RESUMO
Circulating uremic toxin indoxyl sulfate (IS), endothelial cell (EC) dysfunction, and decreased nitric oxide (NO) bioavailability are found in chronic kidney disease patients. NO nitrosylates/denitrosylates a specific protein's cysteine residue(s), forming S-nitrosothios (SNOs), and the decreased NO bioavailability could interfere with NO-mediated signaling events. We were interested in investigating the underlying mechanism(s) of the reduced NO and how it would regulate the S-nitrosylation of tissue transglutaminase (TG2) and its substrates on glycolytic, redox and inflammatory responses in normal and IS-induced EC injury. TG2, a therapeutic target for fibrosis, has a Ca2+-dependent transamidase (TGase) that is modulated by S-nitrosylation. We found IS increased oxidative stress, reduced NADPH and GSH levels, and uncoupled eNOS to generate NO. Immunoblot analysis demonstrated the upregulation of an angiotensin-converting enzyme (ACE) and significant downregulation of the beneficial ACE2 isoform that could contribute to oxidative stress in IS-induced injury. An in situ TGase assay demonstrated IS-activated TG2/TGase aminylated eNOS, NFkB, IkBα, PKM2, G6PD, GAPDH, and fibronectin (FN), leading to caspases activation. Except for FN, TGase substrates were all differentially S-nitrosylated either with or without IS but were denitrosylated in the presence of a specific, irreversible TG2/TGase inhibitor ZDON, suggesting ZDON-bound TG2 was not effectively transnitrosylating to TG2/TGase substrates. The data suggest novel roles of TG2 in the aminylation of its substrates and could also potentially function as a Cys-to-Cys S-nitrosylase to exert NO's bioactivity to its substrates and modulate glycolysis, redox, and inflammation in normal and IS-induced EC injury.
Assuntos
Indicã , Proteína 2 Glutamina gama-Glutamiltransferase , Humanos , Células Endoteliais , Estresse Oxidativo , Glicólise , SulfatosRESUMO
Human Leukocyte Antigen (HLA)-DQ2 and HLA-DQ8 are genetic risk factors for Type 1 Diabetes Mellitus (T1DM) and Celiac disease (CD) in Caucasians, but their association with Taiwanese Han population is unknown. We screened 532 Taiwanese T1DM patients for CD biomarkers including anti-tissue transglutaminase (TGM2), anti-gliadin and anti-neoepitope antibodies (Abs), sequencing DQB1 genotypes, and characterized the TGM2 Abs. We report that 3.76% of Taiwanese patients had TGM2-Abs and all had no CD's symptoms. In contrast to Caucasian's CD patients, DQ2/DQ8 only constituted ~4/5 of TGM2-Abs positive patients, while the other ~1/5 patients belonged to different HLA genotypes. Either anti-gliadin or anti-neoepitope Abs coexisted with ~3/4 of TGM2-Abs positive patients that were likely due to gluten-ingestion, while the cause of TGM2-Abs production for other ~1/4 of patients was unknown. Purified anti-TGM2 IgA (TGA) and anti-TGM2 IgG (TGG) could bind on endothelial cells surface, recognized native better than denatured forms of TGM2, and TGA inhibited TGM2's transamidation activity by up to 80% but TGG had no effects. Epitope mapping of all TGM2-Abs positive sera demonstrated that TGM2-Abs had heterogeneity in specificities. This is the first study on the differences between Taiwanese Han group and Caucasian in HLA genotypes and properties of TGM2-Abs.
Assuntos
Autoanticorpos/genética , Diabetes Mellitus Tipo 1/genética , Proteínas de Ligação ao GTP/genética , Antígenos HLA-DQ/genética , Transglutaminases/genética , Adolescente , Doença Celíaca/genética , Criança , Pré-Escolar , Células Endoteliais/metabolismo , Feminino , Genótipo , Gliadina/genética , Humanos , Imunoglobulina A/genética , Lactente , Masculino , Proteína 2 Glutamina gama-Glutamiltransferase , TaiwanRESUMO
BACKGROUND/AIMS: Fabry disease (FD), a rare x-lined genetic disorder is a cause of renal deterioration. The phenotype of FD is highly variable and nonspecific, and correct diagnosis has always been delayed. We aimed to explore the prevalence and clinical presentation of FD in this high-risk male population in a Northern Taiwan medical center. METHODS: This is the first study to survey the incidence of FD in this high-risk population through the platform of a chronic kidney disease (CKD) education program in Asia. A total of 1,012 male patients with unknown CKD causes were screened using an assay of alpha-galactosidase A activity (α-Gal A) by dried blood spots (DBS). A final GLA gene analysis was also done for those with low enzyme activity. RESULTS: We identified two new patients with classic FD and four patients with late-onset FD. One novel GLA mutation with c.413 G>A was found in one classic FD patient (index 5). The prevalence of FD is about 0.59 % (6 in 1,012) in the high-risk population group with CKD. The clinical symptoms of FD patients are nonspecific except in those with various degrees of renal failure. Those patients' correct diagnosis was delayed, taking years and even decades. Three patients received enzyme replacement therapy and one started regular hemodialysis due to persistent renal function deterioration. Another two patients were found from family screening through a new index. In addition, a false negative result occurred in one patient who was proved to have FD by his kidney pathology as determined by this screening. CONCLUSION: FD is not such as rare a disease and its prevalence is greater in this high-risk male population. Clinicians need to be aware that FD should be included in the differential diagnosis in CKD with unknown etiology.
Assuntos
Doença de Fabry/diagnóstico , Falência Renal Crônica/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Doença de Fabry/epidemiologia , Humanos , Isoenzimas/sangue , Isoenzimas/genética , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Prevalência , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Insuficiência Renal Crônica , Taiwan , Adulto Jovem , alfa-Galactosidase/sangue , alfa-Galactosidase/genéticaRESUMO
Post-translational modification (PTM) is an important mechanism in modulating a protein's structure and can lead to substantial diversity in biological function. Compared to other forms of PTMs such as phosphorylation, acetylation and glycosylation, the physiological significance of aminylation is limited. Aminylation refers to the covalent incorporation of biogenic/polyamines into target protein by calcium-dependent transglutaminases (TGs). The development of novel and more sensitive techniques has led to more proteins identified as tissue transglutaminase (TG2) substrates and potential targets for aminylation. Many of these substrate proteins play a role in cell signaling, cytoskeleton organization, muscle contraction, and inflammation. TG2 is well studied and widely expressed in a variety of tissues and will be the primary focus of this review on recent advance in transglutaminase-mediated aminylation.
Assuntos
Aminas Biogênicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Agregação Patológica de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transglutaminases/metabolismo , Aminação , Animais , Aminas Biogênicas/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Ligação ao GTP/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Agregação Patológica de Proteínas/genética , Domínios Proteicos , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais , Especificidade por Substrato , Transglutaminases/genéticaRESUMO
Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to unactivated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 7590 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.
Assuntos
Fibrinogênio/química , Fibrinogênio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Histamina/metabolismo , Inflamação/metabolismo , Transglutaminases/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Histamina/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transglutaminases/biossíntese , Transglutaminases/isolamento & purificaçãoRESUMO
Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC(50) of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K(3), were found to have an IC(50) in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Transglutaminases/antagonistas & inibidores , Animais , Cálcio/farmacologia , Técnicas de Química Combinatória , Modelos Animais de Doenças , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Avaliação Pré-Clínica de Medicamentos , Fator XIIIa/antagonistas & inibidores , Fator XIIIa/metabolismo , Proteínas de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Humanos , Doença de Machado-Joseph/enzimologia , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Octoxinol , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/metabolismo , Tirfostinas/químicaRESUMO
[This corrects the article DOI: 10.18632/oncotarget.18789.].
RESUMO
Tissue transglutaminase (tTG) is a multifunctional enzyme with transglutaminase crosslinking (TGase), GTP binding, and hydrolysis activities that play a role in many different disorders. We identified, characterized, and investigated the function and stability of two alternatively spliced forms of tTG using biochemical, cellular, and molecular biological approaches. Using a human aortic vascular smooth muscle cells (VSMC) cDNA library, we identified two cDNAs encoding C-terminal truncated forms, tTG(V1) and tTG(V2). tTG(V1,2) mRNAs were synthesized by a rare splicing event using alternate splice sites within exons 12 and 13 of the tTG gene, respectively. Quantitative PCR and immunoblotting demonstrated that there was unique expression and localization of tTG(V1,2) compared with tTG in human umbilical vein endothelial cells (HUVECs), VSMC, and leukocytes. The loss of C-terminal 52 amino acid residues (AAs) in tTG(V1,2) altered GTP binding, enhanced GTP hydrolysis, rendered the variants insensitive to GTP inhibition, and resulted in <10% residual Ca(+2)-dependent TGase activity. Transfection in HEK293 demonstrated a 28- and 5-fold reduction in the expression of tTG(V1) and tTG(V2), respectively, demonstrating that the C-terminal GTP-binding domain is important in stabilizing and promoting the half-life of tTG. The altered affinity for GTP allowed tTG(V1,2) to exhibit enhanced TGase activity when there is a transient increase in Ca(+2) levels. The abundance of tTG(V1,2) and its distinct intracellular expression patterns in human vascular cells and leukocytes indicate these isoforms likely have unique physiological functions.
Assuntos
Processamento Alternativo/genética , Células Endoteliais/enzimologia , Proteínas de Ligação ao GTP/fisiologia , Leucócitos/enzimologia , Músculo Liso Vascular/enzimologia , Transglutaminases/fisiologia , Sequência de Aminoácidos , Aorta/citologia , Aorta/enzimologia , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Regulação Enzimológica da Expressão Gênica/fisiologia , Guanosina Trifosfato/fisiologia , Células HL-60 , Humanos , Líquido Intracelular/enzimologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/fisiologia , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética , Transglutaminases/isolamento & purificação , Veias Umbilicais/enzimologiaRESUMO
Mitochondria are the cell's power plant to satisfy the energy demands. However, dysfunctional mitochondria can cause overproduction of reactive oxygen species (ROS), oxidative stress, and alteration of calcium homeostasis, which are the hallmarks of mitochondrial diseases. Under prolong oxidative stress, repeated cytosolic calcium elevations even only transiently, can lead to activation of some enzymes. One calcium-activated enzyme with demonstrated pathophysiological important in mitochondrial disease is tissue transglutaminase (TG2). TG2 is known as a post-translational modification (PTM) enzyme that is induced by oxidative stress. Compared to other types of PTMs, the physiological significance of TG2 mediated PTM is just beginning to be understood. Once activated, TG2 can modulate transcription, inactivate metabolic enzymes, and cause aggregation of critical proteins. Recent data indicate that TG2's activity not only can modulate the assembly of respiratory chain complexes but can also modulate the transcription of critical genes including PGC-1alpha and cytochrome C that are important for function and biogenesis of mitochondria. Here, we summarize dysfunctional mitochondria in diseases such as in neurodegenerative disorders can modulate TG2's activity and function. TG2 is also important for normal function of mitochondria.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Transglutaminases/metabolismo , Doença de Alzheimer/enzimologia , Animais , Autoimunidade , Transição Epitelial-Mesenquimal , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Doença de Huntington/enzimologia , Doenças Neurodegenerativas/enzimologia , Doença de Parkinson/enzimologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/química , Transglutaminases/genética , CicatrizaçãoRESUMO
Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neutrófilos/metabolismo , Óxido Nítrico/metabolismo , Transglutaminases/metabolismo , Adesão Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Neutrófilos/citologia , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
Renal anemia is a common complication in patients with advanced chronic kidney disease. In vitro studies have shown that indoxyl sulfate decreases erythropoietin production. Whether this effect is seen in vivo remains unclear. Our goal was to explore the role of indoxyl sulfate in renal anemia. We found serum indoxyl sulfate levels are significantly and negatively associated with erythropoietin levels in human. A multiple stepwise linear regression analyses after adjustment for other independent parameters revealed that free indoxyl sulfate, and total indoxyl sulfate were significantly associated with erythropoietin levels. In animal studies, erythropoietin gene and protein expression were markedly inhibited in rats with chronic kidney disease; however, this effect was significantly reversed by lowering serum indoxyl sulfate with AST-120. Indoxyl sulfate may also inhibit erythropoietin expression in animal models with chronic kidney disease. These findings further support the role of indoxyl sulfate in the development of renal anemia.
RESUMO
Alternative splicing is an important mechanism for modulating gene function that accounts for a considerable proportion of proteomic complexity in higher eukaryotes. Alternative splicing is often tightly regulated in a cell-type- or developmental-stage- specific manner and can cause a single gene to have multiple functions. Human Tissue transglutaminase (TGM2) is a multifunctional enzyme with transglutaminase crosslinking (TGase), G protein signaling and kinase activities that are postulated to play a role in many disease states. TGM2 mRNA is regulated by alternative splicing, producing C-terminal truncated forms of TGM2 that are predicted to have distinct biochemical properties and biological functions. In this review, we will discuss how alternatively spliced forms of TGM2 could modulate its roles in cancer, neurodegeneration, inflammation and wound healing.
Assuntos
Processamento Alternativo , Transglutaminases/genética , Animais , Autoimunidade/fisiologia , Cálcio/farmacologia , Adesão Celular/fisiologia , Transição Epitelial-Mesenquimal , Proteínas de Ligação ao GTP , Guanosina Trifosfato/farmacologia , Humanos , Modelos Moleculares , Doenças Neurodegenerativas/fisiopatologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/antagonistas & inibidores , Transglutaminases/química , Transglutaminases/metabolismo , Cicatrização/fisiologiaRESUMO
Human tissue transglutaminase (TGM2) is implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's, Parkinson's and expanded polyglutamine (polyQ) diseases. TGM2 promotes formation of soluble and insoluble high molecular weight aggregates by catalyzing a covalent linkage between peptide-bound Q residues in polyQ proteins and a peptide-bound Lys residue. Therapeutic approaches to modulate the activity of TGM2 are needed to proceed with studies to test the efficacy of TGM2 inhibition in disease processes. We investigated whether acetylation of Lys-residues by sulfosuccinimidyl acetate (SNA) or aspirin (ASA) would alter the crosslinking activity of TGM2. Acetylation by either SNA and/or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys(444), Lys(468), and Lys(663). Hence, acetylation of Lys-residues may modulate the enzymatic function of TGM2 in vivo and offer a novel approach to treatment of TGM2 mediated disorders.