RESUMO
Under conditions that involve a high risk of competition for light among neighbouring plants, shade-intolerant species often display increased shoot elongation and greater susceptibility to pathogens and herbivores. The functional links between morphological and defence responses to crowding are not well understood. In Arabidopsis, the protein JAZ10 is thought to play a key role connecting the inactivation of the photoreceptor phytochrome B (phyB), which takes place under competition for light, with the repression of jasmonate-mediated plant defences. Here, we show that a null mutation of the JAZ10 gene in Arabidopsis did not affect plant growth nor did it suppress the shade-avoidance responses elicited by phyB inactivation. However, the jaz10 mutation restored many of the defence traits that are missing in the phyB mutant, including the ability to express robust responses to jasmonate and to accumulate indolic glucosinolates. Furthermore, the jaz10phyB double mutant showed a significantly increased resistance to the pathogenic fungus Botrytis cinerea compared with the phyB parental line. Our results demonstrate that, by inactivating JAZ10, it is possible to partially uncouple shade avoidance from defence suppression in Arabidopsis. These findings may provide clues to improve plant resistance to pathogens in crops that are planted at high density.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fitocromo B/metabolismo , Imunidade Vegetal , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/fisiologia , Botrytis/fisiologia , Ciclopentanos/metabolismo , Resistência à Doença/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas , Glucosinolatos/metabolismo , Luz , Mutação/genética , Oxilipinas/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos da radiação , Regulação para Cima/genética , Regulação para Cima/efeitos da radiaçãoRESUMO
Agriculture accounts for about 70% of the fresh water use in the world, dominating rainfed production systems. As meeting future food demand will require an increase in crop production, new techniques are necessary to monitor the spatial variability of agricultural water use. However, the use of remote sensing for the water footprint estimation is limited. This study aims at evaluating the spatial variability of the soil-water consumption in soybean crops, also termed as green water footprint (WFgreen), in a sector of the Argentine Pampas using satellite data. WFgreen was evaluated at spatial resolution of 250 m, estimating the soil water availability through the evaporative fraction and crop yield from Moderate-Resolution Imaging Spectroradiometer (MODIS/Aqua) data. In the analysed soybean plots, the WFgreen varied from 900 m3 t-1 to 1800 m3 t-1. The preliminary comparison of the method with field measurements showed a RMSE = 494 m3 t-1 and Bias = -410 m3 t-1, respectively. The high spatial variability reflected the heterogeneity of soil-water use efficiency. The proposed technique can be useful to obtain WFgreen maps at medium spatial resolutions (250 m-1000 m). Also, it can be applied in regions with poor ground data coverage to estimate the WFgreen, after a parameterization of the model. The contribution to our understanding of the relationship between soil-water availability, rainfed-crop productivity and then WFgreen is expected.
RESUMO
The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the alpha and beta chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the MHC alpha helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral putative antigen-binding groove beneath the CD1 alpha helices. Here, we demonstrate that alpha/beta TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences permit a model for recognition in which the TCR CDR3 loops containing charged residues project between the CD1 alpha helices, contacting the lipid antigen hydrophilic head moieties as well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.
Assuntos
Antígenos/imunologia , Lipídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD1/química , Antígenos CD1/imunologia , Células Clonais/imunologia , Clonagem Molecular , Humanos , Camundongos , Modelos Moleculares , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Transfecção/genéticaRESUMO
The development of personalized therapies represents an urgent need owing to the high rate of cancer recurrence and systemic toxicity of conventional drugs. So far, targeted toxins have shown promising results as potential therapeutic compounds. Specifically, toxins conjugated to antibodies or fused to growth factors/enzymes have been largely demonstrated to selectively address and kill cancer cells. We investigated the anti-tumor potential of a chimeric recombinant fusion protein formed by the Ribosome Inactivating Protein saporin (SAP) and the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), whose receptor has been shown to be over-expressed on the surface of aggressive tumors. ATF-SAP was recombinantly produced by the P. pastoris yeast and its activity was assessed on a panel of bladder and breast cancer cell lines. ATF-SAP resulted to be highly active in vitro, as nano-molar concentrations were sufficient to impair viability on tumor cell lines. In contrast to untargeted toxins, the chimeric fusion protein displayed a significantly improved toxic effect in uPAR-expressing cells, demonstrating that the selective activity was due to the presence of the targeting moiety. Fibroblasts were not sensitive to ATF-SAP despite uPAR expression, indicating that cell-specific receptor-mediated internalization pathway(s) might be considered. The in vivo anti-tumor effect of the chimera was shown in a bladder cancer xenograft model. Current findings indicate ATF-SAP as a suitable anti-tumoral therapeutic option to cope with cancer aggressiveness, as a single treatment or in combination with traditional therapeutic approaches, to appropriately address the intra- and inter- tumor heterogeneity.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Saporinas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Proteínas Recombinantes de Fusão/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologiaRESUMO
Murine T10 and T22 are highly related nonclassical major histocompatibility complex (MHC) class Ib proteins that bind to certain gammadelta T cell receptors (TCRs) in the absence of other components. The crystal structure of T22b at 3.1 angstroms reveals similarities to MHC class I molecules, but one side of the normal peptide-binding groove is severely truncated, which allows direct access to the beta-sheet floor. Potential gammadelta TCR-binding sites can be inferred from functional mapping of T10 and T22 point mutants and allelic variants. Thus, T22 represents an unusual variant of the MHC-like fold and indicates that gammadelta and alphabeta TCRs interact differently with their respective MHC ligands.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Proteínas/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Alelos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Glicosilação , Ligação de Hidrogênio , Ligantes , Camundongos , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/imunologia , Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Propriedades de Superfície , Microglobulina beta-2/químicaRESUMO
The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide-major histocompatibility complex (pMHC) antigen H-2Kb-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR beta chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC alpha helices suggests a generalized orientation that is mediated by the Valpha domain of the TCR and rationalizes how TCRs can effectively "scan" different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.
Assuntos
Antígenos H-2/química , Antígenos H-2/imunologia , Oligopeptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Cristalização , Cristalografia por Raios X , Antígenos H-2/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutação , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas RecombinantesRESUMO
The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the alphabeta T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated alphabeta TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Valpha and Vbeta domains but deviates in the constant Calpha domain and in the interdomain pairing of Calpha with Cbeta. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Calpha-Cbeta interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the alpha and beta chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Valpha CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vbeta chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Valpha and Vbeta CDR3s straddle the peptide between the helices around the central position of the peptide.
Assuntos
Antígenos H-2/química , Peptídeos/química , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Linfócitos T Citotóxicos/imunologia , Animais , Sequência de Carboidratos , Células Cultivadas , Cristalização , Cristalografia por Raios X , Drosophila melanogaster , Glicosilação , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Ligação de Hidrogênio , Complexo Principal de Histocompatibilidade , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/imunologia , Peptídeos/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas RecombinantesRESUMO
Transgenic crops producing Bacillus thuringiensis- (Bt) insecticidal proteins (Bt crops) have provided useful pest management tools to growers for the past 20 years. Planting Bt crops has reduced the use of synthetic insecticides on cotton, maize and soybean fields in 11 countries throughout Latin America. One of the threats that could jeopardize the sustainability of Bt crops is the development of resistance by targeted pests. Governments of many countries require vigilance in measuring changes in Bt-susceptibility in order to proactively implement corrective measures before Bt-resistance is widespread, thus prolonging the usefulness of Bt crops. A pragmatic approach to obtain information on the effectiveness of Bt-crops is directly asking growers, crop consultants and academics about Bt-resistance problems in agricultural fields, first-hand information that not necessarily relies on susceptibility screens performed in laboratories. This type of information is presented in this report. Problematic pests of cotton and soybeans in five Latin American countries currently are effectively controlled by Bt crops. Growers that plant conventional (non-Bt) cotton or soybeans have to spray synthetic insecticides against multiple pests that otherwise are controlled by these Bt crops. A similar situation has been observed in six Latin American countries where Bt maize is planted. No synthetic insecticide applications are used to control corn pests because they are controlled by Bt maize, with the exception of Spodoptera frugiperda. While this insect in some countries is still effectively controlled by Bt maize, in others resistance has evolved and necessitates supplemental insecticide applications and/or the use of Bt maize cultivars that express multiple Bt proteins. Partial control of S. frugiperda in certain countries is due to its natural tolerance to the Bt bacterium. Of the 31 pests targeted and controlled by Bt crops in Latin America, only S. frugiperda has shown tolerance to certain Bt proteins in growers' fields, the most reliable indication of the status of Bt-susceptibility in most of the American continent.
Assuntos
Insetos/fisiologia , Controle Biológico de Vetores/estatística & dados numéricos , Animais , Bacillus thuringiensis/química , Resistência a Inseticidas/genética , América Latina , Plantas Geneticamente ModificadasRESUMO
We performed an immunogenetic analysis of 345 IGHV-IGHD-IGHJ rearrangements from 337 cases with primary splenic small B-cell lymphomas of marginal-zone origin. Three immunoglobulin (IG) heavy variable (IGHV) genes accounted for 45.8% of the cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23, 8.1%). Particularly for the IGHV1-2 gene, strong biases were evident regarding utilization of different alleles, with 79/86 rearrangements (92%) using allele (*)04. Among cases more stringently classified as splenic marginal-zone lymphoma (SMZL) thanks to the availability of splenic histopathological specimens, the frequency of IGHV1-2(*)04 peaked at 31%. The IGHV1-2(*)04 rearrangements carried significantly longer complementarity-determining region-3 (CDR3) than all other cases and showed biased IGHD gene usage, leading to CDR3s with common motifs. The great majority of analyzed rearrangements (299/345, 86.7%) carried IGHV genes with some impact of somatic hypermutation, from minimal to pronounced. Noticeably, 75/79 (95%) IGHV1-2(*)04 rearrangements were mutated; however, they mostly (56/75 cases; 74.6%) carried few mutations (97-99.9% germline identity) of conservative nature and restricted distribution. These distinctive features of the IG receptors indicate selection by (super)antigenic element(s) in the pathogenesis of SMZL. Furthermore, they raise the possibility that certain SMZL subtypes could derive from progenitor populations adapted to particular antigenic challenges through selection of VH domain specificities, in particular the IGHV1-2(*)04 allele.
Assuntos
Regiões Determinantes de Complementaridade/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Cadeia Pesada de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Linfoma de Zona Marginal Tipo Células B/genética , Neoplasias Esplênicas/genética , Estudos de Coortes , Humanos , Modelos Moleculares , Mutação/genética , PrognósticoAssuntos
Aleitamento Materno , Saúde Bucal , Cárie Dentária/etiologia , Cárie Dentária/prevenção & controle , Fluoretos/administração & dosagem , Humanos , Lactente , Recém-Nascido , Má Oclusão/prevenção & controle , Leite Humano/química , Higiene Bucal/métodos , Higiene Bucal/normas , Desmineralização do Dente/etiologia , Desmineralização do Dente/prevenção & controle , Escovação DentáriaRESUMO
BACKGROUND: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date. METHODS: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations. RESULTS: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein. CONCLUSIONS: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.
Assuntos
Predisposição Genética para Doença/genética , Cardiopatias/genética , Laminas/genética , Mutação/genética , Doenças Neuromusculares/genética , Adulto , Idade de Início , Criança , Pré-Escolar , Análise por Conglomerados , Estudos de Coortes , Análise Mutacional de DNA , Progressão da Doença , Mutação da Fase de Leitura/genética , Marcadores Genéticos/genética , Haplótipos/genética , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Humanos , Lamina Tipo A/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Mutação de Sentido Incorreto/genética , Miocárdio/metabolismo , Miocárdio/patologia , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/fisiopatologia , FenótipoRESUMO
Nucleoside N-ribohydrolases are targets for disruption of purine salvage in the protozoan parasites. The structure of a trypanosomal N-ribohydrolase in complex with a transition-state inhibitor is reported at 2.3 A resolution. The nonspecific nucleoside hydrolase from Crithidia fasciculata cocrystallized with p-aminophenyliminoribitol reveals tightly bound Ca2+ as a catalytic site ligand. The complex with the transition-state inhibitor is characterized by (1) large protein conformational changes to create a hydrophobic leaving group site (2) C3'-exo geometry for the inhibitor, typical of a ribooxocarbenium ion (3) stabilization of the ribooxocarbenium analogue between the neighboring group 5'-hydroxyl and bidentate hydrogen bonds to Asn168; and (4) octacoordinate Ca2+ orients a catalytic site water and is liganded to two hydroxyls of the inhibitor. The mechanism is ribooxocarbenium stabilization with weak leaving group activation and is a departure from glucohydrolases which use paired carboxylates to achieve the transition state.
Assuntos
Crithidia fasciculata/enzimologia , N-Glicosil Hidrolases/química , Sequência de Aminoácidos , Animais , Cálcio/análise , Ligantes , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Conformação Proteica , Difração de Raios XRESUMO
The crystal structures of catalytically competent soybean beta-amylase, unliganded and bathed with small substrates (beta-maltose, maltal), were determined at 1.9-2.2-A resolution. Two molecules of beta-maltose substrate bind to the protein in tandem, with some maltotetraose enzymic condensation product sharing the same binding sites. The beta-amylase soaked with maltal shows a similar arrangement of two bound molecules of 2-deoxymaltose, the enzymic hydration product. In each case the nonreducing ends of the saccharide ligands are oriented toward the base of the protein's active site pocket. The catalytic center, located between the bound disaccharides and found deeper in the pocket than where the inhibitor alpha-cyclodextrin binds, is characterized by the presence of oppositely disposed carboxyl groups of two conserved glutamic acid residues. The OE2 carboxyl of Glu 186 is below the plane of the penultimate glucose residue (Glc 2) of bound maltotetraose, 2.6 A from the oxygen atom of that ligand's penultimate alpha-1,4-glucosidic linkage. The OE2 carboxyl of Glu 380 lies above the plane of Glc 2, 2.8 A from the O-1 atom of the more deeply bound beta-maltose. Saccharide binding does not alter the spatial coordinates of these two carboxyl groups or the overall conformation of the 57-kDa protein. However, the saccharide complexes of the active enzyme are associated with a significant (10 A) local conformational change in a peptide segment of a loop (L3) that borders the active site pocket.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
beta-Amilase/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Maltose/análogos & derivados , Maltose/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Glycine max , beta-Amilase/ultraestruturaRESUMO
Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.
Assuntos
Crithidia fasciculata/enzimologia , Histidina/química , N-Glicosil Hidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Clonagem Molecular , DNA Complementar , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucleoside hydrolase (IU-NH) from C. fasciculata determined by X-ray crystallography at a nominal resolution of 2.5 A. The enzyme has an open (alpha, beta) structure which differs from the classical dinucleotide binding fold. IU-nucleoside hydrolase is composed of a mixed eight-stranded beta sheet surrounded by six alpha helices and a small C-terminal lobe composed of four alpha helices. Two short antiparallel beta strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of beta strands beta 1 and beta 4. Four aspartate residues are located at the bottom of the cavity in a geometry which suggests interaction with the ribose moiety of the nucleoside. These groups could provide the catalytically important interactions to the ribosyl hydroxyls and the stabilizing anion for the oxycarbonium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates purine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchettini, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The substrate binding site is unlike that from purine nucleoside phosphorylase, phosphoribosyltransferases, or uracil DNA glycosylase and thus represents a novel architecture for general acid-base catalysis. This detailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], allows analysis of the interactions leading to catalysis and an explanation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262].
Assuntos
Crithidia fasciculata/enzimologia , N-Glicosil Hidrolases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The structural basis of antigen recognition in cellular immunity has been elucidated through the determination of crystal structures of major histocompatibility complex (MHC) molecules bound to antigenic peptides, T cell receptors (TCR), CD8 and CD4 co-receptors and, most recently, TCRs in complex with peptide-MHC (pMHC). The mechanisms that generate the diversity of the immune response to invading microorganisms were first realized at a genetic level and are necessary in order to cope with the enormous number of potential antigens. This diversity is manifested in the protein products of the genes which code for the components of the TCR signalling complex. The structure of the TCR reveals both striking similarities with and fundamental differences from its functional counterpart, the antibody, in the humoral immune system. The conserved manner in which the TCR recognizes and interacts with its peptide-MHC ligand allows the TCR great latitude in its potential to form productive interactions with antigen-presenting cells that bear numerous ligands to which the TCR has not been previously exposed. This phenomenon of cross-, or alloreactivity arises from a combination of conserved structural features across all MHC molecules, both self and foreign, and some degree of molecular mimicry. Non-classical MHC ligands presenting either modified or specialized peptides, lipids, carbohydrates, or no ligand at all, are now thought to play increasingly important roles in cellular immunity. We review some of the recent structural results and our current state of knowledge about TCR structure, and how this relates to its function.
Assuntos
Apresentação de Antígeno/imunologia , Imunidade Celular , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Cristalografia por Raios X , Humanos , Polimorfismo Genético , Conformação ProteicaRESUMO
The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid alphagalactosylceramide (alpha-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the alpha helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and alpha-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that alpha-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A' pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) alpha chains and different beta chains respond differently to alpha-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR beta in defining natural killer T cell specificity, despite the more restricted diversity of the alpha chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR alpha and beta chains survey a surface composed of both mCD1.1 and the carbohydrate portion of alpha-GalCer.
Assuntos
Antígenos CD1/imunologia , Galactosilceramidas/imunologia , Glicoesfingolipídeos/imunologia , Linfócitos T/imunologia , Substituição de Aminoácidos , Animais , Antígenos/imunologia , Antígenos CD1/genética , Hibridomas/imunologia , Interleucina-2/biossíntese , Camundongos , Mutagênese Sítio-Dirigida , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Proteínas Recombinantes/imunologia , TransfecçãoRESUMO
MHC class I H-2Ld complexed with peptide QL9 (or p2Ca) is a high-affinity alloantigen for the 2C TCR. We used the crystal structure of H-2Ld with a mixture of bound peptides at 3.1 A to construct a model of the allogeneic 2C-Ld/QL9 complex for comparison with the syngeneic 2C-Kb/dEV8 structure. A prominent ridge on the floor of the Ld peptide-binding groove, not present in Kb, creates a C-terminal bulge in Ld peptides that greatly increases interactions with the 2C beta-chain. Furthermore, weak electrostatic complementarity between Asp77 on the alpha1 helix of Kb and 2C is enhanced in the allogeneic complex by closer proximity of QL9 peptide residue AspP8 to the 2C HV4 loop.
Assuntos
Antígenos H-2/imunologia , Isoantígenos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Apresentação de Antígeno , Células Cultivadas , Simulação por Computador , Cristalografia por Raios X , Drosophila melanogaster , Antígenos H-2/química , Antígeno de Histocompatibilidade H-2D , Substâncias Macromoleculares , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross-reactivity.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos H-2/imunologia , Oligopeptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Antígenos H-2/química , Ligantes , Camundongos , Oligopeptídeos/química , Conformação ProteicaRESUMO
CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A' and F'. We investigated the function of the amino acid residues that line the A' and F' pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A' and F' pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A' pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F' pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A' and F' pockets of human CD1b are required for the presentation of lipid Ags to T cells.