RESUMO
Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain nongenomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes. A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here, we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets. Our research herein shows that these HIPs form spontaneously in beta-cells through a mechanism involving an aspartic anhydride intermediate. This mechanism leads to the formation of a regular HIP containing a standard peptide bond as well as a HIP-isomer containing an isopeptide bond by linkage to the carboxylic acid side chain of the aspartic acid residue. We used mass spectrometric analyses to confirm the presence of both HIP isomers in islets, thereby validating the occurrence of this novel reaction mechanism in beta-cells. The spontaneous formation of new peptide bonds within cells may lead to the development of neoepitopes that contribute to the pathogenesis of type 1 diabetes as well as other autoimmune diseases.
Assuntos
Células Secretoras de Insulina , Insulina , Peptídeos , Animais , Humanos , Camundongos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , Técnicas In Vitro , Espectrometria de MassasRESUMO
Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.
Assuntos
Autoantígenos/imunologia , Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Fragmentos de Peptídeos/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Antígenos HLA-A , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Dados de Sequência MolecularRESUMO
BDC-6.9, a diabetogenic CD4 T cell clone isolated from a non-obese diabetic (NOD) mouse, responds to pancreatic islet cells from NOD but not BALB/c mice. We recently reported that a hybrid insulin peptide (HIP), 6.9HIP, formed by linkage of an insulin C-peptide fragment and a fragment of islet amyloid polypeptide (IAPP), is the antigen for BDC-6.9. We report here that the core 12-mer peptide from 6.9HIP, centered on the hybrid peptide junction, is also highly antigenic for BDC-6.9. In agreement with the observation that BALB/c islet cells fail to stimulate the T cell clone, a single amino acid difference in the BALB/c IAPP sequence renders the BALB/c version of the HIP only weakly antigenic. Mutant peptide analysis indicates that each parent molecule-insulin C-peptide and IAPP-donates residues critical for antigenicity. Through mass spectrometric analysis, we determine the distribution of naturally occurring 6.9HIP across chromatographic fractions of proteins from pancreatic beta cells. This distribution closely matches the profile of the T cell response to the fractions, confirming that 6.9HIP is the endogenous islet antigen for the clone. Using a new MHC II tetramer reagent, 6.9HIP-tet, we show that T cells specific for the 6.9HIP peptide are prevalent in the pancreas of diabetic NOD mice. Further study of HIPs and HIP-reactive T cells could yield valuable insight into key factors driving progression to diabetes and thereby inform efforts to prevent or reverse this disease.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Insulina/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Sequência de Aminoácidos , Animais , Autoantígenos/química , Peptídeo C/química , Peptídeo C/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Epitopos de Linfócito T/química , Insulina/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos KnockoutRESUMO
Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.
Assuntos
Cloro/toxicidade , Cardiopatias/enzimologia , Exposição por Inalação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose , Sinalização do Cálcio , Cardiotônicos/farmacologia , Células Cultivadas , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacologia , Cardiopatias/induzido quimicamente , Masculino , Mitocôndrias Cardíacas , Miocárdio/enzimologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ranolazina/farmacologia , Ratos Sprague-Dawley , Tiocianatos/farmacologiaRESUMO
It has been recognized for nearly 80 years that insoluble aluminum salts are good immunologic adjuvants and that they form long-lived nodules in vivo. Nodule formation has long been presumed to be central for adjuvant activity by providing an antigen depot, but the composition and function of these nodules is poorly understood. We show here that aluminum salt nodules formed within hours of injection and contained the clotting protein fibrinogen. Fibrinogen was critical for nodule formation and required processing to insoluble fibrin by thrombin. DNase treatment partially disrupted the nodules, and the nodules contained histone H3 and citrullinated H3, features consistent with extracellular traps. Although neutrophils were not essential for nodule formation, CD11b(+) cells were implicated. Vaccination of fibrinogen-deficient mice resulted in normal CD4 T-cell and antibody responses and enhanced CD8 T-cell responses, indicating that nodules are not required for aluminum's adjuvant effect. Moreover, the ability of aluminum salts to retain antigen in the body, the well-known depot effect, was unaffected by the absence of nodules. We conclude that aluminum adjuvants form fibrin-dependent nodules in vivo, that these nodules have properties of extracellular traps, and the nodules are not required for aluminum salts to act as adjuvants.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Alumínio/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Alumínio/farmacologia , Animais , Fibrina/metabolismo , Fibrinogênio/metabolismo , Histonas/metabolismo , Camundongos , Camundongos Knockout , Sais , Linfócitos T/imunologia , Trombina/metabolismo , VacinaçãoRESUMO
Hybrid insulin peptides (HIPs) form in pancreatic ß-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs have been identified in pancreatic islets by mass spectrometry and are targeted by CD4 T cells in patients with type 1 diabetes (T1D) as well as by pathogenic CD4 T-cell clones in nonobese diabetic (NOD) mice. The mechanism of HIP formation is currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient in cathepsin L (CatL), another protease implicated in the formation of disease-relevant HIPs, contain elevated levels of HIPs, indicating a role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow the autoimmune destruction of ß-cells mediated by HIP-reactive CD4 T cells in T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Insulina , Catepsina D , Proteômica , Camundongos Endogâmicos NOD , Peptídeos , Linfócitos T CD4-Positivos , Insulina Regular HumanaRESUMO
DJ-1 is a multifunctional protein with cytoprotective functions. It is localized in the cytoplasm, nucleus, and mitochondria. The conserved cysteine residue at position 106 (Cys106) within DJ-1 serves as a sensor of redox state and can be oxidized to both the sulfinate (-SO2-) and sulfonate (-SO3-) forms. DJ-1 with Cys106-SO2- has cytoprotective activity but high levels of reactive oxygen species can induce its overoxidation to Cys106-SO3-. We found increased oxidative stress in alveolar type II (ATII) cells isolated from emphysema patients as determined by 4-HNE expression. DJ-1 with Cys106-SO3- was detected in these cells by mass spectrometry analysis. Moreover, ubiquitination of Cys106-SO3- DJ-1 was identified, which suggests that this oxidized isoform is targeted for proteasomal destruction. Furthermore, we performed controlled oxidation using H2O2 in A549 cells with DJ-1 knockout generated using CRISPR-Cas9 strategy. Lack of DJ-1 sensitized cells to apoptosis induced by H2O2 as detected using Annexin V and propidium iodide by flow cytometry analysis. This treatment also decreased both mitochondrial DNA amount and mitochondrial ND1 (NADH dehydrogenase 1, subunit 1) gene expression, as well as increased mitochondrial DNA damage. Consistent with the decreased cytoprotective function of overoxidized DJ-1, recombinant Cys106-SO3- DJ-1 exhibited a loss of its thermal unfolding transition, mild diminution of secondary structure in CD spectroscopy, and an increase in picosecond-nanosecond timescale dynamics as determined using NMR. Altogether, our data indicate that very high oxidative stress in ATII cells in emphysema patients induces DJ-1 overoxidation to the Cys106-SO3- form, leading to increased protein flexibility and loss of its cytoprotective function, which may contribute to this disease pathogenesis.
Assuntos
Células Epiteliais Alveolares/metabolismo , Cisteína/metabolismo , Proteína Desglicase DJ-1/metabolismo , Idoso , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Estresse Oxidativo/fisiologia , TransfecçãoRESUMO
Emphysema is characterized by alveolar wall destruction induced mainly by cigarette smoke. Oxidative damage of DNA may contribute to the pathophysiology of this disease. We studied the impairment of the non-homologous end joining (NHEJ) repair pathway and DNA damage in alveolar type II (ATII) cells and emphysema development. We isolated primary ATII cells from control smokers, nonsmokers, and patients with emphysema to determine DNA damage and repair. We found higher reactive oxygen species generation and DNA damage in ATII cells obtained from individuals with this disease in comparison with controls. We also observed low phosphorylation of H2AX, which activates DSBs repair signaling, in emphysema. Our results indicate the impairement of NHEJ, as detected by low XLF expression. We also analyzed the role of DJ-1, which has a cytoprotective activity. We detected DJ-1 and XLF interaction in ATII cells in emphysema, which suggests the impairment of their function. Moreover, we found that DJ-1 KO mice are more susceptible to DNA damage induced by cigarette smoke. Our results suggest that oxidative DNA damage and ineffective the DSBs repair via the impaired NHEJ may contribute to ATII cell death in emphysema.
Assuntos
Células Epiteliais Alveolares/metabolismo , Reparo do DNA por Junção de Extremidades , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Animais , Biomarcadores , Dano ao DNA , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Expressão Gênica , Humanos , Camundongos , Estresse Oxidativo , Ligação Proteica , Enfisema Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Fumar/efeitos adversosRESUMO
Chronic obstructive pulmonary disease (COPD) comprises multiple phenotypes such as airflow obstruction, emphysema, and frequent episodes of acute worsening of respiratory symptoms, known as exacerbations. The goal of this pilot study was to test the usefulness of unbiased metabolomics and transcriptomics approaches to delineate biological pathways associated with COPD phenotypes and outcomes. Blood was collected from 149 current or former smokers with or without COPD and separated into peripheral blood mononuclear cells (PBMC) and plasma. PBMCs and plasma were analyzed using microarray and liquid chromatography mass spectrometry, respectively. Statistically significant transcripts and compounds were mapped to pathways using IMPaLA. Results showed that glycerophospholipid metabolism was associated with worse airflow obstruction and more COPD exacerbations. Sphingolipid metabolism was associated with worse lung function outcomes and exacerbation severity requiring hospitalizations. The strongest associations between a pathway and a certain COPD outcome were: fat digestion and absorption and T cell receptor signaling with lung function outcomes; antigen processing with exacerbation frequency; arginine and proline metabolism with exacerbation severity; and oxidative phosphorylation with emphysema. Overlaying transcriptomic and metabolomics datasets across pathways enabled outcome and phenotypic differences to be determined. Findings are relevant for identifying molecular targets for animal intervention studies and early intervention markers in human cohorts.
Assuntos
Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Actinas/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Glicerofosfolipídeos/metabolismo , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Projetos Piloto , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Fumar , Transcriptoma , Capacidade VitalRESUMO
High density oligonucleotide microarrays (OMAs) have been used recently to profile gene expression in lung carcinoma tissue homogenates. The length of the lists of potentially interesting genes generated by these studies is daunting, and biological and clinical relevance of these lists remains to be validated. Moreover, specific identification of individual biomarkers that might be used for early detection and surveillance has not been the objective of these early studies. We have developed a schema for combining the data derived from the OMA analysis of a few lung cancer cell lines with immunohistochemical testing of tissue microarrays to rapidly identify biomarkers of potential clinical relevance. Initially, we profiled gene expression in lung tumor cell lines using the Affymetrix HG-U95Av2 OMA. RNA from 2 non-small cell lung cancer (NSCLC) cell lines (A549 and H647) and 2 small cell lung cancer (SCLC) cell lines (SHP-77 and UMC-19) were tested. Cells from 1 histologically and cytogenetically normal bronchial epithelial primary culture from a volunteer who had never smoked and 10 samples of histologically unremarkable lung tissue from resection specimens served as normalization controls. Array results were analyzed with Gene Spring software. Results were confirmed by reverse transcription-PCR in an expanded number of cell lines. We then validated the cell line data by immunohistochemical testing for protein using a tissue microarray containing 187 NSCLC clinical samples. Of the 20 most highly expressed genes in the tumor lines, 6 were members of the cancer/testis antigen (CTAG) gene group including 5 MAGE-A subfamily members and NY-ESO-1. SCLC lines strongly expressed all of the MAGE-A genes as well as NY-ESO-1, whereas NSCLC lines expressed a subset of MAGE-A genes at a lower level of intensity and failed to express NY-ESO-1. Reverse transcription-PCR of an extended series of 25 lung cancer cell lines including 13 SCLC, 9 NSCLC, and 3 mesothelioma lines indicated that MAGE-A10 and NY-ESO-1 were expressed only by SCLC, and that MAGE-A1, 3, 6, 12, and 4b were expressed by both SCLC and NSCLC. By immunohistochemistry using the monoclonal antibody 6C1 that recognizes several MAGE-A gene subfamily members, 44% of NSCLC clearly expressed MAGE-A proteins in cytoplasm and/or nucleus. Expression of MAGE-A genes did not correlate with survival but did correlate with histological classification with squamous carcinomas more frequently MAGE-A positive than other NSCLC types (P < 0.00002). We conclude that expression of CTAG gene products, whereas apparently not of prognostic importance, may be useful for early detection and surveillance because of a high level of specificity for central airway squamous and small cell carcinomas.
Assuntos
Antígenos de Neoplasias/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Testículo/imunologia , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Análise por Conglomerados , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
T cell-mediated destruction of insulin-producing ß cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in ß cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in ß cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in ß cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.
Assuntos
Peptídeo C/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Células Secretoras de Insulina/imunologia , Sequência de Aminoácidos , Animais , Peptídeo C/química , Células Clonais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Tolerância Imunológica , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologiaRESUMO
OBJECTIVE: To investigate autoantigens in ß-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9. RESEARCH DESIGN AND METHODS: To purify ß-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of ß-cell tumors. The presence of antigen was monitored by measuring the interferon-γ production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice. RESULTS: Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9. CONCLUSIONS: IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos NODRESUMO
RATIONALE: Lung cancer is a multistep process that is preceded and often accompanied by molecular cytogenetic lesions in benign bronchial epithelium, the precise character, extent and timing of which are not well defined. OBJECTIVES: In this study we comprehensively defined molecular cytogenetic changes in bronchial cells that may precede lung carcinoma using spectral karyotyping (SKY). METHODS: SKY was applied to cultured benign bronchial cells from 43 high-risk smokers without carcinoma, 14 patients with concurrent lung carcinoma, and 14 never-smoker healthy volunteers. MEASUREMENTS AND MAIN RESULTS: The proportion of cells displaying numeric or structural anomalies/total number of metaphase cells was calculated for each case and was referred to as the chromosomal abnormality index. Mean chromosomal abnormality indices were 15.8, 10.1, and 0.7% for patients with cancer, high-risk smokers, and never-smokers, respectively. Clonal abnormalities were found in 17 (40%) of the high-risk smokers without carcinoma and 7 (50%) of the patients with carcinoma, but in none of 14 (0%) never-smokers. Chromosomal gains observed by SKY were confirmed in interphase cultured cells or paraffin sections of biopsy specimens by fluorescence in situ hybridization in 11 of 13 cases for which appropriate probes were available. In 6 of 57 high-risk patients or those with carcinoma, identical clonal abnormalities were dispersed at multiple bronchial sites and were admixed with nonclonal cells. CONCLUSIONS: Clonal and single-cell chromosomal abnormalities are frequent in benign bronchial epithelium during lung carcinogenesis, indicating that chromosomal missegregation and other chromosomal rearrangements occur before overt malignancy.