RESUMO
T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.
Assuntos
Autoimunidade , Beriliose/imunologia , Berílio/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Cadeias beta de HLA-DP/metabolismo , Hipersensibilidade/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Cristalografia por Raios X , Cadeias beta de HLA-DP/química , Humanos , Pulmão/patologia , Modelos Moleculares , Sódio/química , Sódio/metabolismoRESUMO
Testicular nuclear receptor 4 (TR4) modulates the transcriptional activation of genes and plays important roles in many diseases. The regulation of TR4 on target genes involves direct interactions with DNA molecules via the DNA-binding domain (DBD) and recruitment of coregulators by the ligand-binding domain (LBD). However, their regulatory mechanisms are unclear. Here, we report high-resolution crystal structures of TR4DBD, TR4DBD-DNA complexes and the TR4LBD-JAZF1 complex. For DNA recognition, multiple factors come into play, and a specific mutual selectivity between TR4 and target genes is found. The coactivators SRC-1 and CREBBP can bind at the interface of TR4 originally occupied by the TR4 activation function region 2 (AF-2); however, JAZF1 suppresses the binding through a novel mechanism. JAZF1 binds to an unidentified surface of TR4 and stabilizes an α13 helix never reported in the nuclear receptor family. Moreover, the cancer-associated mutations affect the interactions and the transcriptional activation of TR4 in vitro and in vivo, respectively. Overall, our results highlight the crucial role of DNA recognition and a novel mechanism of how JAZF1 reinforces the autorepressed conformation and influences the transcriptional activation of TR4, laying out important structural bases for drug design for a variety of diseases, including diabetes and cancers.
Assuntos
Proteínas Correpressoras , Regulação da Expressão Gênica , Receptores de Esteroides , Humanos , Proteínas de Transporte/genética , Proteínas Correpressoras/metabolismo , DNA , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Ativação TranscricionalRESUMO
Tumors frequently express unmutated self-tumor-associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA-specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I-TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA-specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.
Assuntos
Antígenos de Neoplasias/imunologia , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Experimentais/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/prevenção & controle , Células Sf9 , SpodopteraRESUMO
N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation.
Assuntos
Ácidos Nucleicos , Animais , Caenorhabditis elegans/genética , Meiose , DNA , Desmetilação , MamíferosRESUMO
Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy.
Assuntos
Enzimas AlkB , Proteínas de Ciclo Celular , Proteínas Correpressoras , Proteínas de Ligação a DNA , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Correpressoras/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas/metabolismo , RNA/metabolismoRESUMO
Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus, which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus, the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. IMPORTANCE The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.
Assuntos
Modelos Moleculares , Conformação Proteica , RNA Polimerase Dependente de RNA/química , Vírus da Febre do Vale do Rift/enzimologia , Motivos de Aminoácidos , Domínio Catalítico , Fenômenos Químicos , Sequência Conservada , Microscopia Crioeletrônica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão , Proteínas Virais/químicaRESUMO
IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.
Assuntos
Antígenos/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Fator de Transcrição GATA2/imunologia , Interleucina-13/imunologia , Mastócitos/imunologia , Fatores de Transcrição NFATC/imunologia , Elementos de Resposta/imunologia , Fator de Transcrição STAT5/imunologia , Fator de Transcrição AP-1/imunologia , Transcrição Gênica/imunologia , Animais , Linhagem Celular , Mastócitos/citologia , CamundongosRESUMO
A polymorphism at ß57 in some major histocompatibility complex class II (MHCII) alleles of rodents and humans is associated with a high risk for developing type 1 diabetes (T1D). However, a highly diabetogenic insulin B chain epitope within the B:9-23 peptide is presented poorly by these alleles to a variety of mouse and human CD4 T cells isolated from either nonobese diabetic (NOD) mice or humans with T1D. We have shown for both species that mutations at the C-terminal end of this epitope dramatically improve presentation to these T cells. Here we present the crystal structures of these mutated peptides bound to mouse IAg7 and human HLA-DQ8 that show how the mutations function to improve T-cell activation. In both peptide binding grooves, the mutation of B:22R to E in the peptide changes a highly unfavorable side chain for the p9 pocket to an optimal one that is dependent on the ß57 polymorphism, accounting for why these peptides bind much better to these MHCIIs. Furthermore, a second mutation of the adjacent B:21 (E to G) removes a side chain from the surface of the complex that is highly unfavorable for a subset of NOD mouse CD4 cells, thereby greatly enhancing their response to the complex. These results point out the similarities between the mouse and human responses to this B chain epitope in T1D and suggest there may be common posttranslational modifications at the C terminus of the peptide in vivo to create the pathogenic epitopes in both species.
Assuntos
Diabetes Mellitus Tipo 1 , Epitopos , Antígenos HLA-DQ , Antígenos de Histocompatibilidade Classe II , Insulina , Processamento de Proteína Pós-Traducional/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Insulina/química , Insulina/genética , Insulina/imunologia , Camundongos , Camundongos Endogâmicos NOD , Ligação ProteicaRESUMO
RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Animais , Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Ativação Enzimática , Genes Bacterianos , Guanosina Tetrafosfato/metabolismo , Proteínas de Choque Térmico HSP40/química , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Camundongos , Modelos Moleculares , Oxirredução , Estresse Oxidativo , Domínios e Motivos de Interação entre Proteínas , RNA Bacteriano/metabolismo , Salmonella/efeitos dos fármacos , Salmonella/genética , Salmonella/metabolismo , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
Nickel (Ni2+) is one of the most common allergens, affecting around 10-15% of the general population. As the demand for orthopedic implant surgery rises, the number of surgical revisions due to joint implant failure also increases. There is evidence that some patients develop joint failure due to an immune response to a component of the implant, and we have found that Ni2+ is an especially important cause. Hence, understanding the mechanisms by which Ni2+ allergy induces joint implant failure becomes a critical research question. The structural basis of Ni2+ activation of pathogenic T cells is still not clear. The purpose of this study was to characterize Ni2+-reactive T cell repertoires derived from the peripheral blood of joint failure patients due to Ni2+ sensitization using single-cell sequencing techniques. We stimulated the proliferation of Ni2+ -reactive T cells from two implant failure patients in vitro, and sorted them for single-cell VDJ sequencing (10× genomics). We identified 2650 productive V-J spanning pairs. Both TCR α chains and ß chains were enriched. TRBV18 usage is the highest in the P7 CD4+ population (18.1%), and TRBV5-1 usage is the highest in the P7 CD8+ population (12.1%). TRBV19 and TRBV20-1 segments are present in a high percentage of both P7 and P9 sequenced T cells. Remarkably, the alpha and beta chain combination of TRAV41-TRBV18 accounts for 13.5% of the CD4+ population of P7 patient. Compared to current Ni specific T cell repertoire studies of contact dermatitis, the Vα and Vß usages of these joint implant failure patients were different. This could be due to the different availability of self-peptides in these two different tissues. However, TRBV19 (Vß17) was among frequently used TCR ß chains, which are common in previous reports. This implies that some pathogenic T cells could be similar in Ni2+ hypersensitivities in skin and joints. The alignment of the TCR CDR3ß sequences showed a conserved glutamic acid (Glu) that could potentially interact with Ni2+. The study of these Ni2+ specific TCRs may shed light on the molecular mechanism of T cell activation by low molecular weight chemical haptens.
Assuntos
Haptenos , Hipersensibilidade/metabolismo , Prótese Articular , Níquel/imunologia , Falha de Prótese/etiologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Feminino , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Análise de Sequência de DNA , Análise de Célula Única , Linfócitos T/metabolismo , Éxons VDJ/genéticaRESUMO
Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα ß strand, switching Vα's position relative to Vß. In several other structures, either Vα or Vß undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands.
Assuntos
Antígenos/metabolismo , Glicoproteínas/metabolismo , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Antígenos/genética , Antígenos/imunologia , Proliferação de Células , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Reações Cruzadas/imunologia , Cristalografia por Raios X , Mapeamento de Epitopos , Glicoproteínas/genética , Glicoproteínas/imunologia , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos TRESUMO
Chronic beryllium (Be) disease is a granulomatous lung disorder that results from Be exposure in a genetically susceptible host. The disease is characterized by the accumulation of Be-responsive CD4(+) T cells in the lung, and genetic susceptibility is primarily linked to HLA-DPB1 alleles possessing a glutamic acid at position 69 of the ß-chain. Recent structural analysis of a Be-specific TCR interacting with a Be-loaded HLA-DP2-peptide complex revealed that Be is coordinated by amino acid residues derived from the HLA-DP2 ß-chain and peptide and showed that the TCR does not directly interact with the Be(2+) cation. Rather, the TCR recognizes a modified HLA-DP2-peptide complex with charge and conformational changes. Collectively, these findings provide a structural basis for the development of this occupational lung disease through the ability of Be to induce posttranslational modifications in preexisting HLA-DP2-peptide complexes, resulting in the creation of neoantigens.
Assuntos
Beriliose/genética , Beriliose/imunologia , Berílio/imunologia , Linfócitos T CD4-Positivos/imunologia , Cadeias beta de HLA-DP/imunologia , Predisposição Genética para Doença , Cadeias beta de HLA-DP/genética , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Pulmão/imunologia , Pulmão/patologia , Processamento de Proteína Pós-Traducional/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Chromogranin A (ChgA) is an autoantigen for CD4(+) T cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). The natural ChgA-processed peptide, WE14, is a weak agonist for the prototypical T cell, BDC-2.5, and other ChgA-specific T-cell clones. Mimotope peptides with much higher activity share a C-terminal motif, WXRM(D/E), that is predicted to lie in the p5 to p9 position in the mouse MHC class II, IA(g7) binding groove. This motif is also present in WE14 (WSRMD), but at its N terminus. Therefore, to place the WE14 motif into the same position as seen in the mimotopes, we added the amino acids RLGL to its N terminus. Like the other mimotopes, RLGL-WE14, is much more potent than WE14 in T-cell stimulation and activates a diverse population of CD4(+) T cells, which also respond to WE14 as well as islets from WT, but not ChgA(-/-) mice. The crystal structure of the IA(g7)-RLGL-WE14 complex confirmed the predicted placement of the peptide within the IA(g7) groove. Fluorescent IA(g7)-RLGL-WE14 tetramers bind to ChgA-specific T-cell clones and easily detect ChgA-specific T cells in the pancreas and pancreatic lymph nodes of NOD mice. The prediction that many different N-terminal amino acid extensions to the WXRM(D/E) motif are sufficient to greatly improve T-cell stimulation leads us to propose that such a posttranslational modification may occur uniquely in the pancreas or pancreatic lymph nodes, perhaps via the mechanism of transpeptidation. This modification could account for the escape of these T cells from thymic negative selection.
Assuntos
Autoantígenos/imunologia , Cromogranina A/química , Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Modelos Moleculares , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Cromogranina A/genética , Cristalização , Epitopos/genética , Citometria de Fluxo , Hibridomas/imunologia , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Processamento de Proteína Pós-Traducional/genéticaRESUMO
T cell mediated hypersensitivity to nickel (Ni2+) is one of the most common causes of allergic contact dermatitis. Ni2+ sensitization may also contribute to the failure of Ni2+ containing joint implants, and revision to non-Ni2+ containing hardware can be costly and debilitating. Previously, we identified Ni2+ mimotope peptides, which are reactive to a CD4+ T cell clone, ANi2.3 (Vα1, Vß17), isolated from a Ni2+ hypersensitive patient with contact dermatitis. This T cell is restricted to the major histocompatibility complex class II (MHCII) molecule, Human Leukocyte Antigen (HLA)-DR52c (DRA, DRB3*0301). However, it is not known if Ni2+ induced T cell responses in sensitized joint replacement failure patients are similar to subjects with Ni2+ induced contact dermatitis. Here, we generated DR52c/Ni2+ mimotope tetramers, and used them to test if the same Ni2+ T cell activation mechanism could be generalized to Ni2+ sensitized patients with associated joint implant failure. We confirmed the specificity of these tetramers by staining of ANi2.3T cell transfectomas. The DR52c/Ni2+ mimotope tetramer detected Ni2+ reactive CD4+ T cells in the peripheral blood mononuclear cells (PBMC) of patients identified as Ni2+ sensitized by patch testing and a positive Ni2+ LPT. When HLA-typed by a DR52 specific antibody, three out of four patients were DR52 positive. In one patient, Ni2+ stimulation induced the expansion of Vß17 positive CD4+ T cells from 0.8% to 13.3%. We found that the percentage of DR52 positivity and Vß17 usage in Ni2+ sensitized joint failure patients are similar to Ni sensitized skin allergy patients. Ni2+ independent mimotope tetramers may be a useful tool to identify the Ni2+ reactive CD4+ T cells.
Assuntos
Artroplastia de Substituição/efeitos adversos , Linfócitos T CD4-Positivos/imunologia , Hipersensibilidade/imunologia , Níquel/toxicidade , Falha de Prótese/efeitos adversos , Adulto , Idoso , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Estudos de Coortes , Feminino , Humanos , Hipersensibilidade/metabolismo , Insetos , Masculino , Pessoa de Meia-Idade , Níquel/administração & dosagem , Falha de Prótese/efeitos dos fármacosRESUMO
To test whether highly crossreactive alphabeta T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IAb-3K). The TCRs had similar affinities for IAb-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including Vbeta8 48Y and 54E and Valpha4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Sequência Conservada , Reações Cruzadas , Humanos , Ativação Linfocitária/imunologia , Estrutura Quaternária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability-increasing (BPI) fold-containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid-binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.
Assuntos
Glicoproteínas/química , Imunidade Inata , Lipídeos/química , Fosfoproteínas/química , Sequência de Bases , Primers do DNA , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Ligantes , Fosfoproteínas/metabolismo , Conformação ProteicaRESUMO
T cell-mediated allergy to Ni(++) is one of the most common forms of allergic contact dermatitis, but how the T-cell receptor (TCR) recognizes Ni(++) is unknown. We studied a TCR from an allergic patient that recognizes Ni(++) bound to the MHCII molecule DR52c containing an unknown self-peptide. We identified mimotope peptides that can replace both the self-peptide and Ni(++) in this ligand. They share a p7 lysine whose εNH(2) group is surface-exposed when bound to DR52c. Whereas the TCR uses germ-line complementary-determining region (CDR)1/2 amino acids to dock in the conventional diagonal mode on the mimotope-DR52c complex, the interface is dominated by the TCR Vß CDR3 interaction with the p7 lysine. Mutations in the TCR CDR loops have similar effects on the T-cell response to either the mimotope or Ni(++) ligand. We suggest that the mimotope p7 lysine mimics Ni(++) in the natural TCR ligand and that MHCII ß-chain flexibility in the area around the peptide p7 position forms a common site for cation binding in metal allergies.
Assuntos
Dermatite Alérgica de Contato/imunologia , Níquel/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação/imunologia , Epitopos/química , Epitopos/imunologia , Subtipos Sorológicos de HLA-DR/imunologia , Humanos , Cinética , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Estrutura Secundária de Proteína , Linfócitos T/imunologiaRESUMO
Oxygen-evolving photosynthetic organisms regulate carbon metabolism through a light-dependent redox signalling pathway. Electrons are shuttled from photosystem I by means of ferredoxin (Fdx) to ferredoxin-thioredoxin reductase (FTR), which catalyses the two-electron-reduction of chloroplast thioredoxins (Trxs). These modify target enzyme activities by reduction, regulating carbon flow. FTR is unique in its use of a [4Fe-4S] cluster and a proximal disulphide bridge in the conversion of a light signal into a thiol signal. We determined the structures of FTR in both its one- and its two-electron-reduced intermediate states and of four complexes in the pathway, including the ternary Fdx-FTR-Trx complex. Here we show that, in the first complex (Fdx-FTR) of the pathway, the Fdx [2Fe-2S] cluster is positioned suitably for electron transfer to the FTR [4Fe-4S] centre. After the transfer of one electron, an intermediate is formed in which one sulphur atom of the FTR active site is free to attack a disulphide bridge in Trx and the other sulphur atom forms a fifth ligand for an iron atom in the FTR [4Fe-4S] centre--a unique structure in biology. Fdx then delivers a second electron that cleaves the FTR-Trx heterodisulphide bond, which occurs in the Fdx-FTR-Trx complex. In this structure, the redox centres of the three proteins are aligned to maximize the efficiency of electron transfer from the Fdx [2Fe-2S] cluster to the active-site disulphide of Trxs. These results provide a structural framework for understanding the mechanism of disulphide reduction by an iron-sulphur enzyme and describe previously unknown interaction networks for both Fdx and Trx (refs 4-6).
Assuntos
Ferredoxinas/química , Proteínas Ferro-Enxofre/química , Oxirredutases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Transporte de Elétrons , Ferredoxinas/metabolismo , Ferro/química , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Proteínas Recombinantes , Spinacia oleracea , Relação Estrutura-Atividade , Synechocystis , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMO
Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.