ABSTRACT
A nanohybrid-modified glassy carbon electrode based on conducting polypyrrole doped with carbon quantum dots (QDs) was developed and used for the electrochemical detection of anti-tissue transglutaminase (anti-tTG) antibodies. To improve the polypyrrole conductivity, carrier mobility, and carrier concentration, four types of carbon nanoparticles were tested. Furthermore, a polypyrrole-modified electrode doped with QDs was functionalized with a PAMAM dendrimer and transglutaminase 2 protein by cross-linking with N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The steps of electrode surface modification were surveyed via electrochemical measurements (differential pulse voltammetry (DPV), impedance spectroscopy, and X-ray photoelectron spectroscopy (XPS)). The surface characteristics were observed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and contact angle measurements. The obtained modified electrode exhibited good stability and repeatability. DPV between - 0.1 and 0.6 V (vs. Ag/AgCl 3 M KCl reference electrode) was used to evaluate the electrochemical alterations that occur after the antibody interacts with the antigen (transglutaminase 2 protein), for which the limit of detection was 0.79 U/mL. Without the use of a secondary label, (anti-tTG) antibodies may be detected at low concentrations because of these modified electrode features.
Subject(s)
Dendrimers , Protein Glutamine gamma Glutamyltransferase 2 , Pyrroles , Quantum Dots , Transglutaminases , Humans , Antibodies/immunology , Antibodies/chemistry , Biosensing Techniques/methods , Carbon/chemistry , Dendrimers/chemistry , Electrochemical Techniques/methods , Electrodes , GTP-Binding Proteins/immunology , Polymers/chemistry , Pyrroles/chemistry , Quantum Dots/chemistry , Transglutaminases/immunology , Transglutaminases/chemistryABSTRACT
Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.
Subject(s)
Cell Survival , GTP-Binding Proteins , Protein Conformation , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Humans , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Transglutaminases/metabolism , Transglutaminases/chemistry , Transglutaminases/genetics , Cell Survival/drug effects , Cryoelectron Microscopy , Cell Line, Tumor , Cell Death/drug effects , Scattering, Small Angle , X-Ray Diffraction , Calcium/metabolismABSTRACT
Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.
Subject(s)
Epigenesis, Genetic , Histones , Neoplasms , Protein Glutamine gamma Glutamyltransferase 2 , Protein Processing, Post-Translational , Humans , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Neoplasms/metabolism , Neoplasms/genetics , Neoplasms/enzymology , Neoplasms/pathology , Histones/metabolism , Animals , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Transglutaminases/metabolism , Transglutaminases/genetics , Gene Expression Regulation, Neoplastic , Signal TransductionABSTRACT
Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.
Subject(s)
Coumaric Acids , GTP-Binding Proteins , Glioblastoma , Nanoparticles , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Coumaric Acids/pharmacology , Humans , Transglutaminases/metabolism , Transglutaminases/genetics , Glioblastoma/metabolism , Glioblastoma/drug therapy , Glioblastoma/pathology , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Cell Line, Tumor , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Nanoparticles/chemistry , Drug Carriers/chemistry , Apoptosis/drug effects , Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon, and its pathogenesis remains unclear. Polyamine metabolic enzymes play a crucial role in UC. In this study, we aimed to identify pivotal polyamine-related genes (PRGs) and explore the underlying mechanism between PRGs and the disease status and therapeutic response of UC. We analyzed mRNA-sequencing data and clinical information of UC patients from the GEO database and identified NNMT, PTGS2, TRIM22, TGM2, and PPARG as key PRGs associated with active UC using differential expression analysis and weighted gene co-expression network analysis (WCGNA). Receiver operator characteristic curve (ROC) analysis confirmed the accuracy of these key genes in UC and colitis-associated colon cancer (CAC) diagnosis, and we validated their relationship with therapeutic response in external verification sets. Additionally, single-cell analysis revealed that the key PRGs were specific to certain immune cell types, emphasizing the vital role of intestinal tissue stem cells in active UC. The results were validated in vitro and in vivo experiments, including the colitis mice model and CAC mice model. In conclusion, these key PRGs effectively predict the progression of UC patients and could serve as new pharmacological biomarkers for the therapeutic response of UC.
Subject(s)
Biomarkers , Colitis, Ulcerative , Polyamines , Single-Cell Analysis , Colitis, Ulcerative/genetics , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/therapy , Animals , Humans , Mice , Biomarkers/metabolism , Single-Cell Analysis/methods , Polyamines/metabolism , Disease Models, Animal , Protein Glutamine gamma Glutamyltransferase 2 , Male , Female , Colitis-Associated Neoplasms/genetics , Colitis-Associated Neoplasms/pathology , Colitis-Associated Neoplasms/metabolism , Transglutaminases/genetics , Transglutaminases/metabolismABSTRACT
Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.
Subject(s)
Calcium , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Transglutaminases/metabolism , Transglutaminases/chemistry , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Humans , Calcium/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , Crystallography, X-Ray , Glutens/metabolism , Glutens/chemistry , Models, Molecular , Protein Conformation , Celiac Disease/metabolism , Protein BindingABSTRACT
Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.
Subject(s)
Bleomycin , Lung , Protein Glutamine gamma Glutamyltransferase 2 , Pulmonary Fibrosis , Transglutaminases , Animals , Male , Mice , Glycolysis , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Lung/pathology , Lung/metabolism , Lung/drug effects , Mice, Inbred C57BL , Mice, Knockout , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/pathology , Transglutaminases/metabolism , Transglutaminases/geneticsABSTRACT
In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.
Subject(s)
Celiac Disease , Duodenum , Interleukin-7 , Intestinal Mucosa , Models, Biological , Organoids , Humans , Autoantibodies/immunology , Autoimmunity , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biopsy , Celiac Disease/immunology , Celiac Disease/pathology , Celiac Disease/metabolism , Duodenum/immunology , Duodenum/pathology , Duodenum/metabolism , Epitopes/immunology , Glutens/immunology , Glutens/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/immunology , HLA-DQ Antigens/immunology , HLA-DQ Antigens/metabolism , Interleukin-7/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Killer Cells, Natural/immunology , Myeloid Cells/immunology , Organoids/immunology , Organoids/metabolism , Organoids/pathology , Protein Glutamine gamma Glutamyltransferase 2/immunology , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Pulmonary hypertension (PH) is a severe cardiovascular disease characterised by pulmonary vascular remodelling. The pivotal role of cellular senescence in vascular remodelling has been acknowledged. Transglutaminase type 2 (TG2), a calcium-dependent enzyme, is intricately linked to both cellular senescence and PH. However, the precise mechanisms underlying the involvement of TG2 in PH remain unclear. In this study, we explored the expression of TG2 and the cellular senescence marker p16INK4a in the pulmonary vasculature of mice with PH induced by hypoxia combined with SU5416. Our findings revealed upregulation of both TG2 and p16INK4a expression in the pulmonary vasculature of PH mice. Additionally, a notable increase in TG2 expression was observed in senescent pulmonary artery smooth muscle cells (PASMC). To delve deeper, we employed proteomic sequencing to reveal seven genes associated with cellular senescence, with a subsequent focus on MAPK14. Our investigation revealed that TG2 regulates senescence in PASMC by modulating the phosphorylation levels of MAPK14. Additionally, in the context of hypoxia combined with SU5416, our observations revealed a noteworthy reduction in both pulmonary vascular remodelling and senescent manifestations in smooth muscle-specific TG2 knockout mice compared with their wild-type counterparts. In summary, our findings indicate that TG2 deficiency lowers the senescence levels of PASMC by inhibiting the activity of MAPK14. This inhibition of senescence in the pulmonary vasculature of PH mice helps to decelerate the progression of pulmonary vascular remodelling and consequently hinders the onset and development of PH.
Subject(s)
Cellular Senescence , Hypertension, Pulmonary , Myocytes, Smooth Muscle , Protein Glutamine gamma Glutamyltransferase 2 , Pulmonary Artery , Vascular Remodeling , Animals , Pulmonary Artery/metabolism , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Myocytes, Smooth Muscle/metabolism , Mice , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Transglutaminases/metabolism , Transglutaminases/genetics , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Mice, Knockout , Mice, Inbred C57BL , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Male , Hypoxia/metabolism , Muscle, Smooth, Vascular/metabolism , Indoles , PyrrolesABSTRACT
Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.
Subject(s)
Catalytic Domain , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Transglutaminases/metabolism , Transglutaminases/chemistry , Transglutaminases/antagonists & inhibitors , Humans , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/antagonists & inhibitors , Computer Simulation , Protein Binding , Models, Molecular , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Molecular Docking Simulation , Ligands , Protein ConformationSubject(s)
Celiac Disease , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Celiac Disease/genetics , Celiac Disease/immunology , Humans , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , Transglutaminases/genetics , Transglutaminases/metabolism , Transcriptome , Gene Expression Profiling , Enzyme Inhibitors/therapeutic use , Enzyme Inhibitors/pharmacologyABSTRACT
Chronic spontaneous urticaria (CSU) is an immunological disease that is depicted by high prevalence and eminent burden for patients and society that is attributable to the arbitrary nature of symptoms and inconsistent tools for assessment of activity and severity. Transglutaminase-2 (TG2) is a posttranslational enzyme that is pervasively expressed in many cells and tissue types including mast cells. It has various biological functions, and its role in allergic disorders has been highlighted and delineated through several postulated mechanisms. This case-control study aimed at determining the relationship between serum levels TG2 and severity of CSU. To the best of our knowledge, this is the first study in Egypt to determine the relationship between serum TG2 and severity of CSU. We enrolled 60 adult patients with confirmed diagnosis of CSU. According to urticaria activity score (UAS), patients were categorized into three groups [20 with mild disease; UAS = 0, 20 with moderate disease; UAS = 1-3, 20 with severe disease; UAS = 4-6]. Another 20 healthy individuals (age and gender matched) served as a control group. All patients were subjected to detailed medical history, clinical examination, complete blood count with differential, serum total IgE, CRP, ESR, TSH, ANA, liver and renal function tests. Serum level of TG2 was done by quantitative ELISA for all enrolled patients and controls. Serum TG2 is significantly higher in patients group compared to control group (P value < 0.001). Serum TG2 levels were significantly higher in patients with severe disease compared to patients with moderate or mild disease. This is illustrated by the significant positive correlation between serum TG2 and UAS (r 0.814 and P value 0.000). Moreover, serum TG2 accurately classified CSU patients into mild, moderate and severe subgroups: as regards differentiation between mild and moderate cases (sensitivity 70%, specificity 80%, PPV 77.8, NPV 72.7) and as for the differentiation between moderate and severe cases (sensitivity 95%, specificity 90%, PPV 90.5, NPV 94.7). Serum TG2 may have a pivotal role as a marker of severity in patients with CSU.
Subject(s)
Biomarkers , Protein Glutamine gamma Glutamyltransferase 2 , Urticaria , Protein Glutamine gamma Glutamyltransferase 2/blood , Urticaria/blood , Urticaria/pathology , Chronic Disease , Patient Acuity , Biomarkers/blood , Humans , Male , Female , Young Adult , Adult , Predictive Value of Tests , Immunoglobulin E/blood , C-Reactive Protein/metabolism , Blood Cell CountABSTRACT
Celiac disease (CD) is an autoimmune disease of the small intestine triggered by the consumption of gluten-containing foods in individuals with a genetic predisposition. CD was a rare disease until 20 years ago, when the prevalence increased. Currently, there is no data on the prevalence of CD in high-risk adult populations in Indonesia, even though there is a trend of increasing gluten consumption. Therefore, basic research is needed to determine the magnitude of CD in high-risk adult patients in Indonesia while identifying clinical signs/symptoms, illness history, and lifestyle to determine factors associated with CD in Indonesia. This study is an observational study with a cross-sectional method.Two hundred eighty-three patients who fulfilled the selection and signed the informed consent were recruited from the gastroenterology clinic of Dr. Cipto Mangunkusumo General Hospital. Patients were asked to fill out a celiac disease-related questionnaire and then given anthropometry measurement and blood test for serologic examination with ELISA, consisting of IgA anti-tissue transglutaminase (anti-TTG) and IgG anti-deaminated gliadin peptide (anti-DGP). Statistical analysis was performed using Chi-square and Multivariate logistic regression tests with SPSS software ver. 26. Statistical test differences were considered significant if the p-values were < 0.05. Eight of 283 patients are serologically confirmed with CD (2,83%). On bivariate analysis, the significant variables are age (p < 0,05), constipation (p < 0,05) and history of autoimmune disease (p < 0,05). On multivariate analysis, the only significant variable is the history of autoimmune disease (p < 0,05). This study concluded that the prevalence of CD in high-risk patients with functional gastrointestinal disorder at Dr. Cipto Mangunkusumo Hospital is relatively high (2.83%). CD-associated factors are age, constipation, and history of autoimmune disease in patients. On simultaneous interaction between these factors, autoimmune is the only significant variable associated with CD.
Subject(s)
Celiac Disease , Humans , Celiac Disease/epidemiology , Celiac Disease/complications , Female , Male , Adult , Prevalence , Indonesia/epidemiology , Middle Aged , Cross-Sectional Studies , Risk Factors , Gastrointestinal Diseases/epidemiology , Young Adult , Aged , Transglutaminases/immunology , Adolescent , Protein Glutamine gamma Glutamyltransferase 2ABSTRACT
Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.
Subject(s)
Celiac Disease , Diet, Gluten-Free , GTP-Binding Proteins , Gene Expression Profiling , Glutens , Intestinal Mucosa , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Celiac Disease/immunology , Humans , Glutens/immunology , Transglutaminases/metabolism , Transglutaminases/antagonists & inhibitors , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/genetics , Intestinal Mucosa/pathology , Intestinal Mucosa/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/drug effects , Female , Male , Adult , Transcriptome , Duodenum/pathology , Duodenum/immunology , Duodenum/metabolism , Interferon-gamma/metabolism , Middle Aged , HLA-DQ Antigens/genetics , HLA-DQ Antigens/immunology , Young Adult , Adaptive Immunity/drug effectsABSTRACT
Up to 30% of patients with celiac disease (CD) suffer from concurrent autoimmune disease, compared to 3% of the general population. The association between CD and the current clinical phenotypes of inflammatory myopathies (IIM) patients has not been thoroughly addressed. Assess the CD features among patients with IIM and their relationship with the clinical phenotype and the myositis specific (MSA) and associated antibodies (MAA). For this cross-sectional study, we recruited 99 adult patients classified as IIM from a tertiary center in Mexico. We assessed serum MSA, MAA, and CD-associated autoantibodies (IgA anti-tissue transglutaminase (tTG) and both IgA and IgG anti-deaminated gliadin peptide (DGP)). Patients with highly suggestive serology for CD were then tested for IgG anti-endomysium antibodies, and a duodenal biopsy was performed. 70.7% of patients were positive for at least one antibody. Nine duodenal biopsies were taken, revealing findings compatible with celiac disease in two cases. Subjects with anti-MDA5 antibodies were more likely to have positive anti-tTG IgA antibodies (OR 6.76, 95% CI 1.85-24.62, P = 0.013) and suggestive CD serology (OR 6.41, 95% CI 1.62-25.29, P = 0.009). Patients with anti-Mi2 antibodies were more likely to have positive anti-DGP IgG antibodies (OR 3.35, 95% CI 1.12-9.96, P = 0.039), while positivity for these autoantibodies was less frequent in patients with anti-NXP2 antibodies (OR 0.22, 95% CI 0.06-0.80, P = 0.035). There is a higher prevalence of serologic and definite CD in patients with IIM compared to the general population. Identifying this subgroup of patients may have prognostic and therapeutic implications. Key points ⢠The study estimated a serological celiac disease (CD) prevalence of 70.7% in patients with idiopathic inflammatory myopathies (IIM) and a biopsy-confirmed prevalence of 2%, suggesting that IIM patients should be considered a high-risk population for CD. ⢠We identified a significant association between serological CD and the presence of anti-MDA5 and anti-Mi2 antibodies, suggesting a potential justification for celiac disease screening in this specific subgroup of patients. ⢠The impact of gluten-free diets on IIM patients with serological markers of CD remains untested and warrants further investigation through prospective, randomized studies.
Subject(s)
Autoantibodies , Celiac Disease , Myositis , Humans , Celiac Disease/epidemiology , Celiac Disease/immunology , Celiac Disease/blood , Celiac Disease/diagnosis , Celiac Disease/complications , Cross-Sectional Studies , Female , Male , Middle Aged , Adult , Prevalence , Autoantibodies/blood , Myositis/immunology , Myositis/epidemiology , Myositis/blood , Mexico/epidemiology , Transglutaminases/immunology , Aged , Immunoglobulin A/blood , Gliadin/immunology , Immunoglobulin G/blood , Protein Glutamine gamma Glutamyltransferase 2ABSTRACT
Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.
Subject(s)
Colorectal Neoplasms , Histones , Protein Glutamine gamma Glutamyltransferase 2 , Proteomics , Humans , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/genetics , Proteomics/methods , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Histones/metabolism , Transglutaminases/metabolism , Transglutaminases/genetics , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Cell Line, Tumor , Proteome/analysis , Proteome/metabolism , Protein Processing, Post-Translational , Glutamine/metabolism , Glutamine/chemistry , Epigenesis, GeneticABSTRACT
Potential celiac disease (PCD) is a clinical condition characterised by the presence of a positive CD-specific serology and a normal intestinal architecture. Asymptomatic PCD patients are generally advised to continue on a gluten-containing diet (GCD), but long-term risks of this approach have never been explored. In the present study, we aimed to investigate nutritional and autoimmune complications possibly developing overtime in a cohort of asymptomatic PCD children on a GCD. We compared children's parameters of growth, nutritional status, and autoimmunity between the time of diagnosis and on the occasion of their last medical check, after a long-term gluten-containing diet. Altogether, we collected data from 171 PCD children with a mean follow-up time of 3 years (range 0.35-15.3 years). During follow-up, although patients did not reduce their amount of daily gluten intake, their anti-tissue transglutaminase (anti-TG2) antibodies spontaneously and significantly decreased. Most parameters analysed had not changed during follow-up (height centile, ferritin, albumin, cholesterol, calcium, alkaline phosphatase, parathormone, and vitamin D) or even improved significantly (weight and BMI centile, haemoglobin, blood iron, HDL, glycaemia, and HbA1C, p < 0.05), always remaining within the limit of normality. Equally, autoantibodies for other concomitant autoimmune disorders did not increase overtime. Similar results were obtained excluding from analysis patients who had stopped producing anti-TG2 and those with a follow-up time < 3 years. Our pilot study has provided reassuring results regarding the maintenance of a gluten-containing diet in asymptomatic PCD children, even when long-term follow-up was considered.
Subject(s)
Autoantibodies , Celiac Disease , Diet, Gluten-Free , Nutritional Status , Humans , Celiac Disease/diet therapy , Celiac Disease/immunology , Child , Male , Female , Child, Preschool , Adolescent , Autoantibodies/blood , Protein Glutamine gamma Glutamyltransferase 2 , GTP-Binding Proteins/immunology , Transglutaminases/immunology , Glutens/adverse effects , Glutens/immunology , Health Status , Infant , Follow-Up Studies , AutoimmunityABSTRACT
OBJECTIVES: European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines enable the diagnosis of celiac disease (CD) without biopsies in patients with immunoglobulin A (IgA)-antibodies against tissue transglutaminase (TGA-IgA) ≥ 10× the upper limit of normal (ULN) and positivity of endomysial antibodies in a second blood sample. Limited data exist comparing the biopsy versus the nonbiopsy diagnostic approach regarding long-term outcomes in CD patients. Our study aimed to investigate the influence of the diagnostic approach on adherence to gluten-free diet (GFD), serological remission (defined as normalization of TGA-IgA during follow-up (FU)) and clinical remission in CD patients with TGA-IgA ≥ 10× ULN. METHODS: Retrospective multicenter study. Patients with CD and TGA-IgA ≥ 10× ULN at diagnosis were included in the study. Patients with confirmed diagnosis by biopsy were compared to patients diagnosed by nonbiopsy approach using univariate analysis, Kaplan-Meier survival curve, and logistic regression models. RESULTS: A total of 282 CD patients (192 [68.1%] in the biopsy group; 90 [31.9%] in the nonbiopsy group) were analyzed. The median time to normalization of TGA-IgA was 16.5 months [interquartile range, IQR: 13, 28] in the biopsy and 15 months [IQR: 12, 26] in the nonbiopsy group; p = 0.14). Rates of normalized TGA-IgA at first to third-year FU were comparable between both groups. Adherence to GFD did not seem to be influenced by the diagnostic approach. CONCLUSIONS: The nonbiopsy approach is not inferior to the biopsy approach in terms of adherence to GFD and serological remission in patients with CD.
Subject(s)
Celiac Disease , Diet, Gluten-Free , Immunoglobulin A , Transglutaminases , Humans , Celiac Disease/diagnosis , Celiac Disease/diet therapy , Celiac Disease/blood , Celiac Disease/immunology , Retrospective Studies , Male , Child , Female , Biopsy , Transglutaminases/immunology , Child, Preschool , Adolescent , Immunoglobulin A/blood , Autoantibodies/blood , Protein Glutamine gamma Glutamyltransferase 2 , GTP-Binding Proteins/immunology , Treatment Outcome , Follow-Up Studies , Infant , Patient ComplianceABSTRACT
Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-ß1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.
Subject(s)
Fibrosis , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Renal Insufficiency, Chronic , Animals , Humans , Male , Rabbits , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacology , Disease Models, Animal , Fibrosis/drug therapy , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/immunology , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Macaca fascicularis , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology , Transglutaminases/antagonists & inhibitors , Transglutaminases/metabolismABSTRACT
Olmesartan is an angiotensin II receptor blocker licensed for the treatment of hypertension. It can cause a sprue-like enteropathy (SLE), characterised by chronic diarrhoea, weight loss and villous atrophy. Transiently raised anti-tissue transglutaminase (ATTG) antibody has also been rarely reported in the literature.We describe the case of a woman in her mid-50s, who presented with a history of intermittent loose stools over 1 year, associated with significant weight loss. She had two marginally raised serum ATTG antibody tests during her work-up.After extensive investigations, she was diagnosed with olmesartan-induced enteropathy. On subsequent follow-up, her symptoms had resolved with cessation of her olmesartan therapy.This case adds to existing literature, highlighting the importance of considering olmesartan as a possible differential diagnosis for SLE. It also reports the presence of a raised ATTG antibody which is infrequently reported in this context.