RESUMO
Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. Here we report that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. We found that the glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 (or its orthologs) in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. Our work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.
Assuntos
Glucose/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Animais , Biomarcadores , Metabolismo dos Carboidratos , Cromatografia Líquida , Drosophila melanogaster , Técnicas de Silenciamento de Genes , Ácidos Glicéricos/metabolismo , Glicólise , Humanos , Espectrometria de Massas , Redes e Vias Metabólicas , Metaboloma , Metabolômica/métodos , Camundongos , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteína Desglicase DJ-1/químicaRESUMO
Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.
Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Bactérias/patogenicidade , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Evolução Biológica , Linhagem Celular , Regulação Viral da Expressão Gênica/genética , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Proteínas Imediatamente Precoces/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Viroses/genética , Viroses/metabolismo , Replicação Viral/fisiologia , Vírus/patogenicidadeRESUMO
By tying peptide fragments originally distant in parental proteins, the proteasome can generate spliced peptides that are recognized by CTL. This occurs by transpeptidation involving a peptide-acyl-enzyme intermediate and another peptide fragment present in the catalytic chamber. Four main subtypes of proteasomes exist: the standard proteasome (SP), the immunoproteasome, and intermediate proteasomes ß1-ß2-ß5i (single intermediate proteasome) and ß1i-ß2-ß5i (double intermediate proteasome). In this study, we use a tandem mass tag-quantification approach to study the production of six spliced human antigenic peptides by the four proteasome subtypes. Peptides fibroblast growth factor-5172-176/217-220, tyrosinase368-373/336-340, and gp10040-42/47-52 are better produced by the SP than the other proteasome subtypes. The peptides SP110296-301/286-289, gp100195-202/191or192, and gp10047-52/40-42 are better produced by the immunoproteasome and double intermediate proteasome. The current model of proteasome-catalyzed peptide splicing suggests that the production of a spliced peptide depends on the abundance of the peptide splicing partners. Surprisingly, we found that despite the fact that reciprocal peptides RTK_QLYPEW (gp10040-42/47-52) and QLYPEW_RTK (gp10047-52/40-42) are composed of identical splicing partners, their production varies differently according to the proteasome subtype. These differences were maintained after in vitro digestions involving identical amounts of the splicing fragments. Our results indicate that the amount of splicing partner is not the only factor driving peptide splicing and suggest that peptide splicing efficiency also relies on other factors, such as the affinity of the C-terminal splice reactant for the primed binding site of the catalytic subunit.
Assuntos
Peptídeos , Complexo de Endopeptidases do Proteassoma , Antígenos/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Splicing de RNARESUMO
Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four-amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.
Assuntos
Triptofano Oxigenase/metabolismo , Triptofano/sangue , Triptofano/metabolismo , Ubiquitinação , Animais , Células HEK293 , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Triptofano/análogos & derivadosRESUMO
Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules.
Assuntos
Apresentação de Antígeno , Antígenos de Neoplasias/metabolismo , Insulisina/metabolismo , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Anticorpos Bloqueadores/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Antígenos de Neoplasias/imunologia , Fracionamento Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Células Clonais , Citosol , Glicopeptídeos/farmacologia , Antígeno HLA-A1/metabolismo , Humanos , Insulisina/genética , Insulisina/imunologia , Interferon gama/metabolismo , Espectrometria de Massas , Metaloendopeptidases/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/imunologia , Fenantrolinas/farmacologia , Inibidores de Proteassoma , RNA Interferente Pequeno/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologiaRESUMO
Indoleamine 2,3-dioxygenase (IDO1) and tryptophane 2,3-dioxygenase (TDO) are two heme-containing enzymes which catalyze the conversion of tryptophan to N-formylkynurenine. Both enzymes are well establish therapeutic targets as important factors in the tumor immune evasion mechanism. A number of analogues of the marine pyrroloquinoline alkaloids tsitsikammamines or wakayin have been synthesized, two of them were synthesized using an original method to build the bispyrroloquinone framework. All the derivatives were evaluated in a cellular assay for their capacity to inhibit the enzymes. Six compounds have shown a significant potency on HEK 293-EBNA cell lines expressing hIDO1 or hTDO.
Assuntos
Alcaloides/síntese química , Inibidores Enzimáticos/síntese química , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Pirroliminoquinonas/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Triptofano Oxigenase/antagonistas & inibidores , Alcaloides/metabolismo , Organismos Aquáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Alcaloides Indólicos/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Pirróis/química , Pirroliminoquinonas/metabolismo , Quinolinas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeRESUMO
The intracellular pathway of cross-presentation, which allows MHC class I-restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2-restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1-restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2-restricted peptide, cross-presentation of the HLA-A1-restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos de Neoplasias/metabolismo , Células Dendríticas/imunologia , Retículo Endoplasmático/metabolismo , Antígeno HLA-A1/metabolismo , Proteínas de Neoplasias/metabolismo , Linfócitos T Citotóxicos/imunologia , Vacúolos/metabolismo , Apresentação de Antígeno , Linhagem Celular , Apresentação Cruzada , Citosol/metabolismo , Antígeno HLA-A2/metabolismo , Humanos , Peptídeos/metabolismo , Antígeno gp100 de Melanoma/metabolismoRESUMO
Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.
Assuntos
Glicemia/efeitos dos fármacos , Flavonoides/química , Flavonoides/farmacologia , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Moraceae/química , Quinases Proteína-Quinases Ativadas por AMP , Animais , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Masculino , Camundongos , Proteínas Quinases/metabolismo , Ratos , Ratos WistarRESUMO
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being â¼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.
Assuntos
Aminoidrolases/metabolismo , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Aminoidrolases/fisiologia , Animais , Desaminação , Humanos , Hidrólise , Camundongos , Camundongos Knockout , Especificidade por SubstratoRESUMO
KRAS is a powerful oncogene responsible for the development of many cancers. Despite the great progress in understanding its function during the last decade, the study of KRAS expression, subcellular localization, and post-translational modifications remains technically challenging. Accordingly, many facets of KRAS biology are still unknown. Antibodies could be an effective and easy-to-use tool for in vitro and in vivo research on KRAS. Here, we generated a novel rabbit polyclonal antibody that allows immunolabeling of cells and tissues overexpressing KRAS. Cell transfection experiments with expression vectors for the members of the RAS family revealed a preferential specificity of this antibody for KRAS. In addition, KRAS was sensitively detected in a mouse tissue electroporated with an expression vector. Interestingly, our antibody was able to detect endogenous forms of unprenylated (immature) and prenylated (mature) KRAS in mouse organs. We found that KRAS prenylation was increased ex vivo and in vivo in a model of KRASG12D-driven tumorigenesis, which was concomitant with an induction of expression of essential KRAS prenylation enzymes. Therefore, our tool helped us to put the light on new regulations of KRAS activation during cancer initiation. The use of this tool by the RAS community could contribute to discovering novel aspects of KRAS biology.
Assuntos
Carcinogênese/patologia , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Animais , Formação de Anticorpos , Carcinogênese/imunologia , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Coelhos , Células Tumorais CultivadasRESUMO
The proteasome is the major protease responsible for the production of antigenic peptides recognized by CD8+ cytolytic T cells (CTL). These peptides, generally 8-10 amino acids long, are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. Originally, these peptides were believed to be solely derived from linear fragments of proteins, but this concept was challenged several years ago by the isolation of anti-tumor CTL that recognized spliced peptides, i.e. peptides composed of fragments distant in the parental protein. The splicing process was shown to occur in the proteasome through a transpeptidation reaction involving an acyl-enzyme intermediate. Here, we review the steps that led to the discovery of spliced peptides as well as the recent advances that uncover the unexpected importance of spliced peptides in the composition of the MHC class I repertoire.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Modelos Biológicos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína , Animais , Biocatálise , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Membrana Celular/metabolismo , Biologia Computacional , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Conformação Proteica , Multimerização Proteica , Proteômica/métodos , Proteômica/tendências , Propriedades de SuperfícieRESUMO
Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, 'metabolite repair enzymes' eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.
Assuntos
Glicolatos/metabolismo , Glicólise , Lactatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Açúcares Ácidos/metabolismo , Glicolatos/química , Glicolatos/toxicidade , Glicólise/efeitos dos fármacos , Células HCT116 , Humanos , Lactatos/química , Lactatos/toxicidade , Via de Pentose Fosfato/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/deficiência , Fosforilação , Piruvato Quinase/metabolismo , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Açúcares Ácidos/química , Açúcares Ácidos/toxicidadeRESUMO
Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vacúolos/imunologia , Antígeno gp100 de Melanoma/imunologia , Linhagem Celular , Células Cultivadas , Humanos , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
By manipulating the various physicochemical properties of amino acids, the design of peptides with specific self-assembling properties has been emerging for more than a decade. In this context, short peptides possessing detergent properties (so-called "peptergents") have been developed to self-assemble into well-ordered nanostructures that can stabilize membrane proteins for crystallization. In this study, the peptide with "peptergency" properties, called ADA8 and extensively described by Tao et al., is studied by molecular dynamic simulations for its self-assembling properties in different conditions. In water, it spontaneously forms beta sheets with a ß barrel-like structure. We next simulated the interaction of this peptide with a membrane protein, the bacteriorhodopsin, in the presence or absence of a micelle of dodecylphosphocholine. According to the literature, the peptergent ADA8 is thought to generate a belt of ß structures around the hydrophobic helical domain that could help stabilize purified membrane proteins. Molecular dynamic simulations are here used to image this mechanism and provide further molecular details for the replacement of detergent molecules around the protein. In addition, we generalized this behavior by designing an amphipathic peptide with beta propensity, which was called ABZ12. Both peptides are able to surround the membrane protein and displace surfactant molecules. To our best knowledge, this is the first molecular mechanism proposed for "peptergency".
Assuntos
Detergentes/química , Simulação de Dinâmica Molecular , Peptídeos/química , Aminoácidos/química , Detergentes/farmacologia , Proteínas de Membrana/química , Peptídeos/farmacologia , Conformação Proteica , Relação Estrutura-Atividade , Água/químicaRESUMO
For several days after antigenic stimulation, human cytolytic T lymphocyte (CTL) clones exhibit a decrease in their effector activity and in their binding to human leukocyte antigen (HLA)-peptide tetramers. We observed that, when in this state, CTLs lose the colocalization of the T cell receptor (TCR) and CD8. Effector function and TCR-CD8 colocalization were restored with galectin disaccharide ligands, suggesting that the binding of TCR to galectin plays a role in the distancing of TCR from CD8. These findings appear to be applicable in vivo, as TCR was observed to be distant from CD8 on human tumor-infiltrating lymphocytes, which were anergic. These lymphocytes recovered effector functions and TCR-CD8 colocalization after ex vivo treatment with galectin disaccharide ligands. The separation of TCR and CD8 molecules could be one major mechanism of anergy in tumors and other chronic stimulation conditions.
Assuntos
Antígenos CD8/metabolismo , Anergia Clonal/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismo , Antígenos CD8/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo , Galectinas/metabolismo , Antígenos HLA/imunologia , Humanos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Microscopia Confocal , Microscopia Eletrônica de Varredura , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Periostin (POSTN), a secreted homodimeric protein that binds integrins αvß3, αvß5, and α6ß4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over-expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop six mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvß3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN-induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136-51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1-1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136-151 peptide to inhibit integrin-mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status.
Assuntos
Anticorpos Monoclonais , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Motivos de Aminoácidos , Animais , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Neoplasias da Mama/metabolismo , Movimento Celular/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Pessoa de Meia-Idade , Análise Serial de TecidosRESUMO
Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.
Assuntos
Melanoma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína/fisiologia , Antígeno gp100 de Melanoma/genética , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Antígeno HLA-A3/genética , Antígeno HLA-A3/imunologia , Antígeno HLA-A3/metabolismo , Humanos , Melanoma/genética , Melanoma/imunologia , Fragmentos de Peptídeos/genética , Linfócitos T Citotóxicos/imunologia , Antígeno gp100 de Melanoma/imunologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
Carnosine synthase is the ATP-dependent ligase responsible for carnosine (ß-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic ß-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈ 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with ß-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed ß-alanyl-lysine, ß-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on ß-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from ß-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was â¼ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (ß-alanyl-lysine, ß-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.
Assuntos
Carnosina/análogos & derivados , Dipeptidases , Dipeptídeos , Animais , Carnosina/química , Carnosina/genética , Carnosina/metabolismo , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/metabolismo , Dipeptídeos/química , Dipeptídeos/genética , Dipeptídeos/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas , Camundongos , Especificidade de Órgãos/fisiologia , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/fisiologiaRESUMO
Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance, and IDO1 inhibition is an active area of drug development. Tryptophan 2,3-dioxygenase (TDO) is an unrelated hepatic enzyme that also degrades tryptophan along the kynurenine pathway. Here, we show that enzymatically active TDO is expressed in a significant proportion of human tumors. In a preclinical model, TDO expression by tumors prevented their rejection by immunized mice. We developed a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject TDO-expressing tumors. Our results describe a mechanism of tumoral immune resistance based on TDO expression and establish proof-of-concept for the use of TDO inhibitors in cancer therapy.
Assuntos
Neoplasias/imunologia , Triptofano Oxigenase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Triptofano Oxigenase/metabolismoRESUMO
We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3(114-122), MAGE-C2(42-50), MAGE-C2(336-344)) or the standard proteasome (Melan-A(26-35), tyrosinase(369-377), gp100(209-217)). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2(336-344) was only produced by intermediate proteasome ß1i-ß5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2(191-200) is produced only by intermediate proteasome ß1i-ß5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.