RESUMO
Vertebrate cells contain at least 12 different genes for Hsp70 proteins, 3 of which are encoded in the major histocompatibility complex (MHC) class III region. In the human MHC, these are named Hsp70-1, -2, and -Hom. To characterize these proteins, we have determined their substrate binding specificity, their cellular and tissue distribution, and the regulation of their expression. We show for the first time (1) peptide binding specificity of Hsp70-Hom; (2) endogenous expression of Hsp70-Hom in human cell lines; (3) cytoplasmic location of Hsp70-Hom protein under basal conditions and concentration in the nucleus after heat shock; (4) unique RNA expression profiles in human tissues for each of the MHC-encoded Hsp70s, significantly different from that for the constitutive Hsc70; (5) a relative increase in levels of Hsp70-Hom protein, compared with other Hsp70s, in response to interferon gamma; and (6) a specific increase on lipopolysaccharide (LPS) treatment of in vivo messenger RNA levels for the MHC-encoded Hsp70s and the DnaJ homologue, hdj2, relative to other chaperones. The unique tissue distributions and specific up-regulation by LPS of the MHC-encoded Hsp70s suggest some specialization of functions for these members of the Hsp70 family, possibly in the inflammatory response.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Complexo Principal de Histocompatibilidade , Anticorpos/imunologia , Northern Blotting , Fracionamento Celular , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/sangue , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Células HeLa , Humanos , Interferon gama/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
We investigated how the accessory molecule interactions encountered during T cell priming influence T cell-mediated destruction of insulin-producing beta cells and lead to type 1 diabetes. T cell receptor (TCR)-transgenic CD4+ T cells were primed under controlled conditions in vitro before being adoptively transferred into transgenic recipients expressing membrane ovalbumin under the control of the rat insulin promoter (RIP-mOVA). During priming, antigen-presenting cell expression of B7-1 without intracellular adhesion molecule 1 (ICAM-1) led to the generation of effector cells that migrated to the pancreata of RIP-mOVA recipients but did not cause diabetes. In contrast, when T cells were primed with APCs expressing both B7-1 and ICAM-1, pronounced destruction of beta cells and a rapid onset of diabetes were observed. Pathogenicity was associated with T cell production of the macrophage-attracting chemokines CCL3 and CCL4. Thus, interactions of lymphocyte function-associated antigen 1 with ICAM-1 during priming induce both qualitative and quantitative alterations in T effector function and induce potentially autodestructive responses.
Assuntos
Inflamação/etiologia , Molécula 1 de Adesão Intercelular/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Técnicas In Vitro , Inflamação/imunologia , Inflamação/patologia , Insulina/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ratos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The K(bm1) and K(bm8) natural mutants of the murine MHC class I molecule H-2K(b) were originally identified by allograft rejection. They also bind viral peptides VSV8 and SEV9 with high affinity, but their peptide complexes have substantially decreased thermostability, and the K(bm1) complexes do not elicit alloreactive T cell responses. Crystal structures of the four mutant complexes at 1.7-1.9 A resolution are similar to the corresponding wild-type K(b) structures, except in the vicinity of the mutated residues, which alter the electrostatic potential, topology, hydrogen bonding, and local water structure of the peptide binding groove. Thus, these natural K(b) mutations define the minimal perturbations in the peptide environment that alter antigen presentation to T cells and abolish alloreactivity.
Assuntos
Antígenos H-2/química , Antígenos H-2/imunologia , Animais , Apresentação de Antígeno , Sítios de Ligação , Cristalografia por Raios X , Epitopos/imunologia , Antígenos H-2/classificação , Antígenos H-2/genética , Meia-Vida , Camundongos , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Conformação Proteica , Eletricidade Estática , Propriedades de Superfície , Linfócitos T Citotóxicos/imunologia , TermodinâmicaRESUMO
The claudin superfamily consists of at least 18 homologous proteins in humans. These proteins are important structural and functional components of tight junctions in paracellular transport. Complexed with two other integral transmembrane proteins, occludin and junctional adhesion molecule, claudins are located in both epithelial and endothelial cells in all tight junction-bearing tissues. Claudins interact directly with tight junction-specific, membrane-associated guanylate kinase homologues, ZO-1, ZO-2, and ZO-3, and indirectly with AF-6 and the myosin-binding molecule cingulin. These protein-protein interactions promote scaffolding of the tight junction transmembrane proteins and provide a link to the actin cytoskeleton for transducing regulatory signals to and from tight junctions. The distinct permeability properties observed in different epithelia and endothelia seemingly result from the restricted tissue expression, variability of the homopolymer and heteropolymer assembly, regulated transcription and translation, and the subcellular localization of claudin family proteins. Defects in claudins are causatively associated with a variety of human diseases, demonstrating that claudins play important roles in human physiology. In conditions where the cell adhesion function contributed by tight junctions is essential, such as in altered paracellular transport, in proliferative diseases, and during morphogenesis, the claudin superfamily of homologous proteins provides the molecular basis for the uniqueness of tight junctions and emerges as a new target for intervention.
Assuntos
Proteínas de Membrana/metabolismo , Conformação Proteica , Junções Íntimas/fisiologia , Animais , Transporte Biológico , Moléculas de Adesão Celular/metabolismo , Claudina-1 , Humanos , Moléculas de Adesão Juncional , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Biológicos , Família Multigênica , Ocludina , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Proteína da Zônula de Oclusão-1RESUMO
PURPOSE: This article describes work at the Virginia School of the Deaf and Blind in Hampton, Virginia, USA. Disability sensitivity training in businesses and government organizations has become a more important activity in the United States since the Americans with Disability Act (ADA) of 1992 was published. That act of the US Congress required organizations to hire and make reasonable accommodations for the disabled. ISSUE: Lack of sensitivity or awareness of the plight of the disabled in business and government continues to be a distinct problem requiring attitude changes and training, but this problem can become acute even for professionals in organizations dedicated to the care and education of the disabled. CONCLUSION: Professionals tend to become inured to the hardships of others and lose sight of the need for both verbal and non-verbal sensitivity in the workplace.
Assuntos
Cegueira/reabilitação , Surdez/reabilitação , Educação Continuada/métodos , Pessoal de Saúde , Relações Profissional-Paciente , Reabilitação/educação , Adolescente , Adulto , Atitude do Pessoal de Saúde , Conscientização , Criança , Pré-Escolar , Humanos , VirginiaRESUMO
AIMS: Amyloid has recently been shown to accumulate intracellularly in the brains of patients with Alzheimer's disease (AD), yet amyloid plaques are generally thought to arise from gradual extracellular amyloid deposition. We have investigated the possibility of a link between these two apparently conflicting observations. METHODS AND RESULTS: Immunohistochemistry and digital image analysis was used to examine the detailed localization of beta-amyloid(42) (A beta 42), a major component of amyloid plaques, in the entorhinal cortex and hippocampus of AD brains. A beta 42 first selectively accumulates in the perikaryon of pyramidal cells as discrete, granules that appear to be cathepsin D-positive, suggesting that they may represent lysosomes or lysosome-derived structures. AD brain regions abundantly populated with pyramidal neurones exhibiting excessive A beta 42 accumulations also contained evidence of neuronal lysis. Lysis of these A beta 42-burdened neurones apparently resulted in a local, radial dispersion of their cytoplasmic contents, including A beta 42 and lysosomal enzymes, into the surrounding extracellular space. A nuclear remnant was found at the dense core of many amyloid plaques, strengthening the idea that each amyloid plaque represents the end product of a single neuronal cell lysis. The inverse relationship between the amyloid plaque density and pyramidal cell density in the AD brain regions also supports this possibility, as does the close correlation between plaque size and the size of local pyramidal cells. CONCLUSIONS: Our findings suggest that excessive intracellular accumulation of A beta 42-positive material in pyramidal cells can result in cell lysis, and that cell lysis is an important source of amyloid plaques and neuronal loss in AD brains.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Córtex Entorrinal/metabolismo , Hipocampo/metabolismo , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Células Piramidais/metabolismo , Idoso , Doença de Alzheimer/patologia , Córtex Entorrinal/patologia , Exocitose/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Hipocampo/patologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Placa Amiloide/patologia , Células Piramidais/patologiaRESUMO
Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining body's iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.
Assuntos
Doença , Saúde , Ferro/metabolismo , Anemia/metabolismo , Animais , Hemocromatose/genética , Hemocromatose/metabolismo , Humanos , Absorção Intestinal , Deficiências de Ferro , Receptores da Transferrina/metabolismoRESUMO
The mechanisms controlling induction of anergy at the level of naive CD4+ T cells are poorly understood but thought to reflect limited contact with costimulatory molecules during T cell antigen receptor (TCR) ligation. To clarify this question, naive TCR transgenic CD4+ cells were exposed to specific peptide presented by transfected antigen-presenting cells (APC) expressing MHC class II molecules with defined accessory molecules. Significantly, culturing CD4(+) cells with APC expressing MHC II plus peptide alone elicited early TCR signaling but failed to induce either proliferation or anergy. Culture with APC expressing MHC II plus B7 molecules led to strong proliferation and T cell priming but no anergy. In marked contrast, conspicuous induction of anergy occurred after T cell culture with APC expressing MHC class II and intercellular adhesion molecule-1 (ICAM-1). Thus, at the level of naive CD4(+) cells, anergy induction appears to reflect selective contact with APC expressing ICAM-1 in the absence of B7.
Assuntos
Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Anergia Clonal/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígeno B7-2 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Anergia Clonal/efeitos dos fármacos , Drosophila/citologia , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Interleucinas/imunologia , Interleucinas/farmacologia , Lectinas Tipo C , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Testes de Precipitina , Agregação de Receptores , Receptores de Interleucina-2/metabolismo , TransfecçãoRESUMO
The central event in the cellular immune response to invading pathogens is the presentation of non-self antigenic peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). As peptide binding and transport proteins, MHC class I molecules have evolved distinct biochemical and cellular strategies for acquiring antigenic peptides, providing CTLs an extracellular representation of the intracellular antigen content. Whereas efficient generation of MHC class I binding peptides depends on the intracellular, immunoproteasome-mediated proteolysis machinery, translocation of peptides into the lumen of the endoplasmic reticulum requires the endoplasmic reticulum-resident, adenosine 5'-triphosphate (ATP) binding cassette transporter associated with antigen processing (TAP). Here we show, for the first time, that immunoproteasomes, TAP complexes, and MHC class I molecules are physically associated, providing an effective means of transporting MHC class I binding peptides from their sites of generation into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. In this review, we assess the current understanding of the functional regulation of immunoproteasomes and transporter associated with antigen processing.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Apresentação de Antígeno , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico Ativo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/imunologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Tangier disease (TD) and familial HDL deficiency (FHA) have recently been linked to mutations in the human ATP-binding cassette transporter 1 (hABCA1), a member of the ABC superfamily. Both diseases are characterized by the lowering or lack of high-density lipoprotein cholesterol (HDL-C) and low serum cholesterol. The murine ABCA1-/- phenotype corroborates the human TD linkage to ABCA1. Similar to TD in humans, HDL-C is virtually absent in ABCA1-/- mice accompanied by a reduction in serum cholesterol and lipid deposition in various tissues. In addition, the placenta of ABCA1-/- mice is malformed, resulting in severe embryo growth retardation, fetal loss, and neonatal death. The basis for these defects appears to be altered steroidogenesis, a direct result of the lack of HDL-C. By 6 months of age, ABCA1-/- animals develop membranoproliferative glomerulonephritis due to deposition of immunocomplexes followed by cardiomegaly with ventricular dilation and hypertrophy, ultimately succumbing to congestive heart failure. This murine model of TD will be very useful in the study of lipid metabolism, renal inflammation, and cardiovascular disease and may reveal previously unsuspected relationships between them.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , HDL-Colesterol/deficiência , Modelos Animais de Doenças , Glomerulonefrite/etiologia , Placenta/anormalidades , Doença de Tangier/etiologia , Transportador 1 de Cassete de Ligação de ATP , Animais , Animais Recém-Nascidos , Northern Blotting , Southern Blotting , HDL-Colesterol/sangue , DNA/análise , Análise Mutacional de DNA , Primers do DNA/química , Feminino , Morte Fetal , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Mutação , Placenta/metabolismo , Placenta/patologia , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/metabolismo , Doença de Tangier/metabolismo , Doença de Tangier/patologiaRESUMO
HFE is a non-classical major histocompatibility complex class I molecule that complexes with a beta2-microglobulin. A functional link between HFE and iron metabolism has been established by the discovery of a physical association between HFE and the transferrin receptor. By inhibiting transferrin receptor internalization, HFE functions as a negative modulator of transferrin receptor function. In addition, HFE appears to be an iron sensor that directly or indirectly communicates the body's iron status to T cells, which then use cytokines as feedback modulators to achieve iron homeostasis. A working model for the feedback regulatory mechanism between iron metabolism and immune function is proposed.
Assuntos
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Ferro/metabolismo , Proteínas de Membrana , Transporte Biológico Ativo , Citocinas/metabolismo , Retroalimentação , Regulação da Expressão Gênica , Genes MHC Classe I , Antígenos HLA/genética , Hemocromatose/genética , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Homeostase , Humanos , Imunidade , Modelos Biológicos , Mutação , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The ATP-binding cassette transporter associated with antigen processing (TAP) is required for transport of antigenic peptides, generated by proteasome complexes in the cytoplasm, into the lumen of the endoplasmic reticulum where assembly with major histocompatibility complex class I molecules takes place. The TAP transporter is a heterodimer of TAP1 and TAP2. Here we show that both TAP1 and TAP2 are phosphorylated under physiological conditions. Phosphorylation induces formation of high molecular weight TAP complexes that contain TAP1, TAP2, tapasin, and class I heterodimers. In addition, a 43-kDa phosphoprotein, which appears to be a kinase, is contained in the phosphorylated TAP-containing complexes. Phosphorylated TAP complexes are able to bind peptides and ATP, however, they are not capable of transporting peptides. After de-phosphorylation, TAP complexes regain the ability to transport peptides. Interestingly, phosphorylation levels of TAP complexes induced by viral infection inversely correlates with a significant reduction in TAP-dependent peptide transport activity. Enhanced TAP phosphorylation appears to be one of several strategies that viruses have exploited to better escape from host immune surveillance. These results demonstrate that major histocompatibility complex class I antigen processing and presentation is modulated by reversible TAP phosphorylation, and implicate the importance of TAP phosphorylation in the regulation of cytotoxic immune response.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/metabolismo , Fosfoproteínas/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Doenças Autoimunes/etiologia , Transporte Biológico , Membrana Celular/metabolismo , Dimerização , Humanos , Masculino , Ácido Okadáico/farmacologia , Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismoRESUMO
At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos CD/imunologia , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígeno B7-2 , Antígenos CD28/genética , Linhagem Celular , Células Dendríticas/imunologia , Drosophila , Endocitose/imunologia , Molécula 1 de Adesão Intercelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos SCID , Camundongos TransgênicosRESUMO
Alzheimer's disease pathology is characterized by the presence of neuritic plaques and the loss of cholinergic neurons in the brain. The underlying mechanisms leading to these events are unclear, but the 42-amino acid beta-amyloid peptide (Abeta(1-42)) is involved. Immunohistochemical studies on human sporadic Alzheimer's disease brains demonstrate that Abeta(1-42) and a neuronal pentameric cation channel, the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), are both present in neuritic plaques and co-localize in individual cortical neurons. Using human brain tissues and cells that overexpress either alpha7nAChR or amyloid precursor protein as the starting material, Abeta(1-42) and alpha7nAChR can be co-immunoprecipitated by the respective specific antibodies, suggesting that they are tightly associated. The formation of the alpha7nAChR.Abeta(1-42) complex can be efficiently suppressed by Abeta(12-28), implying that this Abeta sequence region contains the binding epitope. Receptor binding experiments show that Abeta(1-42) and alpha7nAChR bind with high affinity, and this interaction can be inhibited by alpha7nAChR ligands. Human neuroblastoma cells overexpressing alpha7nAChR are readily killed by Abeta(1-42), whereas alpha7nAChR agonists such as nicotine and epibatidine offered protection. Because Abeta(1-42) inhibits alpha7nAChR-dependent calcium activation and acetylcholine release, two processes critically involved in memory and cognitive functions, and the distribution of alpha7nAChR correlates with neuritic plaques in Alzheimer's disease brains, we propose that interaction of the alpha7nAChR and Abeta(1-42) is a pivotal mechanism involved in the pathophysiology of Alzheimer's disease.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Estudos de Casos e Controles , Morte Celular , Córtex Cerebral/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Relação Dose-Resposta a Droga , Feminino , Galanina/farmacologia , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neurônios/metabolismo , Agonistas Nicotínicos/farmacologia , Placa Amiloide/metabolismo , Testes de Precipitina , Ligação ProteicaRESUMO
The major histocompatibility complex-encoded gene, Hfe, has been implicated to play a pivotal role in hereditary hemochromatosis, a common autosomal recessive disorder of iron metabolism. The recent finding that a physical interaction between HFE and transferrin receptor establishes a functional link between HFE and transferrin receptor-mediated iron metabolism in the pathophysiology of hereditary hemochromatosis. To elucidate the underlying mechanisms by which HFE interacts with and affects transferrin receptor function, we have systematically investigated the consequences of the HFE-transferrin receptor interaction in cellular iron homeostasis. Herein we show that in HFE-expressing cells, the amount of intracellular transferrin is decreased by approximately 28%, despite a approximately 40% increase in surface-expressed transferrin receptor. Kinetic analysis of receptor-bound transferrin endocytosis reveals that HFE expression not only reduces transferrin binding but also abrogates transferrin receptor endocytosis. As a result, HFE expression leads to an accumulation of non-functional transferrin receptors at the cell surface, and a decrease in iron uptake. Moreover, HFE expression induces hyper-serine phosphorylation of the transferrin receptor. Taken together, these results suggest that HFE negatively modulates cellular iron uptake by impairing transferrin receptor endocytosis via HFE-induced receptor phosphorylation.
Assuntos
Genes MHC Classe I , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Receptores da Transferrina/metabolismo , Sequência de Aminoácidos , Transporte Biológico Ativo , Divisão Celular , Membrana Celular/metabolismo , Endocitose/genética , Endocitose/imunologia , Antígenos HLA/metabolismo , Hemocromatose/genética , Hemocromatose/imunologia , Hemocromatose/metabolismo , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ferro/metabolismo , Cinética , Dados de Sequência Molecular , Fosforilação , Transferrina/metabolismoRESUMO
Human cytomegalovirus (HCMV) interferes with major histocompatibility complex (MHC) class I antigen presentation by a sequential multistep process to escape T cell surveillance. During the immediate early phase of infection, the glycoprotein US3 prevents intracellular transport of MHC class I molecules. Interestingly, US3 displays a significantly shorter half-life than US3-retained MHC class I molecules. Here we show that US3 associates only transiently with MHC class I molecules, exits the ER, and is inefficiently retrieved from the Golgi. US3 was degraded in a post-Golgi compartment, most likely lysosomes, because: i) Brefeldin A treatment prolonged the half-life of US3; and ii) US3 co-localized with the lysosomal marker protein LAMP in chloroquine-treated cells. In contrast, MHC class I molecules remained stable in the ER. Upon inhibition of protein synthesis MHC class I molecules were released suggesting that a continuous supply of newly synthesized US3 molecules is required for inhibition of transport. Thus, US3 does not seem to retain MHC class I molecules by a retrieval mechanism. Instead, our observations are consistent with US3 preventing MHC class I trafficking by blocking forward transport.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Antibacterianos/farmacologia , Apresentação de Antígeno , Antimaláricos/farmacologia , Brefeldina A/farmacologia , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Separação Celular , Cloroquina/farmacologia , Citoplasma/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Epitopos , Citometria de Fluxo , Proteínas Ligadas por GPI , Glicoproteínas , Complexo de Golgi/metabolismo , Células HeLa , Hexosaminidases/metabolismo , Humanos , Lisossomos/metabolismo , Macrolídeos , Proteínas de Membrana , Microscopia Confocal , Testes de Precipitina , Transporte Proteico , Puromicina/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.
Assuntos
Apresentação de Antígeno , Cisteína Endopeptidases/metabolismo , Ativadores de Enzimas/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Autoantígenos , Cisteína Endopeptidases/química , Epitopos de Linfócito T/imunologia , Feminino , Antígeno H-Y/imunologia , Infecções por Herpesviridae/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Interferons/farmacologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Complexos Multienzimáticos/química , Muromegalovirus/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma , Proteínas/genéticaRESUMO
Peptide-major histocompatibility complex protein complexes (pMHCs) on antigen-presenting cells (APCs) are central to T cell activation. Within minutes of peptide-specific T cells interacting with APCs, pMHCs on APCs formed clusters at the site of T cell contact. Thereafter, these clusters were acquired by T cells and internalized through T cell receptor-mediated endocytosis. During this process, T cells became sensitive to peptide-specific lysis by neighboring T cells (fratricide). This form of immunoregulation could explain the "exhaustion" of T cell responses that is induced by high viral loads and may serve to down-regulate immune responses.
Assuntos
Endocitose , Antígenos de Histocompatibilidade/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Drosophila , Citometria de Fluxo , Substâncias Macromoleculares , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.
Assuntos
Sequência Conservada , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Genes de Plantas , Família Multigênica/imunologia , Timo/citologia , Timo/imunologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Biomarcadores/química , Complexo CD3/fisiologia , Diferenciação Celular/imunologia , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Especificidade de Órgãos/genética , Proteínas de Plantas/genética , RNA Mensageiro/biossíntese , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Timo/química , Timo/metabolismoRESUMO
We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.