RESUMO
In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.
Assuntos
Células T Matadoras Naturais , Neoplasias , Humanos , Sulfoglicoesfingolipídeos/metabolismo , Antígenos CD1d/genética , Apresentação de Antígeno , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Sulfatos/metabolismoRESUMO
S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.
Assuntos
Lactonas/análise , Propiolactona/análogos & derivados , Proteômica , Sulfonas/análise , Proteínas ras/metabolismo , Humanos , Lactonas/metabolismo , Lactonas/farmacologia , Estrutura Molecular , Propiolactona/análise , Propiolactona/metabolismo , Propiolactona/farmacologia , Sulfonas/metabolismo , Sulfonas/farmacologia , Proteínas ras/antagonistas & inibidores , Proteínas ras/químicaRESUMO
Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
Assuntos
Membrana Celular/metabolismo , Hidrolases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas ras/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Promielocítica Aguda/patologia , Lipoilação , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura MolecularRESUMO
Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.
Assuntos
Receptores de Antígenos de Linfócitos T , Antígenos CD1d/química , Humanos , Receptores de Antígenos de Linfócitos T/químicaRESUMO
Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.
Assuntos
Apresentação de Antígeno/imunologia , Lipídeos/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Streptococcus pneumoniae/imunologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antígenos CD1d/imunologia , Linhagem Celular , Endossomos/metabolismo , Redes Reguladoras de Genes , Lisossomos/metabolismo , Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Interferente Pequeno/metabolismoRESUMO
Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6â³ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.
Assuntos
Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Galactosilceramidas/química , Galactosilceramidas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Células T Matadoras Naturais/efeitos dos fármacos , Neoplasias/terapia , Adolescente , Adulto , Idoso , Animais , Antígenos CD1d/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Células T Matadoras Naturais/imunologia , Neoplasias/imunologiaRESUMO
Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d-α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1(+) type I NKT cell repertoire. These cells express a range of TCR α- and ß-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7-8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A'-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d-α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition.
Assuntos
Antígenos CD1d/imunologia , Galactosilceramidas/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Cristalografia por Raios X , Gangliosídeos/imunologia , Glucosilceramidas/imunologia , Humanos , Lipídeos/imunologia , Simulação de Acoplamento Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.
Assuntos
Anticorpos Monoclonais/imunologia , Hibridomas/metabolismo , Fragmentos Fab das Imunoglobulinas/imunologia , Nitrosaminas/imunologia , Animais , Sítios de Ligação de Anticorpos , Proteínas de Transporte/química , Linhagem Celular Tumoral , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nicotina/farmacologia , Nitrosaminas/química , Baço/citologiaRESUMO
Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4',4â³-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4'-OH position in comparison with the 3'-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4'-OH > 3'-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.
Assuntos
Antígenos CD1d/metabolismo , Células T Matadoras Naturais/imunologia , Receptores de Antígenos/metabolismo , Motivos de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos/química , Cristalografia por Raios X/métodos , Citometria de Fluxo/métodos , Glicolipídeos/química , Humanos , Leucócitos Mononucleares/citologia , Lipídeos/química , Camundongos , Modelos Moleculares , Conformação Molecular , Células T Matadoras Naturais/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR α-chain is invariant, NKT TCR Vß exhibits greater diversity, with one (Vß11) and three (Vß8, Vß7, and Vß2) Vß chains in humans and mice, respectively. With the exception of the Vß2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2ß that are critical for CD1d binding. Thus, how Vß2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2ß-encoded tyrosine residues, we show that the Vß2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the Vß8.2 and Vß7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ß-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the Vß2 NKT TCR and the Vß8.2 and Vß7 NKT TCRs, with the Vß2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the Vß2 NKT TCR-CD1d-αGalCer complex, the CDR2ß loop mediated fewer contacts with CD1d, whereas the CDR1ß and CDR3ß loops contacted CD1d to a much greater extent compared with most Vß11, Vß8.2, and Vß7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ß-chain of the Vß2 NKT TCR that enables CD1d-Ag ligation.
Assuntos
Antígenos CD1d/imunologia , Glicolipídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Clonagem Molecular , Epitopos/genética , Epitopos/imunologia , Citometria de Fluxo , Glicolipídeos/metabolismo , Camundongos , Mutagênese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.
Assuntos
Antígenos CD1d/imunologia , Epitopos , Células T Matadoras Naturais/imunologia , Animais , Sequência de Carboidratos , Linhagem Celular , Proliferação de Células , Glicolipídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Células T Matadoras Naturais/citologia , Receptores de Células Matadoras Naturais/imunologiaRESUMO
The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.
Assuntos
Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adaptação Biológica/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD1d/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Células Cultivadas , Galactosilceramidas/química , Galactosilceramidas/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Relação Estrutura-Atividade , Especificidade do Receptor de Antígeno de Linfócitos T/imunologiaRESUMO
Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.
Assuntos
Antígenos CD1d/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Antígenos CD1d/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/imunologiaRESUMO
Four 3''- and 4''-deoxy and -fluorogalactosyl ceramides were synthesized, and their ability to stimulate iNKT cells, based on levels of IL-2 production, was assessed in three NKT cell receptor hybridomas. In two of the hybridomas, 1.2 and 2H4, all of the analogs were immunostimulatory, while in the 1.4 hybridoma only the 4''-fluoro analog led to the production of significant levels of IL-2.
Assuntos
Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/farmacologia , Química Farmacêutica/métodos , Galactosilceramidas/síntese química , Galactosilceramidas/farmacologia , Hibridomas/metabolismo , Quinoxalinas/síntese química , Animais , Antígenos CD1d/biossíntese , Desenho de Fármacos , Galactosilceramidas/química , Glicolipídeos/química , Humanos , Interleucina-2/metabolismo , Camundongos , Modelos Químicos , Células T Matadoras Naturais , Poríferos , Quinoxalinas/farmacologiaRESUMO
Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.
Assuntos
Glicolipídeos/química , Células Matadoras Naturais/citologia , Linfócitos/citologia , Sphingomonas/metabolismo , Animais , Antígenos/química , Linhagem Celular , Citocinas/metabolismo , Hibridomas/metabolismo , Lipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Químicos , Oligossacarídeos/químicaRESUMO
Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how alphabeta T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR-CD1d-alpha-galactosylceramide (alpha-GalCer) complex as a guide, we undertook an alanine scanning mutagenesis approach to define the energetic basis of this interaction between the NKT TCR and CD1d. Moreover, we explored how analogues of alpha-GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR-CD1d-alpha-GalCer cross-reactivity. Some, but not all, of the contact residues within the Jalpha18-encoded invariant CDR3alpha loop and Vbeta11-encoded CDR2beta loop were critical for recognizing CD1d. The residues within the Valpha24-encoded CDR1alpha and CDR3alpha loops that contacted the glycolipid Ag played a smaller energetic role compared with the NKT TCR residues that contacted CD1d. Collectively, our data reveal that the region distant to the protruding Ag and directly above the F' pocket of CD1d was the principal factor in the interaction with the NKT TCR. Accordingly, although the structural footprint at the NKT TCR-CD1d-alpha-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.
Assuntos
Antígenos CD1/química , Antígenos CD1/imunologia , Glicolipídeos/química , Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Alanina , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antígenos CD1d , Galactosilceramidas , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
A series of glycolamide naproxen prodrugs containing a nitrate group as a nitric oxide (NO) donor moiety has been synthesized. These compounds were evaluated for their anti-inflammatory activity, naproxen release, and gastric tolerance. Compounds 4a, 4b, 5a, 5b, 7b, and 7c exhibited anti-inflammatory activity equivalent to that of the parent NSAID, naproxen-Na, in the rat carrageenan paw edema model. At equimolar doses relative to naproxen-Na, the NO-donor glycolamide derivatives 4a, 4b, 5a, 5b, 7b, and 7c were gastro-sparing in the rat. Naproxen formation from these NO-donor glycolamides varied among the structures examined, with the N-substituent on the amide group having a particular influence, and demonstrated their prodrug nature. Compound 7b was selected for exemplary demonstration that the glycolamide nitrates can be bioactivated to release NO. These data open the possibility that naproxen glycolamide nitrates may represent a safer alternative to naproxen as anti-inflammatory medicines.
Assuntos
Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Naproxeno/farmacologia , Doadores de Óxido Nítrico/farmacologia , Pró-Fármacos , Amidas/química , Animais , Anti-Inflamatórios/química , Gastrite/induzido quimicamente , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Naproxeno/síntese química , Naproxeno/química , Doadores de Óxido Nítrico/síntese química , Doadores de Óxido Nítrico/química , Ratos , Ratos Sprague-DawleyRESUMO
Valpha14i natural killer T (NKT)-cell function has been implicated in a number of disease conditions. The molecular events that drive Valpha14i NKT-cell development remain elusive. We recently showed that T-bet is required for the terminal maturation of these cells. Here we identify some of the genetic targets of T-bet during Valpha14i NKT-cell lineage development. Microarray gene-expression analyses on developing Valpha14i NKT cells were performed and provide a molecular framework to study these maturation events. In vitro ectopic expression of T-bet in immature Valpha14i NKT cells, which do not yet express T-bet, was sufficient to promote Valpha14i NKT-cell maturation, driving the expression of multiple genes, including those that participate in migration, survival, and effector functions. By regulating the expression of T-helper 1 (Th1)-associated cytokines, chemokines, chemokine receptors, and molecules involved in cytolysis, T-bet defines the unique lineage attributes of mature Valpha14i NKT cells and acts to link these attributes to a developmental process.