Técnicas de Introdução de Genes , Glucose/metabolismo , Glicólise/genética , Heme/metabolismo , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Via de Pentose Fosfato/genética , Especificidade por SubstratoRESUMO
Biliverdin reductase IXß (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1)â elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2)â defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3)â provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Serina/química , Animais , Sítios de Ligação , Domínio Catalítico , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , NAD/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Estabilidade Proteica , Serina/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Human blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRB(S111L)) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRB(S111L) encompassed within the substrate/cofactor [α/ß dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXß tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.
Assuntos
Heme/metabolismo , Redes e Vias Metabólicas , Mutação/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Trombopoese/genética , Alelos , Antígenos CD34/metabolismo , Plaquetas/metabolismo , Linhagem da Célula , Estudos de Coortes , Células Eritroides/citologia , Células Eritroides/enzimologia , Estudos de Associação Genética , Hematopoese , Humanos , Megacariócitos/citologia , Megacariócitos/enzimologia , Oxirredução , Polimorfismo de Nucleotídeo Único/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco , Análise de Sequência de RNA , Trombocitose/genéticaRESUMO
ADAMTS-18 is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of proteases, which are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. Mutations of ADAMTS-18 have been linked to abnormal early eye development and reduced bone mineral density. In this review, we briefly summarize the structural organization and the expression of ADAMTS-18. We will also focus on the emerging role of ADAMTS-18 in several pathophysiological conditions which include: hematological diseases, tumorgenesis, osteogenesis, eye-related diseases, central nervous system disorders, and last but not least a research perspective of ADAMTS-18 and its potential as a promising diagnostic and therapeutic target.
Assuntos
Proteínas ADAM/genética , Predisposição Genética para Doença/genética , Modelos Genéticos , Mutação , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Doenças Ósseas/enzimologia , Doenças Ósseas/genética , Oftalmopatias/enzimologia , Oftalmopatias/genética , Doenças Hematológicas/enzimologia , Doenças Hematológicas/genética , Humanos , Neoplasias/enzimologia , Neoplasias/genéticaRESUMO
We previously reported that C-terminal fragment of ADAMTS-18 induces platelet fragmentation through ROS release. We have shown that thrombin cleaves ADAMTS-18 and that a short form of ADAMTS-18 in in vitro translational assay. However, the exact thrombin cleavage site and whether a short form ADAMTS-18 presents in vivo are not clear. In this study, we first identified that the thrombin cleavage site is between Arg775 and Ser776 by thrombin cleavage of ADAMTS-18 peptide following mass spectrum assay. We then showed that a short form ADAMTS-18 presents in brain, kidney, lung, and testicle from C57BL/6 mouse embryo. Since alternative form of ADAMTS-18 could be a mechanism to regulate its activity, we then investigated the mechanism involves in the generation of ADAMTS-18 short form. However, neither protease inhibitors nor mutations in catalytic domain of ADAMTS-18 have any significant effect on the generation of ADAMTS-18 short form. Thus, our data demonstrate a thrombin cleavage site and confirm a short form of ADAMTS-18 presents in vivo.
Assuntos
Proteínas ADAM/química , Trombina/química , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Proteínas ADAMTS , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/genética , Domínio Catalítico , Códon/genética , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Serina/química , Serina/genética , Distribuição TecidualRESUMO
Thrombocytopenia is a common feature of myelodysplastic syndromes (MDS). 5-aza-2'-deoxycytidine (decitabine) has been used to treat MDS with an approximately 20% response rate in thrombocytopenia. However, the mechanism of how decitabine increases platelet count is not clear. In this study, we investigated the effect of decitabine on megakaryocyte maturation and platelet release in the mouse. The effect of decitabine on megakaryocyte maturation was studied in an in vitro megakaryocyte differentiation model utilising mouse bone marrow cells and mouse megakaryoblastic cell line L8057. Decitabine (2.5 µM) is able to induce L8057 cells to differentiate into a megakaryocyte-like polyploidy cells with positive markers of acetylcholinesterase and αIIb integrin (CD41). Higher expression of αIIb integrin was also found in primary mouse bone marrow cells and human cord blood CD34+ cells cultured with both thrombopoietin and decitabine as compared to thrombopoietin alone. In addition, we noted a 30% platelet count increase in Balb/c mice 12 hours after the injection of decitabine at a clinically relevant dose (15 mg/m2), suggesting a rapid platelet release from the spleen or bone marrow. Our data suggest that decitabine increases platelet counts by enhancing platelet release and megakaryocyte maturation.
Assuntos
Azacitidina/análogos & derivados , Plaquetas/efeitos dos fármacos , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Trombopoese/efeitos dos fármacos , Animais , Azacitidina/farmacologia , Plaquetas/citologia , Plaquetas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Metilação de DNA/efeitos dos fármacos , Decitabina , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Humanos , Técnicas In Vitro , Megacariócitos/metabolismo , Camundongos , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/tratamento farmacológico , Contagem de Plaquetas , Poliploidia , Trombocitopenia/sangue , Trombocitopenia/tratamento farmacológico , Regulação para Cima/efeitos dos fármacosRESUMO
We previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 µM). We therefore conclude that the anti-GPIIIa49-66 antibody inhibits MK differentiation through ß3 integrin signalling independent of ROS release.
Assuntos
Plaquetas/efeitos dos fármacos , Diferenciação Celular , Infecções por HIV/imunologia , HIV-1/imunologia , Megacariócitos/efeitos dos fármacos , Anticorpos Bloqueadores/farmacologia , Autoanticorpos/farmacologia , Plaquetas/metabolismo , Plaquetas/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/fisiologia , HIV-1/patogenicidade , Humanos , Integrina beta3/imunologia , Integrina beta3/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Anti-platelet autoantibodies are frequently found in systemic lupus erythematosus (SLE) patients and contribute to the development of SLE-associated immunologic thrombocytopenia (SLE-ITP). Although the correlation of anti-dsDNA autoantibody with platelet-associated antibody has been reported, the potential mechanism underlying such a correlation is incompletely understood. We have reported that anti-platelet integrin GPIIIa49-66 (CAPESIEFPVSEARVLED) autoantibodies play a major role in the development of HIV-1-related thrombocytopenia (HIV-1-ITP). The strong negative charge of GPIIIa49-66 prompts us to investigate whether GPIIIa49-66 can be an epitope mimicking dsDNA. We report here that anti-GPIIIa49-66 antibodies are found in three out of nine SLE-ITP patients. Double-stranded (ds) DNA competitively inhibited the binding of purified patient anti-dsDNA antibodies to GPIIIa49-66 peptide. Both polyclonal and monoclonal anti-GPIIIa49-66 antibodies are able to cross-react with dsDNA. Consistent with previous reports, the DNA binding activities of anti-GPIIIa49-66 antibodies are mainly dependent on the positively charged amino acid in the heavy-chain complementarity-determining region 3 (HCDR3). The HCDR3 of human SLE anti-dsDNA monoclonal antibody (mAb) 412.67 demonstrates a similar positively charged amino acid chain orientation compared with that of anti-GPIIIa49-66 mAb A11, and it cross-reacts with GPIIIa49-66 peptide. Purified anti-GPIIIa49-66 antibodies from SLE-ITP patients are able to induce platelet fragmentation in vitro and to induce thrombocytopenia in vivo. Thus, our data suggest that specific epitope cross-reaction between GPIIIa49-66 and dsDNA could be a mechanism involved in the development of SLE-associated thrombocytopenia.
Assuntos
Reações Cruzadas/imunologia , DNA/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/imunologia , Plaquetas/imunologia , Regiões Determinantes de Complementaridade/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Contagem de Plaquetas , Distribuição AleatóriaRESUMO
Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11. We show that A11 does not induce significant thrombocytopenia or inhibit platelet function. A11 can prevent the cessation of carotid artery flow produced by induced artery injury and dissolve the induced thrombus 2 hours after cessation of blood flow. In addition, A11 can prevent, as well as ameliorate, murine middle cerebral artery stroke, without thrombocytopenia or brain hemorrhage. To further optimize the antithrombotic activity of A11, we produced a bifunctional A11-plasminogen first kringle agent (SLK), which homes to newly deposited fibrin strands within and surrounding the platelet thrombus, reducing effects on nonactivated circulating platelets. Indeed, SLK is able to completely reopen occluded carotid vessels 4 hours after cessation of blood flow, whereas A11 had no effect at 4 hours. Thus, a new antithrombotic agent was developed for platelet thrombus clearance.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Fibrinolíticos/uso terapêutico , Fragmentos de Peptídeos/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Trombose/sangue , Trombose/terapia , Animais , Anticorpos Biespecíficos/genética , Sequência de Bases , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/terapia , Primers do DNA/genética , Humanos , Técnicas In Vitro , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/prevenção & controle , Infarto da Artéria Cerebral Média/terapia , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.
Assuntos
Proteínas ADAM/farmacologia , Plaquetas/metabolismo , Estenose das Carótidas/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Acidente Vascular Cerebral/prevenção & controle , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Animais , Tempo de Sangramento , Plaquetas/patologia , Estenose das Carótidas/metabolismo , Estenose das Carótidas/patologia , AMP Cíclico/farmacologia , Endotélio Vascular/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biblioteca de Peptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/farmacologia , Reação em Cadeia da Polimerase , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Trombina/farmacologia , Veias Umbilicais/citologiaRESUMO
Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1-seropositive drug abusers are more prone to develop immune thrombocytopenia than non-drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non-drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti-GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 nonconserved peptides from HCV core envelope 1. Reactivity correlated inversely with platelet count (r(2) = 0.7, P < .01). Ab raised against peptide PHC09 in GPIIIa(-/-) mice induced thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as titer of anti-GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab versus PHC09 and significantly decreased their platelet count (P < .001). Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66.
Assuntos
Hepatite C/imunologia , Hepatite C/metabolismo , Integrina beta3/metabolismo , Mimetismo Molecular , Trombocitopenia/imunologia , Trombocitopenia/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Anticorpos Antivirais/imunologia , Plaquetas/metabolismo , Contagem de Células , Reações Cruzadas/imunologia , Feminino , Hepacivirus , Hepatite C/complicações , Hepatite C/genética , Integrina beta3/genética , Camundongos , Camundongos Knockout , Estresse Oxidativo , Biblioteca de Peptídeos , Trombocitopenia/etiologia , Trombocitopenia/genética , Proteínas do Envelope Viral/imunologiaRESUMO
We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.
Assuntos
Anticorpos Monoclonais/química , Plaquetas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Integrina beta3/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Mutação , Biblioteca de Peptídeos , Ativação Plaquetária , Agregação Plaquetária , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the beta3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti-GPIIIa49-66-induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated.
Assuntos
Infecções por HIV/complicações , Infecções por HIV/imunologia , HIV-1/imunologia , Mimetismo Molecular , Púrpura Trombocitopênica Idiopática/etiologia , Púrpura Trombocitopênica Idiopática/imunologia , Proteínas dos Retroviridae/imunologia , Sequência de Aminoácidos , Animais , Epitopos/química , Epitopos/genética , Feminino , Anticorpos Anti-HIV , Antígenos HIV/química , Antígenos HIV/genética , HIV-1/genética , Humanos , Imunização Passiva , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Coelhos , Proteínas dos Retroviridae/química , Proteínas dos Retroviridae/genéticaRESUMO
We show that the microRNA miR-155 can be processed from sequences present in BIC RNA, a spliced and polyadenylated but non-protein-coding RNA that accumulates in lymphoma cells. The precursor of miR-155 is likely a transient spliced or unspliced nuclear BIC transcript rather than accumulated BIC RNA, which is primarily cytoplasmic. By using a sensitive and quantitative assay, we find that clinical isolates of several types of B cell lymphomas, including diffuse large B cell lymphoma (DLBCL), have 10- to 30-fold higher copy numbers of miR-155 than do normal circulating B cells. Similarly, the quantities of BIC RNA are elevated in lymphoma cells, but ratios of the amounts of the two RNAs are not constant, suggesting that the level of miR-155 is controlled by transcription and processing. Significantly higher levels of miR-155 are present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this microRNA may be diagnostically useful.
Assuntos
Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/metabolismo , RNA não Traduzido/metabolismo , Linhagem Celular Tumoral , Humanos , MicroRNAs/análise , RNA Mensageiro/metabolismo , RNA não Traduzido/análiseRESUMO
Antiplatelet GPIIIa49-66 Ab of HIV-related thrombocytopenic patients induces thrombocytopenia and platelet fragmentation by the generation of peroxide and other reactive oxygen species (ROS). Here we report the presence of a functional platelet NADPH oxidase pathway that requires activation by the platelet 12-lipoxygenase (12-LO) pathway to fragment platelets. A new Ab-mediated mechanism is described in which the platelet 12-LO product, 12(S)-HETE activates the NADPH oxidase pathway to generate ROS.
Assuntos
Anticorpos/imunologia , Araquidonato 12-Lipoxigenase/metabolismo , Plaquetas/metabolismo , NADPH Oxidases/metabolismo , Plaquetas/imunologia , Proteínas do Sistema Complemento/metabolismo , Infecções por HIV/metabolismo , HIV-1 , Humanos , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Trombocitopenia/metabolismoRESUMO
Primary effusion lymphoma (PEL) is a unique form of malignant lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). The majority of PELs also contain the EBV genome. Although viral infection is believed to play a critical role in the pathogenesis of PEL, it has been suggested that additional molecular lesions are required for the development of PEL. Alternative splicing of pre-mRNA is an important mechanism in the regulation of cellular and viral gene expression. Deregulation of pre-mRNA splicing may shift the gene expression balance and lead to the development of cancer. In order to investigate mRNA splicing in PELs, we examined mRNA splicing of three genes, DNA polymerase beta (pol beta), Bcl-x and CD45, in eight PEL cell lines. We found that the average variant percentage of pol beta in PEL cell lines is two times higher than in peripheral blood mononuclear cells (PBMC) and that the variant pattern of genes bcl-x and CD45 is quite different in PEL cell lines than in PBMC. In addition, we also found that the percentage of variant pol beta increased two-fold in PBMC following Epstein-Barr virus (EBV) infection. Therefore, viral infection may contribute to mRNA alternative splicing in PEL. In order to explore the mechanism by which viral infection affects mRNA splicing, we also examined the roles of genes KS-SM, SM and EBERs and viral copies in mRNA splicing. Our findings indicate that various factors acting as positive or negative regulators may be involved in mRNA alternative splicing caused by viral infection. In conclusion, mRNA splicing in PEL can be altered by viral infection and this alteration may contribute to the pathogenesis of PEL.
Assuntos
Linfoma/genética , Derrame Pleural Maligno/genética , Splicing de RNA/fisiologia , RNA Neoplásico/genética , Transformação Celular Viral/genética , DNA Polimerase beta/genética , Infecções por Vírus Epstein-Barr/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Virais/genética , Herpesvirus Humano 4/genética , Humanos , Antígenos Comuns de Leucócito/genética , Linfoma/etiologia , Linfoma/virologia , Derrame Pleural Maligno/etiologia , Derrame Pleural Maligno/virologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Tumorais Cultivadas , Proteína bcl-XRESUMO
DNA polymerase mu (pol mu) is a novel error-prone DNA repair enzyme bearing significant structural homology with terminal deoxynucleotidyltransferase. Whereas other human error-prone DNA polymerases identified thus far show no preferential lymphoid tissue distribution, the highest levels of pol mu mRNA have been detected in peripheral lymphoid tissues, particularly germinal center B cells. Conceivably, up-regulation of the pol mu gene may be biologically significant in lymphomagenesis, especially in the development of B-cell non-Hodgkin's lymphomas (B-NHLs), because of enhanced error-prone DNA repair activities. To explore this possibility, we generated a digoxigenin-labeled riboprobe to pol mu mRNA and used the probe and in situ hybridization to examine the expression pattern of the pol mu gene in formalin-fixed, paraffin-embedded tissue sections of 37 B-NHLs. This included eight chronic lymphocytic leukemia/small lymphocytic lymphomas, six mantle cell lymphomas, seven follicular lymphomas, nine diffuse large B-cell lymphomas, three splenic marginal zone lymphomas, two Burkitt's lymphomas, and two precursor B-lymphoblastic lymphomas. We also correlated the pol mu mRNA expression levels with the tumor proliferation index, which was assessed in each case by image analysis of Ki-67 immunostained slides. Nineteen of 21 (90%) B-NHLs arising from postgerminal center B cells (follicular lymphomas, diffuse large B-cell lymphomas, splenic marginal zone lymphomas, and Burkitt's lymphomas) exhibited high expression of pol mu mRNA. In contrast, only 2 of 16 (13%) B-NHLs arising from pregerminal center B cells (chronic lymphocytic leukemia/small lymphocytic lymphomas, mantle cell lymphomas, and precursor B-lymphoblastic lymphomas) expressed significant levels of pol mu mRNA. Pol mu gene expression did not seem to correlate with the proliferation index, especially because a significant level of pol mu mRNA was not detected in either case of precursor B-lymphoblastic lymphomas. In conclusion, pol mu gene expression is highly associated with B-NHLs of postgerminal center B-cell derivation. Furthermore, the expression level is independent of the proliferation rate and thus is unrelated to the biological aggressiveness of the tumors. These findings, along with the error-prone nature of the enzyme, suggest that up-regulation of pol mu gene expression may be a contributing factor to the pathogenesis of a subset of B-NHLs through DNA repair-associated genomic instability.