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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
Nature ; 596(7870): 119-125, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290406

RESUMO

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Especificidade por Substrato/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/sangue , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Transcriptoma/genética , Microambiente Tumoral
3.
Cells ; 9(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878341

RESUMO

Driving nanomaterials to specific cell populations is still a major challenge for different biomedical applications. Several strategies to improve cell binding and uptake have been tried thus far by intrinsic material modifications or decoration with active molecules onto their surface. In the present work, we covalently bound the chemokine CXCL5 on fluorescently labeled amino-functionalized SiO2 nanoparticles to precisely targeting CXCR2+ immune cells. We synthesized and precisely characterized the physicochemical features of the modified particles. The presence of CXCL5 on the surface was detected by z-potential variation and CXCL5-specific electron microscopy immunogold labeling. CXCL5-amino SiO2 nanoparticle cell binding and internalization performances were analyzed in CXCR2+ THP-1 cells by flow cytometry and confocal microscopy. We showed improved internalization of the chemokine modified particles in the absence or the presence of serum. This internalization was reduced by cell pre-treatment with free CXCL5. Furthermore, we demonstrated CXCR2+ cell preferential targeting by comparing particle uptake in THP-1 vs. low-CXCR2 expressing HeLa cells. Our results provide the proof of principle that chemokine decorated nanomaterials enhance uptake and allow precise cell subset localization. The possibility to aim at selective chemokine receptor-expressing cells can be beneficial for the diverse pathological conditions involving immune reactions.


Assuntos
Quimiocina CXCL5/química , Nanopartículas/química , Receptores de Interleucina-8B/química , Quimiocina CXCL5/metabolismo , Endocitose/imunologia , Endocitose/fisiologia , Células HeLa , Humanos , Receptores de Interleucina-8B/metabolismo , Dióxido de Silício/química , Especificidade por Substrato/imunologia , Células THP-1
4.
PLoS Pathog ; 13(10): e1006667, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29077761

RESUMO

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.


Assuntos
Acetiltransferases/metabolismo , Bactérias Gram-Positivas/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Resistência a Medicamentos , Humanos , Peptidoglicano/farmacologia , Staphylococcus aureus/patogenicidade , Especificidade por Substrato/imunologia , Virulência
5.
Mol Cell Biol ; 37(20)2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28716949

RESUMO

Activation-induced cytidine deaminase (AID) is a genome-mutating enzyme that initiates class switch recombination and somatic hypermutation of antibodies in jawed vertebrates. We previously described the biochemical properties of human AID and found that it is an unusual enzyme in that it exhibits binding affinities for its substrate DNA and catalytic rates several orders of magnitude higher and lower, respectively, than a typical enzyme. Recently, we solved the functional structure of AID and demonstrated that these properties are due to nonspecific DNA binding on its surface, along with a catalytic pocket that predominantly assumes a closed conformation. Here we investigated the biochemical properties of AID from a sea lamprey, nurse shark, tetraodon, and coelacanth: representative species chosen because their lineages diverged at the earliest critical junctures in evolution of adaptive immunity. We found that these earliest-diverged AID orthologs are active cytidine deaminases that exhibit unique substrate specificities and thermosensitivities. Significant amino acid sequence divergence among these AID orthologs is predicted to manifest as notable structural differences. However, despite major differences in sequence specificities, thermosensitivities, and structural features, all orthologs share the unusually high DNA binding affinities and low catalytic rates. This absolute conservation is evidence for biological significance of these unique biochemical properties.


Assuntos
Citidina Desaminase/metabolismo , Switching de Imunoglobulina/imunologia , Lampreias/metabolismo , Especificidade por Substrato/imunologia , Sequência de Aminoácidos , Animais , DNA/metabolismo , Humanos , Mutação/genética
6.
PLoS One ; 10(7): e0132818, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26172376

RESUMO

Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.


Assuntos
Neutrófilos/metabolismo , Serina Proteases/metabolismo , Especificidade por Substrato/imunologia , Aminoácidos/metabolismo , Humanos , Peptídeos/metabolismo , Especificidade por Substrato/fisiologia
7.
J Mol Recognit ; 28(10): 614-27, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25867248

RESUMO

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.


Assuntos
Clonagem Molecular , Cadeias Leves de Imunoglobulina/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteína Básica da Mielina/metabolismo , Bacteriófagos/genética , Escherichia coli , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Ligantes , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/enzimologia , Linfócitos/enzimologia , Metais/química , Metais/imunologia , Proteína Básica da Mielina/química , Proteína Básica da Mielina/imunologia , Oligopeptídeos/química , Oligopeptídeos/imunologia , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Especificidade por Substrato/imunologia
8.
Sci Signal ; 7(355): ra118, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25492967

RESUMO

The substrate specificity of Src family kinases (SFKs) is partly determined by their Src homology 2 (SH2) domains. Thus, transient alterations in the SH2 domain of SFKs might change their binding partners and affect intracellular signaling pathways. Lck is an SFK that is central to the initiation of T cell activation in response to ligand binding to the T cell receptor (TCR) and is also critical for later signaling processes. The kinase activity of Lck requires both the phosphorylation of an activating tyrosine residue and the dephosphorylation of an inhibitory tyrosine residue. We found that a third conserved tyrosine phosphorylation site (Tyr(192)) within the SH2 domain of Lck was required for proper T cell activation and formation of cell-cell conjugates between T cells and antigen-presenting cells. Through phosphopeptide arrays and biochemical assays, we identified several regulators of the actin cytoskeleton that preferentially bound to Lck phosphorylated at Tyr(192) compared to Lck that was not phosphorylated at this site. Two of these phosphorylation-dependent binding partners, the kinase Itk (interleukin-2-inducible Tec kinase) and the adaptor protein TSAd (T cell-specific adaptor), promoted the TCR-dependent phosphorylation of Lck at Tyr(192). Our data suggest that phosphorylation transiently alters SH2 domain specificity and provide a potential mechanism whereby SFKs may be rewired from one signaling program to another to enable appropriate cell activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Humanos , Células Jurkat , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Camundongos , Camundongos Knockout , Fosforilação/genética , Fosforilação/imunologia , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/genética , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Linfócitos T/citologia , Tirosina/genética , Tirosina/imunologia , Domínios de Homologia de src/genética , Domínios de Homologia de src/imunologia
9.
Eur J Immunol ; 44(12): 3508-21, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25231383

RESUMO

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard- and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard- and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.


Assuntos
Apresentação do Antígeno/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteólise , Animais , Linhagem Celular Transformada , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Camundongos Mutantes , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/genética , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia
10.
J Immunol ; 193(3): 1344-52, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24973455

RESUMO

Proteolytic shedding of ligands for the NK group 2D (NKG2D) receptor is a strategy used by tumors to modulate immune recognition by NK cells and cytotoxic T cells. A number of metalloproteases, especially those of the A disintegrin and metalloprotease (ADAM) family, can mediate NKG2D ligand cleavage and this process can be modulated by expression of the thiol isomerase ERp5. In this article, we describe that an increased shedding of the NKG2D ligand MICA is observed postinfection with several strains of human CMV due to an enhanced activity of ADAM17 (TNF-α converting enzyme) and matrix metalloprotease 14 caused by a reduction in the expression of the endogenous inhibitor of metalloproteases tissue inhibitors of metalloproteinase 3 (TIMP3). This decrease in TIMP3 expression correlates with increased expression of a cellular miRNA known to target TIMP3, and we also identify a human CMV-encoded microRNA able to modulate TIMP3 expression. These observations characterize a novel viral strategy to influence the shedding of cell-surface molecules involved in immune response modulation. They also provide an explanation for previous reports of increased levels of various ADAM17 substrates in the serum from patients with CMV disease. Consistent with this hypothesis, we detected soluble MICA in serum of transplant recipients with CMV disease. Finally, these data suggest that it might be worthwhile to prospectively study ADAM17 activity in a larger group of patients to assay whether this might be a useful biomarker to identify patients at risk for development of CMV disease.


Assuntos
Infecções por Citomegalovirus/imunologia , Regulação para Baixo/imunologia , Regulação Viral da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , MicroRNAs/genética , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Linhagem Celular Tumoral , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/genética , Regulação para Baixo/genética , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Metaloproteinase 14 da Matriz/sangue , Metaloproteinase 14 da Matriz/metabolismo , MicroRNAs/biossíntese , Cultura Primária de Células , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Inibidor Tecidual de Metaloproteinase-3/sangue , Regulação para Cima/genética , Regulação para Cima/imunologia
11.
Curr Opin Immunol ; 26: 21-31, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24556397

RESUMO

MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Desenho de Fármacos , Retículo Endoplasmático/química , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/imunologia , Interações Hospedeiro-Patógeno , Humanos , Fragmentos de Peptídeos/biossíntese , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Ligação Proteica/imunologia , Especificidade por Substrato/imunologia , Vacinas de Subunidades/síntese química , Vacinas de Subunidades/metabolismo
12.
Curr Opin Immunol ; 26: 76-89, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24556404

RESUMO

Necroptosis describes a pro-inflammatory form of cell death governed by the kinases RIP1 and RIP3. Necroptosis can occur following stimulation of the DNA receptor, DAI, or activation of death receptor, Toll-like receptor, T-cell antigen receptor, or interferon receptor signaling. Analysis of RIP3 deficient mice has implicated necroptosis in several inflammatory-driven diseases, including atherosclerosis, alcoholic liver disease and retinal degeneration. Although studies have demonstrated that mixed lineage kinase domain-like (MLKL) is the only substrate of RIP3 kinase that is essential for necroptotic death, the molecular determinants acting downstream of MLKL remain ambiguous. In addition, RIP3 can signal necroptosis independent of RIP1, may induce apoptosis, and can directly promote pro-inflammatory cytokine production. Therefore it will be important to determine if non-necroptotic RIP3 signaling influences RIP3 dependent pathologies.


Assuntos
Apoptose/imunologia , Regulação para Baixo/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Transdução de Sinais/imunologia , Animais , Apoptose/genética , Caspase 8/fisiologia , Morte Celular/genética , Morte Celular/imunologia , Regulação para Baixo/genética , Proteína Ligante Fas/fisiologia , Humanos , Células Jurkat , Necrose/genética , Necrose/imunologia , Necrose/patologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Transdução de Sinais/genética , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia
13.
Curr Opin Immunol ; 26: 123-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24556408

RESUMO

Classical MHC class I molecules open a window into the cell by presenting intracellular peptides (pMHC I) on the surface. The peptides are used for immune surveillance by circulating CD8+ T and NK cells to detect and eliminate infected or tumor cells. Not surprisingly, viruses and tumor cells have evolved immune evasion mechanisms to keep the window shades down and the cytotoxic cells oblivious to their presence. Here, we review counter mechanisms that nevertheless allow the immune system to detect and eliminate cells unable to properly process antigenic peptides in the endoplasmic reticulum.


Assuntos
Apresentação do Antígeno/imunologia , Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Monitorização Imunológica/métodos , Fragmentos de Peptídeos/metabolismo , Aminopeptidases/deficiência , Aminopeptidases/metabolismo , Animais , Apresentação do Antígeno/genética , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Antígenos de Histocompatibilidade Menor , Fragmentos de Peptídeos/deficiência , Fragmentos de Peptídeos/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células-Tronco/enzimologia , Células-Tronco/imunologia , Células-Tronco/patologia , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia
14.
Curr Opin Immunol ; 26: 128-37, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24556409

RESUMO

For over two decades, we have embraced the cytokine storm theory to explain sepsis, severe sepsis and septic shock. The failure of numerous large-scale clinical trials, which aimed to treat sepsis by neutralizing inflammatory cytokines and LPS, indicates that alternative pathophysiological mechanisms are likely to account for sepsis and the associated immune suppression in patients with severe infection. Recent insights that extricate pyroptotic death from inflammatory cytokine production in vivo have highlighted a need to investigate the consequences of apoptotic and non-apoptotic death in contributing to cytopenia and immune suppression. In this review, we will focus on the biochemical and cellular mechanisms controlling pyroptosis, a Caspase-1/11 dependent form of cell death during infection.


Assuntos
Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Animais , Apoptose/imunologia , Caspase 1/metabolismo , Caspases/metabolismo , Caspases Iniciadoras , Morte Celular/imunologia , Citofagocitose/imunologia , Modelos Animais de Doenças , Ativação Enzimática/imunologia , Células-Tronco Hematopoéticas/enzimologia , Humanos , Hospedeiro Imunocomprometido , Inflamassomos/biossíntese , Inflamassomos/metabolismo , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Camundongos , Necrose , Especificidade por Substrato/imunologia , Síndrome de Resposta Inflamatória Sistêmica/enzimologia
15.
Adv Immunol ; 121: 91-119, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24388214

RESUMO

There is now an abundance of evidence to show that short-chain fatty acids (SCFAs) play an important role in the maintenance of health and the development of disease. SCFAs are a subset of fatty acids that are produced by the gut microbiota during the fermentation of partially and nondigestible polysaccharides. The highest levels of SCFAs are found in the proximal colon, where they are used locally by enterocytes or transported across the gut epithelium into the bloodstream. Two major SCFA signaling mechanisms have been identified, inhibition of histone deacetylases (HDACs) and activation of G-protein-coupled receptors (GPCRs). Since HDACs regulate gene expression, inhibition of HDACs has a vast array of downstream consequences. Our understanding of SCFA-mediated inhibition of HDACs is still in its infancy. GPCRs, particularly GPR43, GPR41, and GPR109A, have been identified as receptors for SCFAs. Studies have implicated a major role for these GPCRs in the regulation of metabolism, inflammation, and disease. SCFAs have been shown to alter chemotaxis and phagocytosis; induce reactive oxygen species (ROS); change cell proliferation and function; have anti-inflammatory, antitumorigenic, and antimicrobial effects; and alter gut integrity. These findings highlight the role of SCFAs as a major player in maintenance of gut and immune homeostasis. Given the vast effects of SCFAs, and that their levels are regulated by diet, they provide a new basis to explain the increased prevalence of inflammatory disease in Westernized countries, as highlighted in this chapter.


Assuntos
Ácidos Graxos Voláteis/fisiologia , Animais , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Citocinas/fisiologia , Ácidos Graxos Voláteis/biossíntese , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Microbiota/imunologia , Microbiota/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de Sinais/imunologia , Especificidade por Substrato/imunologia
16.
Infect Immun ; 81(9): 3128-38, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23774595

RESUMO

Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsE(M1)) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsE(M1) and SsE produced by serotype M28 GAS (SsE(M28)) have a 37% sequence difference. This study aims at determining whether SsE(M28) is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsE(M1), SsE(M28), SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsE(M28) or SeE was converted into lyso-PAF. SsE(M1) and SsE(M28) had kcat values of 373 s(-1) and 467 s(-1), respectively, that were ≥ 30-fold greater than that of hpPAF-AH (12 s(-1)). The comparison of SsE(M1), SsE(M28), and hpPAF-AH in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsE(M28) possesses much lower esterase activities against triglycerides and other esters than SsE(M1) but have similar potency with SsE(M1) in PAF hydrolysis. Deletion of sse(M28) in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsE(M28) in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Streptococcus/imunologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Esterases/genética , Esterases/imunologia , Esterases/metabolismo , Feminino , Humanos , Hidrólise , Evasão da Resposta Imune/genética , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fator de Ativação de Plaquetas/genética , Fator de Ativação de Plaquetas/imunologia , Fator de Ativação de Plaquetas/metabolismo , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Streptococcus/genética , Streptococcus/metabolismo , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Triglicerídeos/genética , Triglicerídeos/imunologia , Triglicerídeos/metabolismo
17.
J Immunol ; 191(1): 35-43, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23733883

RESUMO

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides for MHC class I presentation, influencing the degree and specificity of CD8(+) T cell responses. Single-nucleotide polymorphisms within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that single-nucleotide polymorphisms occur as distinct haplotypes in the human population and that these haplotypes encode functionally distinct ERAP1 alleles. Using a wide range of substrates, we are able to demonstrate that for any given substrate distinct ERAP1 alleles can be "normal," "hypofunctional," or "hyperfunctional" and that each allele has a trend bias toward one of these three activities. Thus, the repertoire of peptides presented at the cell surface for recognition by CTL is likely to depend on the precise combination of both MHC class I and ERAP1 alleles expressed within an individual, and has important implications for predisposition to disease.


Assuntos
Alelos , Aminopeptidases/genética , Haplótipos/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linhagem Celular , Feminino , Haplótipos/imunologia , Humanos , Antígenos de Histocompatibilidade Menor , Peptídeos/genética , Peptídeos/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/metabolismo
18.
J Immunol ; 190(8): 4236-44, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23479224

RESUMO

Despite expanded definition of the leukocyte adhesion cascade and mechanisms underlying individual steps, very little is known about regulatory mechanisms controlling sequential shifts between steps. We tested the hypothesis that metalloproteinases provide a mechanism to rapidly transition monocytes between different steps. Our study identifies diapedesis as a step targeted by metalloproteinase activity. Time-lapse video microscopy shows that the presence of a metalloproteinase inhibitor results in a doubling of the time required for human monocytes to complete diapedesis on unactivated or inflamed human endothelium, under both static and physiological-flow conditions. Thus, diapedesis is promoted by metalloproteinase activity. In contrast, neither adhesion of monocytes nor their locomotion over the endothelium is altered by metalloproteinase inhibition. We further demonstrate that metalloproteinase inhibition significantly elevates monocyte cell surface levels of integrins CD11b/CD18 (Mac-1), specifically during transendothelial migration. Interestingly, such alterations are not detected for other endothelial- and monocyte-adhesion molecules that are presumed metalloproteinase substrates. Two major transmembrane metalloproteinases, a disintegrin and metalloproteinase (ADAM)17 and ADAM10, are identified as enzymes that control constitutive cleavage of Mac-1. We further establish that knockdown of monocyte ADAM17, but not endothelial ADAM10 or ADAM17 or monocyte ADAM10, reproduces the diapedesis delay observed with metalloproteinase inhibition. Therefore, we conclude that monocyte ADAM17 facilitates the completion of transendothelial migration by accelerating the rate of diapedesis. We propose that the progression of diapedesis may be regulated by spatial and temporal cleavage of Mac-1, which is triggered upon interaction with endothelium.


Assuntos
Proteínas ADAM/fisiologia , Metaloproteases/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Migração Transendotelial e Transepitelial/imunologia , Proteínas ADAM/deficiência , Proteínas ADAM/metabolismo , Proteína ADAM17 , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Antígeno de Macrófago 1/metabolismo , Metaloproteases/antagonistas & inibidores , Monócitos/enzimologia , Especificidade por Substrato/imunologia , Imagem com Lapso de Tempo/métodos
19.
J Immunol ; 190(6): 2527-35, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23396948

RESUMO

CD45 is a receptor-like tyrosine phosphatase that positively regulates BCR signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases. We showed previously that a single point mutation, E613R, introduced into the cytoplasmic membrane-proximal "wedge" domain of CD45 is sufficient to drive a lupus-like autoimmune disease on a susceptible genetic background. To clarify the molecular mechanism of this disease, we took advantage of a unique allelic series of mice in which the expression of CD45 is varied across a broad range. Although both E613R B cells and those with supraphysiologic CD45 expression exhibited hyperresponsive BCR signaling, they did so by opposite regulation of the Src family kinase Lyn. We demonstrated that the E613R allele of CD45 does not function as a hyper- or hypomorphic allele but rather alters the substrate specificity of CD45 for Lyn. Despite similarly enhancing BCR signaling, only B cells with supraphysiologic CD45 expression became anergic, whereas only mice harboring the E613R mutation developed frank autoimmunity on a susceptible genetic background. We showed that selective impairment of a Lyn-dependent negative-regulatory circuit in E613R B cells drove autoimmunity in E613R mice. This demonstrates that relaxing negative regulation of BCR signaling, rather than enhancing positive regulation, is critical for driving autoimmunity in this system.


Assuntos
Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Tolerância Imunológica , Antígenos Comuns de Leucócito/fisiologia , Proteínas Tirosina Fosfatases Semelhantes a Receptores/fisiologia , Alelos , Animais , Subpopulações de Linfócitos B/citologia , Variação Genética/imunologia , Tolerância Imunológica/genética , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Quinases da Família src/genética , Quinases da Família src/metabolismo
20.
J Immunol ; 189(8): 4112-22, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22984079

RESUMO

Posttranslational modifications regulate physiology either by directly modulating protein function or by impacting immune recognition of self-proteins. Citrullination is a posttranslational modification formed by the conversion of arginine residues into the citrulline amino acid by protein arginine deiminase (PAD) family members. We have identified mast cells as a major source of the PAD2 enzyme. Activation of the P2X7 purinergic receptor (P2X7) by the inflammatory "danger" signal ATP induces PAD2 activity and robust protein citrullination. P2X7-mediated activation of PAD2 is sensitive to p38 MAPK and protein kinase C inhibitors, and PAD2 regulates the expression of the TNFR2, Adamts-9, and Rab6b transcripts in mast cells. Further, the PAD2 enzyme and its citrullinated substrate proteins are released from mast cells on activation with ATP. PAD2 expression is closely linked with inflammation in rheumatoid arthritis (RA) synovial tissue, and PAD2 and citrullinated proteins are found in the synovial fluid of RA patients. In addition, RA is associated with the development of autoantibodies to citrullinated self-proteins. Our results suggest that P2X7 activation of mast cells may play a role in inflammation by providing PAD2 and PAD2 substrates access to the extracellular space.


Assuntos
Trifosfato de Adenosina/fisiologia , Citrulina/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Proteína-Arginina N-Metiltransferases/fisiologia , Receptores Purinérgicos P2X7/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Espaço Extracelular/enzimologia , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Mastócitos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P2X7/metabolismo , Especificidade por Substrato/imunologia
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