RESUMO
Transaldolase (TAL) is an enzyme in the pentose phosphate pathway (PPP) that generates NADPH for protection against oxidative stress. While deficiency of other PPP enzymes, such as transketolase (TKT), are incompatible with mammalian cell survival, mice lacking TAL are viable and develop progressive liver disease attributed to oxidative stress. Mice with homozygous or heterozygous TAL deficiency are predisposed to cirrhosis, hepatocellular carcinoma (HCC) and acetaminophen (APAP)-induced liver failure. Both mice and humans with complete TAL deficiency accumulate sedoheptulose 7-phosphate (S7P). Previous human studies relied on screening patients with S7P accumulation, thus excluding potentially pathogenic haploinsufficiency. Of note, mice with TAL haploinsufficiency are also predisposed to HCC and APAP-induced liver failure which are preventable with oral N-acetylcysteine (NAC) administration. Based on TALDO1 DNA sequencing, we detected functional TAL deficiency due to novel, heterozygous variations in two of 94 healthy adults and four of 27 subjects with APAP-induced liver failure (P = .022). The functional consequences of these variations were individually validated by site-directed mutagenesis of normal cDNA and loss of activity by recombinant enzyme. All four patients with TAL haplo-insufficiency with APAP-induced liver failure were successfully treated with NAC. We also document two novel variations in two of 15 children with previously unexplained liver cirrhosis. Examination of the National Center for Biotechnology Information databases revealed 274 coding region variations have been documented in 1125 TALDO1 sequences relative to 25 variations in 2870 TKT sequences (P < .0001). These findings suggest an unexpected prevalence and variety of genetic changes in human TALDO1 with relevance for liver injury that may be preventable by treatment with NAC.
Assuntos
Acetilcisteína/farmacologia , Haploinsuficiência/efeitos dos fármacos , Falência Hepática/induzido quimicamente , Transaldolase/deficiência , Adulto , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato , Transaldolase/metabolismo , Adulto JovemRESUMO
PURPOSE: In the late1990s, reacting to the outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom that caused a new variant of Creutzfeldt-Jakob disease (vCJD) in humans, manufacturers withdrew bovine heparin from the market in the United States. There have been growing concerns about the adequate supply and safety of porcine heparin. Since the BSE epidemic has been declining markedly, the US Food and Drug Administration reevaluates the vCJD risk via use of bovine heparin. METHODS: We developed a computational model to estimate the vCJD risk to patients receiving bovine heparin injections. The model incorporated information including BSE prevalence, infectivity levels in the intestines, manufacturing batch size, yield of heparin, reduction in infectivity by manufacturing process, and the dose-response relationship. RESULTS: The model estimates a median risk of vCJD infection from a single intravenous dose (10 000 USP units) of heparin made from US-sourced bovine intestines to be 6.9 × 10-9 (2.5-97.fifth percentile: 1.5 × 10-9 -4.3 × 10-8 ), a risk of 1 in 145 million, and 4.6 × 10-8 (2.5-97.fifth percentile: 1.1 × 10-8 -2.6 × 10-7 ), a risk of 1 in 22 million for Canada-sourced products. The model estimates a median risk of 1.4 × 10-7 (2.5-97.fifth percentile: 2.9 × 10-8 -9.3 × 10-7 ) and 9.6 × 10-7 (2.5-97.fifth percentile: 2.1 × 10-7 -5.6 × 10-6 ) for a typical treatment for venous thromboembolism (infusion of 2-4 doses daily per week) using US-sourced and Canada-sourced bovine heparin, respectively. CONCLUSIONS: The model estimates the vCJD risk from use of heparin when appropriately manufactured from US or Canadian cattle is likely small. The model and conclusions should not be applied to other medicinal products manufactured using bovine-derived materials.
Assuntos
Anticoagulantes/efeitos adversos , Síndrome de Creutzfeldt-Jakob/etiologia , Heparina/efeitos adversos , Animais , Bovinos , Aprovação de Drogas , Encefalopatia Espongiforme Bovina/epidemiologia , Humanos , Modelos Teóricos , Fatores de Risco , Reino Unido/epidemiologia , Estados Unidos , United States Food and Drug AdministrationRESUMO
Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8(+) CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2-restricted TAL 168-176-specific CD8(+) T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28-337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8(+) T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.
Assuntos
Autoantígenos/imunologia , Granzimas/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Transaldolase/imunologia , Sequência de Aminoácidos , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/metabolismo , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodendroglia/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transaldolase/metabolismoRESUMO
Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinADeltaE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinADeltaE when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinADeltaE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinADeltaE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinADeltaE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinADeltaE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.
Assuntos
Distonia Muscular Deformante/genética , Inativação Gênica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , Sequência de Bases , Técnicas de Cultura de Células , Células Cultivadas , Distonia Muscular Deformante/metabolismo , Distonia Muscular Deformante/patologia , Fibroblastos/metabolismo , Genes Reporter , Humanos , Luciferases/metabolismo , Chaperonas Moleculares/química , Dados de Sequência Molecular , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.
Assuntos
Apoptose/fisiologia , Homeostase/fisiologia , Mitocôndrias/fisiologia , Via de Pentose Fosfato/fisiologia , Transaldolase/deficiência , Linhagem Celular Transformada , Células Cultivadas , Feminino , Glucose-6-Fosfato/metabolismo , Humanos , Microscopia Eletrônica , Transdução de Sinais , Fosfatos Açúcares/metabolismo , Transaldolase/genéticaRESUMO
Homozygous deletion of three nucleotides coding for Ser-171 (S171) of TAL-H (human transaldolase) has been identified in a female patient with liver cirrhosis. Accumulation of sedoheptulose 7-phosphate raised the possibility of TAL (transaldolase) deficiency in this patient. In the present study, we show that the mutant TAL-H gene was effectively transcribed into mRNA, whereas no expression of the TALDeltaS171 protein or enzyme activity was detected in TALDeltaS171 fibroblasts or lymphoblasts. Unlike wild-type TAL-H-GST fusion protein (where GST stands for glutathione S-transferase), TALDeltaS171-GST was solubilized only in the presence of detergents, suggesting that deletion of Ser-171 caused conformational changes. Recombinant TALDeltaS171 had no enzymic activity. TALDeltaS171 was effectively translated in vitro using rabbit reticulocyte lysates, indicating that the absence of TAL-H protein in TALDeltaS171 fibroblasts and lymphoblasts may be attributed primarily to rapid degradation. Treatment with cell-permeable proteasome inhibitors led to the accumulation of TALDeltaS171 in whole cell lysates and cytosolic extracts of patient lymphoblasts, suggesting that deletion of Ser-171 led to rapid degradation by the proteasome. Although the TALDeltaS171 protein became readily detectable in proteasome inhibitor-treated cells, it displayed no appreciable enzymic activity. The results suggest that deletion of Ser-171 leads to inactivation and proteasome-mediated degradation of TAL-H. Since TAL-H is a regulator of apoptosis signal processing, complete deficiency of TAL-H may be relevant for the pathogenesis of liver cirrhosis.
Assuntos
Complexo de Endopeptidases do Proteassoma/genética , Deleção de Sequência/genética , Serina/genética , Transaldolase/deficiência , Transaldolase/genética , Células Cultivadas , Criança , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Fibroblastos/química , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Cirrose Hepática/enzimologia , Cirrose Hepática/genética , Linfócitos/química , Linfócitos/enzimologia , Linfócitos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/genética , Complexo de Endopeptidases do Proteassoma/fisiologia , Conformação Proteica , RNA/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Deleção de Sequência/fisiologia , Serina/fisiologia , Transaldolase/biossíntese , Transaldolase/metabolismoRESUMO
Dehydroascorbate (DHA), the oxidized form of vitamin C (ascorbate), enhanced antioxidant defenses of human T cells preferentially importing DHA over ascorbate. In itself, DHA did not affect cytosolic or mitochondrial reactive oxygen intermediate levels as monitored by flow cytometry using oxidation-sensitive fluorescent probes. DHA at 200-1,000 microM stimulated activity of pentose phosphate pathway enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase, elevated intracellular glutathione levels, and inhibited H(2)O(2)-induced changes in mitochondrial transmembrane potential and cell death. With respect to the CD4 antigen, DHA selectively enhanced cell-surface expression of the Fas receptor and increased susceptibility of Jurkat and H9 human T cells to Fas-mediated cell death. The data identify DHA as a selective regulator of H(2)O(2)- and Fas-dependent apoptosis pathways.
Assuntos
Antioxidantes/metabolismo , Apoptose/fisiologia , Ácido Ascórbico/metabolismo , Ácido Desidroascórbico/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxidantes/metabolismo , Receptor fas/metabolismo , Animais , Antioxidantes/química , Ácido Ascórbico/química , Ácido Desidroascórbico/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/metabolismo , Oxirredução , Fosfogluconato Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transaldolase/metabolismo , Células Tumorais CultivadasRESUMO
Transaldolase is a target of autoimmunity mediated by T cells and antibody (Ab) in patients with multiple sclerosis. Functional T-cell assays, T- and B-cell epitope mapping, and detection of transaldolase-specific antibodies in patients with multiple sclerosis are described. Recombinant transaldolase was produced in a prokaryotic expression vector for use in Western blot analysis of sera of these patients. Overlapping transaldolase peptides 15 amino acids (aa) long were synthesized onto cellulose membranes to map immunodominant B-cell epitopes. Amino acid sequence homologies between viral peptides and immunodominant B-cell epitopes of transaldolase were identified using a computer-based algorithm. Direct assessment of molecular mimicry between transaldolase B-cell epitopes and related viral peptides is also shown. T-cell epitopes are mapped in a T-cell proliferation assay using multiple sclerosis patient and control donor cells. Autoantigen-specific T cells are identified by MHC-peptide tetramer staining using flow cytometry analysis.
Assuntos
Autoimunidade , Esclerose Múltipla/enzimologia , Esclerose Múltipla/imunologia , Transaldolase/imunologia , Sequência de Aminoácidos , Autoanticorpos/sangue , Autoantígenos , Linfócitos B/imunologia , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Epitopos Imunodominantes/genética , Mimetismo Molecular , Dados de Sequência Molecular , Esclerose Múltipla/genética , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Transaldolase/genéticaRESUMO
Immunotherapeutics include drugs and biologics that render therapeutic benefit by harnessing the power of the immune system. The promise of immune-mediated therapies is target specificity with a consequent reduction in off-target side effects. Recent scientific advances have led to clinical trials of both active and passive immunotherapeutic products that have the potential to convert life-ending diseases into chronic but manageable conditions. Clinical trials investigating immunotherapeutics are ongoing with some trials at advanced stages of development. However, as with many products involving novel mechanisms of action, major regulatory and scientific issues arising with clinical use of immunotherapeutic products remain to be addressed. In this review, we address issues related to different immunotherapeutics and provide recommendations for the characterization and evaluation of these products during various stages of product and clinical development.
RESUMO
Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1-/- and Taldo1+/- mice spontaneously developed HCC, and Taldo1-/- mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1-/- livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced beta-catenin phosphorylation and enhanced c-Jun expression in Taldo1-/- livers reflected adaptation to oxidative stress. Taldo1-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1-/- mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.
Assuntos
Acetilcisteína/farmacologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/prevenção & controle , Transformação Celular Neoplásica/metabolismo , Falência Hepática/induzido quimicamente , Neoplasias Hepáticas/enzimologia , Transaldolase/deficiência , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosforilação , Transaldolase/metabolismo , alfa-Fetoproteínas/metabolismo , beta Catenina/metabolismo , Receptor fas/metabolismoRESUMO
A specific mutation (DeltaE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3alpha co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3alpha. This association with human nesprin-3 appeared to be stronger for torsinADeltaE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3alpha was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular/genética , Proteínas do Citoesqueleto , Distonia Muscular Deformante/genética , Distonia Muscular Deformante/metabolismo , Embrião de Mamíferos/metabolismo , Retículo Endoplasmático/genética , Fibroblastos/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Chaperonas Moleculares/genética , Mutação , Proteínas do Tecido Nervoso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Plectina/genética , Plectina/metabolismo , Estrutura Terciária de Proteína/fisiologia , Vimentina/genética , Vimentina/metabolismo , Cicatrização/genéticaRESUMO
TorsinA is an AAA(+) protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope responsible for early onset torsion dystonia (DYT1). Most cases of this dominantly inherited movement disorder are caused by deletion of a glutamic acid in the carboxyl terminal region of torsinA. We used a sensitive reporter, Gaussia luciferase (Gluc) to evaluate the role of torsinA in processing proteins through the ER. In primary fibroblasts from controls and DYT1 patients most Gluc activity (95%) was released into the media and processed through the secretory pathway, as confirmed by inhibition with brefeldinA and nocodazole. Fusion of Gluc to a fluorescent protein revealed coalignment and fractionation with ER proteins and association of Gluc with torsinA. Notably, fibroblasts from DYT1 patients were found to secrete markedly less Gluc activity as compared with control fibroblasts. This decrease in processing of Gluc in DYT1 cells appear to arise, at least in part, from a loss of torsinA activity, because mouse embryonic fibroblasts lacking torsinA also had reduced secretion as compared with control cells. These studies demonstrate the exquisite sensitivity of this reporter system for quantitation of processing through the secretory pathway and support a role for torsinA as an ER chaperone protein.
Assuntos
Distonia/metabolismo , Distonia/patologia , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Contagem de Células , Retículo Endoplasmático/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Solubilidade , Fatores de TempoRESUMO
OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown origin. Current treatment options are often ineffective or poorly tolerated. Recent observations have revealed mitochondrial hyperpolarization and enhanced Ca2+ fluxing in T cells from SLE patients. Rapamycin, a lipophilic macrolide antibiotic that regulates mitochondrial transmembrane potential and Ca2+ fluxing, has been used safely and effectively to treat renal transplant rejection since 1999. In addition, rapamycin has been shown to ameliorate T cell function and to prolong survival in lupus-prone MRL/lpr mice. We therefore undertook the present study to investigate whether rapamycin is beneficial in patients with SLE. METHODS: Nine patients with clinically active SLE that had been treated unsuccessfully with other immunosuppressive medications began therapy with rapamycin, 2 mg/day orally. Disease activity was assessed with the British Isles Lupus Assessment Group (BILAG) score, SLE Disease Activity Index (SLEDAI), and requirement for prednisone therapy. Mitochondrial transmembrane potential and Ca2+ fluxing were assessed by flow cytometry. RESULTS: In patients treated with rapamycin, the BILAG score was reduced by a mean +/- SEM of 1.93 +/- 0.9 (P = 0.0218), the SLEDAI by 5.3 +/- 0.8 (P = 0.00002), and concurrent prednisone use by 26.4 +/- 6.7 mg/day (P = 0.0062) compared with pre-rapamycin treatment. While mitochondrial hyperpolarization persisted, pretreatment cytosolic and mitochondrial Ca2+ levels and T cell activation-induced rapid Ca2+ fluxing were normalized in rapamycin-treated patients. CONCLUSION: Rapamycin appears to be a safe and effective therapy for SLE that has been refractory to traditional medications. Mitochondrial dysfunction and Ca2+ fluxing could serve as biomarkers to guide decisions regarding future therapeutic interventions in SLE.
Assuntos
Sinalização do Cálcio/fisiologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Sirolimo/uso terapêutico , Linfócitos T/imunologia , Adolescente , Adulto , Antibacterianos/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Técnicas de Cultura de Células , Citometria de Fluxo , Humanos , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Linfócitos T/efeitos dos fármacosRESUMO
Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.
Assuntos
Encéfalo/metabolismo , Citoesqueleto/metabolismo , Chaperonas Moleculares/metabolismo , Mutação/fisiologia , Neuritos/metabolismo , Vimentina/metabolismo , Actinas/metabolismo , Encéfalo/fisiopatologia , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Forma Celular/fisiologia , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Corrente Citoplasmática/fisiologia , Distonia Muscular Deformante/metabolismo , Distonia Muscular Deformante/fisiopatologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Cinesinas , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Neuritos/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
A novel 2986-base transcript encoded by the antisense strand of the HRES-1 human endogenous retrovirus was isolated from peripheral blood lymphocytes. This transcript codes for a 218-amino acid protein, termed HRES-1/Rab4, based on homology to the Rab4 family of small GTPases. Antibody 13407 raised against recombinant HRES-1/Rab4 detected a native protein of identical molecular weight in human T cells. HRES-1 nucleotides 2151-1606, located upstream of HRES-1/Rab4 exon 1, have promoter activity when oriented in the direction of HRES-1/Rab4 transcription. The human immunodeficiency virus, type 1 (HIV-1), tat gene stimulates transcriptional activity of the HRES-1/Rab4 promoter via trans-activation of the HRES-1 long terminal repeat. Transfection of HIV-1 tat into HeLa cells or infection of H9 and Jurkat cells by HIV-1 increased HRES-1/Rab4 protein levels. Overexpression of HRES-1/Rab4 in Jurkat cells abrogated HIV infection, gag p24 production, and apoptosis, whereas dominant-negative HRES-1/Rab4(S27N) had the opposite effects. HRES-1/Rab4 inhibited surface expression of CD4 and targeted it for lysosomal degradation. HRES-1/Rab4(S27N) enhanced surface expression, recycling, and total cellular CD4 content. Infection by HIV elicited a coordinate down-regulation of CD4 and up-regulation of HRES-1/Rab4 in PBL. Moreover, overexpression of HRES-1/Rab4 reduced CD4 expression on peripheral blood CD4+ T cells. Stimulation by HIV-1 of HRES-1/Rab4 expression and its regulation of CD4 recycling reveal novel coordinate interactions between an infectious retrovirus and the human genome.
Assuntos
Antígenos CD4/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tat/genética , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Proteínas rab4 de Ligação ao GTP/metabolismo , Antígenos CD/metabolismo , Apoptose , Sequência de Bases , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Cloranfenicol O-Acetiltransferase/metabolismo , Dependovirus/genética , Suscetibilidade a Doenças , Éxons/genética , Citometria de Fluxo , Produtos do Gene tat/farmacologia , Genes Dominantes , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , Células HeLa , Humanos , Íntrons/genética , Células Jurkat , Lisossomos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico , Transfecção , Proteínas rab4 de Ligação ao GTP/genética , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168-176 (LLFSFAQAV, TALpep) exhibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8+ T cells specific for TALpep was increased in PBMC of HLA-A2+ MS patients, as compared with HLA-A2- MS patients, HLA-A2+ other neurological disease patients, and HLA-A2+ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8+ T cells, and precursor frequency of TAL-specific IFN-gamma-producing T cells was increased in each of seven HLA-A2+ MS patients tested. Stimulation by TALpep or rTAL of PBMC from HLA-A2+ MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lymphoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8+ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR Vbeta repertoire analysis revealed use of the TCR Vbeta14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR Vbeta14 on the cell surface. Moreover, Ab to TCR Vbeta14 abrogated cytotoxicity by HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in patients with MS.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Antígenos HLA-A/imunologia , Esclerose Múltipla/imunologia , Transaldolase/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Estudos de Casos e Controles , Feminino , Antígeno HLA-A2 , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Oligodendroglia/enzimologia , Fragmentos de Peptídeos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Abnormal death signaling in lymphocytes of systemic lupus erythematosus (SLE) patients has been associated with elevation of the mitochondrial transmembrane potential (Delta psi(m)) and increased production of reactive oxygen intermediates (ROI). The resultant ATP depletion sensitizes T cells for necrosis that may significantly contribute to inflammation in patients with SLE. In the present study, the role of mitochondrial signal processing in T cell activation was investigated. CD3/CD28 costimulation of PBL elicited transient mitochondrial hyperpolarization and intracellular pH (pH(i)) elevation, followed by increased ROI production. Baseline Delta psi(m), ROI production, and pH(i) were elevated, while T cell activation-induced changes were blunted in 15 patients with SLE in comparison with 10 healthy donors and 10 rheumatoid arthritis patients. Similar to CD3/CD28 costimulation, treatment of control PBL with IL-3, IL-10, TGF-beta(1), and IFN-gamma led to transient Delta psi(m) elevation. IL-10 had diametrically opposing effects on mitochondrial signaling in lupus and control donors. Unlike healthy or rheumatoid arthritis PBL, cells of lupus patients were resistant to IL-10-induced mitochondrial hyperpolarization. By contrast, IL-10 enhanced ROI production and cell death in lupus PBL without affecting ROI levels and survival of control PBL. Ab-mediated IL-10 blockade or stimulation with antagonistic lymphokine IL-12 normalized baseline and CD3/CD28-induced changes in ROI production and pH(i) with no impact on Delta psi(m) of lupus PBL. The results suggest that mitochondrial hyperpolarization, increased ROI production, and cytoplasmic alkalinization play crucial roles in altered IL-10 responsiveness in SLE.
Assuntos
Citoplasma/metabolismo , Interleucina-10/fisiologia , Membranas Intracelulares/fisiologia , Lúpus Eritematoso Sistêmico/metabolismo , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/imunologia , Regulação para Cima/imunologia , Adolescente , Adulto , Apoptose/imunologia , Células Cultivadas , Citoplasma/fisiologia , Regulação para Baixo/imunologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Soros Imunes/farmacologia , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/farmacologia , Membranas Intracelulares/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Permeabilidade , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
OBJECTIVE: Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (deltapsim) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE. METHODS: We assessed the deltapsim with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin-luciferase assay. RESULTS: Both deltapsim and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased deltapsim and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and deltapsim elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the deltapsim elevation in lupus. CD3:CD28 costimulation led to transient elevation of the deltapsim, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1-ATPase, had similar effects. CONCLUSION: T cell activation and apoptosis are mediated by deltapsim elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE.