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1.
Amino Acids ; 53(7): 1051-1063, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34059947

RESUMO

Celiac disease (CeD) is a T-cell-dependent enteropathy with autoimmune features where tissue transglutaminase (TG2)-mediated posttranslational modification of gliadin peptides has a decisive role in the pathomechanism. The humoral immune response is reported to target mainly TG2-deamidated γ-gliadin peptides. However, α-gliadin peptides, like p57-68, playing a crucial role in the T-cell response, and p31-43, a major trigger of innate responses, also contain B-cell gliadin epitopes and γ-gliadin like motifs. We aimed to identify if there are anti-gliadin-specific antibodies in CeD patients targeting the p31-43 and p57-68 peptides and to examine whether deamidation of these peptides could increase their antigenicity. We explored TG2-mediated deamidation of the p31-43 and p57-68 peptides, and investigated serum antibody reactivity toward the native and deamidated α and γ-gliadin peptides in children with confirmed CeD and in prospectively followed infants at increased risk for developing CeD. We affinity-purified antibody populations utilizing different single peptide gliadin antigens and tested their binding preferences for cross-reactivity in real-time interaction assays based on bio-layer interferometry. Our results demonstrate that there is serum reactivity toward p31-43 and p57-68 peptides, which is due to cross-reactive γ-gliadin specific antibodies. These γ-gliadin specific antibodies represent the first appearing antibody population in infancy and they dominate the serum reactivity of CeD patients even later on and without preference for deamidation. However, for the homologous epitope sequences in α-gliadins shorter than the core QPEQPFP heptapeptide, deamidation facilitates antibody recognition. These findings reveal the presence of cross-reactive antibodies in CeD patients recognizing the disease-relevant α-gliadins.


Assuntos
Autoanticorpos/imunologia , Doença Celíaca/metabolismo , Gliadina/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase/imunologia , Adolescente , Amidas/química , Autoanticorpos/metabolismo , Doença Celíaca/imunologia , Criança , Pré-Escolar , Reações Cruzadas , Epitopos/imunologia , Gliadina/imunologia , Humanos , Lactente , Fragmentos de Peptídeos/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase/química , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo
2.
Int J Mol Sci ; 22(22)2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34830327

RESUMO

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.


Assuntos
Colo/enzimologia , Miocárdio/enzimologia , Próstata/enzimologia , Transglutaminases/metabolismo , Bexiga Urinária/enzimologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Clonagem Molecular , Estabilidade Enzimática , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dodecilsulfato de Sódio/química , Especificidade por Substrato , Distribuição Tecidual , Transglutaminases/genética
3.
Anal Biochem ; 600: 113699, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32335063

RESUMO

Blood coagulation factor XIII-A (FXIII-A), a member of the transglutaminase enzyme family, is best known for its fibrin clot stabilizing function during blood coagulation. It possesses amine incorporating and protein crosslinking transamidase activities, but it is also able to cleave the previously formed isopeptide bond by its isopeptidase activity. Our aim was to develop a protein-based assay for better characterization of FXIII-A isopeptidase activity. The first attempt applying the crosslinked D-dimer of fibrin as a substrate was not successful because of poor reproducibility. Then, the principle of an earlier published anisotropy based activity assay was adapted for the measurement of FXIII-A isopeptidase activity. After crosslinking the fluorescently labelled α2-antiplasmin derived peptide and S100A4(GST) lysine donor protein, this protease-resistant γ-glutamyl-ε-lysine isopeptide bond containing protein-peptide product was applied as a substrate for FXIII-A. Using this substrate and detecting decreasing anisotropy, kinetic measurement of FXIII-A isopeptidase activity was achieved at high sensitivity even in a complex biological sample and in the presence of inhibitor.


Assuntos
Carbono-Nitrogênio Liases/metabolismo , Fator XIIIa/metabolismo , Anisotropia , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/isolamento & purificação , Fator XIIIa/química , Fluorescência , Humanos
4.
Int J Mol Sci ; 20(21)2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31717806

RESUMO

Huntington's disease (HD) is an inherited neurodegenerative disorder, caused by an abnormal polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Mitochondrial dysfunction and impairment of the ubiquitin-proteasome system (UPS) are hallmarks of HD neurons. The extraneural manifestations of HD are still unclear. We investigated the crosstalk between mitochondria and proteolytic function in skin fibroblasts from juvenile HD patients. We found reduced mitosis, increased cell size, elevated ROS and increased mitochondrial membrane potential in juvenile HD fibroblasts, while cellular viability was maintained. Mitochondrial OXPHOS analysis did not reveal significant differences compared to control. However, the level of mitochondrial fusion and fission proteins was significantly lower and branching in the mitochondria network was reduced. We hypothesized that juvenile HD fibroblasts counterbalance cellular damage and mitochondrial network deficit with altered proteasome activity to promote cell survival. Our data reveal that juvenile HD fibroblasts exhibit higher proteasome activity, which was associated with elevated gene and protein expression of parkin. Moreover, we demonstrate elevated proteasomal degradation of the mitochondrial fusion protein Mfn1 in diseased cells compared to control cells. Our data suggest that juvenile HD fibroblasts respond to mutant polyQ expansion of Htt with enhanced proteasome activity and faster turnover of specific UPS substrates to protect cells.


Assuntos
Fibroblastos/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , GTP Fosfo-Hidrolases/metabolismo , Glicólise , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Neurônios/metabolismo , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pele/citologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
5.
Amino Acids ; 49(3): 605-614, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27627884

RESUMO

Transglutaminase 2 (TGM2) is a unique protein of a nine member family with several enzymatic and non-enzymatic activities and interacting partners. Its physiological and pathological roles, however, are not fully understood. Comparative genomic and computational analysis reported here have revealed phylogenetic changes of TGM2 resulting in novel amino acid clusters in humans and other primates, which may impact secondary structure and increase protein stability. These clusters are located in intrinsically disordered regions and via short linear motifs influence interactions with TGM2 partners directly, or through post-translation modification (phosphorylation and N-glycosylation sites). Our data shed new light on the structural background and evolution of TGM2 multi-functionality and points to so far unrevealed biological roles of the enzyme.


Assuntos
Aminoácidos/química , Evolução Molecular , Proteínas de Ligação ao GTP/química , Proteínas Intrinsicamente Desordenadas/química , Processamento de Proteína Pós-Traducional , Transglutaminases/química , Motivos de Aminoácidos , Aminoácidos/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Ligação ao GTP/metabolismo , Glicosilação , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteína 2 Glutamina gama-Glutamiltransferase , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Transglutaminases/metabolismo
6.
Biochem J ; 473(1): 31-42, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26487698

RESUMO

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.


Assuntos
Aminas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Metástase Neoplásica , Proteínas S100/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proteínas de Ligação ao GTP/genética , Humanos , Dados de Sequência Molecular , Metástase Neoplásica/genética , Ligação Proteica/fisiologia , Proteína 2 Glutamina gama-Glutamiltransferase , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , Especificidade por Substrato/fisiologia , Transglutaminases/genética
7.
Anal Biochem ; 505: 36-42, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27131890

RESUMO

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca(2+)-dependent transamidase activity forming protease-resistant N(ε)-(γ-glutamyl) lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet, mainly because of the lack of a protein-based method for its characterization. Here we report the development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarization of a protein substrate previously formed by crosslinking fluorescently labeled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds that influence isopeptidase activity of TG2.


Assuntos
Carbono-Nitrogênio Liases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Carbono-Nitrogênio Liases/química , Reagentes de Ligações Cruzadas/química , Polarização de Fluorescência , Corantes Fluorescentes/química , Proteínas de Ligação ao GTP/química , Humanos , Cinética , Proteína 2 Glutamina gama-Glutamiltransferase , Fatores de Tempo , Transglutaminases/química
8.
Amino Acids ; 48(1): 31-40, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26250429

RESUMO

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca(2+)-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the K m and the V max kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild-type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2-driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of crosslinked proteins correlates with the manifestation of degenerative disorders.


Assuntos
Aminas/metabolismo , Carbono-Nitrogênio Liases/química , Carbono-Nitrogênio Liases/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/química , Transglutaminases/metabolismo , Cálcio/metabolismo , Carbono-Nitrogênio Liases/genética , Domínio Catalítico , Proteínas de Ligação ao GTP/genética , Humanos , Cinética , Mutação de Sentido Incorreto , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética
9.
Proc Natl Acad Sci U S A ; 109(2): 431-6, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22198767

RESUMO

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1-2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/genética , Doença Celíaca/imunologia , Epitopos/genética , Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Transglutaminases/genética , Análise de Variância , Animais , Autoanticorpos/metabolismo , Autoantígenos/metabolismo , Células Cultivadas , Cristalografia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Gliadina/metabolismo , Humanos , Imunoterapia/métodos , Ativação Linfocitária , Camundongos , Proteína 2 Glutamina gama-Glutamiltransferase , Linfócitos T/imunologia , Transglutaminases/química , Transglutaminases/metabolismo
10.
Biochem J ; 455(3): 261-72, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23941696

RESUMO

TG2 (transglutaminase 2) is a calcium-dependent protein cross-linking enzyme which is involved in a variety of cellular processes. The threshold level of calcium needed for endogenous and recombinant TG2 activity has been controversial, the former being more sensitive to calcium than the latter. In the present study we address this question by identifying a single amino acid change from conserved valine to glycine at position 224 in recombinant TG2 compared with the endogenous sequence present in the available genomic databases. Substituting a valine residue for Gly224 in the recombinant TG2 increased its calcium-binding affinity and transamidation activity 10-fold and isopeptidase activity severalfold, explaining the inactivity of widely used recombinant TG2 at physiological calcium concentrations. ITC (isothermal titration calorimetry) measurements showed 7-fold higher calcium-binding affinities for TG2 valine residues which could be activated inside cells. The two forms had comparable substrate- and GTP-binding affinities and also bound fibronectin similarly, but coeliac antibodies had a higher affinity for TG2 valine residues. Structural analysis indicated a higher stability for TG2 valine residues and a decrease in flexibility of the calcium-binding loop resulting in improved metal-binding affinity. The results of the present study suggest that Val224 increases TG2 activity by modulating its calcium-binding affinity enabling transamidation reactions inside cells.


Assuntos
Cálcio/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Valina/genética , Sítios de Ligação , Carbono-Nitrogênio Liases/metabolismo , Glicina/genética , Glicina/metabolismo , Células HEK293 , Humanos , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Amino Acids ; 44(1): 215-25, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22160262

RESUMO

Transglutaminase 2 (TG2) is a multifunctional member of an enzyme family: it modifies glutamine residues by cross-linking proteins and incorporating primary amines into them, has protein disulphide isomerase and protein kinase activities, mediates trans-membrane signal transduction and interactions between cell surface proteins and the extracellular matrix. These unusual multiple roles encoded into one polypeptide chain suggest that genomic variations in the TGM2 gene should be limited. Indeed, the available information in databases shows that unlike in the case of most other transglutaminases there are no common single nucleotide polymorphisms in exons of human TGM2. We collected data on and produced some of the rare genetic variants of TGM2 by site directed mutagenesis and found that some were less stable than the most abundant (wild type) enzyme variant and the majority had deficient transamidating activity. Further studies are required to clarify the pathologic significance of these rare TGM2 alleles in the human population.


Assuntos
Polimorfismo de Nucleotídeo Único , Transglutaminases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Estabilidade Enzimática , Fibronectinas/química , Proteínas de Ligação ao GTP , Frequência do Gene , Genoma Humano , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Análise de Sequência de DNA , Transglutaminases/química
12.
Front Immunol ; 14: 1139204, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36936920

RESUMO

Macrophage polarization is a process whereby macrophages develop a specific phenotype and functional response to different pathophysiological stimuli and tissue environments. In general, two main macrophage phenotypes have been identified: inflammatory (M1) and alternatively activated (M2) macrophages characterized specifically by IL-1ß and IL-10 production, respectively. In the cardiotoxin-induced skeletal muscle injury model bone marrow-derived macrophages (BMDMs) play the central role in regulating tissue repair. Bone marrow-derived monocytes arriving at the site of injury differentiate first to M1 BMDMs that clear cell debris and trigger proliferation and differentiation of the muscle stem cells, while during the process of efferocytosis they change their phenotype to M2 to drive resolution of inflammation and tissue repair. The M2 population is formed from at least three distinct subsets: antigen presenting, resolution-related and growth factor producing macrophages, the latest ones expressing the transcription factor PPARγ. Nuclear receptor subfamily 4 group A member 1 (NR4A1; also termed Nur77) transcription factor is expressed as an early response gene, and has been shown to suppress the expression of pro-inflammatory genes during efferocytosis. Here we demonstrate that (1) Nur77 null BMDMs are characterized by elevated expression of PPARγ resulting in enhanced efferocytosis capacity; (2) Nur77 and PPARγ regulate transcription in different subsets of M2 skeletal muscle macrophages during muscle repair; (3) the loss of Nur77 prolongs M1 polarization characterized by increased and prolonged production of IL-1ß by the resolution-related macrophages normally expressing Nur77; whereas, in contrast, (4) it promotes M2 polarization detected via the increased number of IL-10 producing CD206+ macrophages generated from the PPARγ-expressing subset.


Assuntos
Interleucina-10 , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , PPAR gama , Humanos , Inflamação/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo
13.
Front Cell Dev Biol ; 9: 737872, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34708041

RESUMO

Thermogenic brown and beige adipocytes might open up new strategies in combating obesity. Recent studies in rodents and humans have indicated that these adipocytes release cytokines, termed "batokines". Irisin was discovered as a polypeptide regulator of beige adipocytes released by myocytes, primarily during exercise. We performed global RNA sequencing on adipocytes derived from human subcutaneous and deep-neck precursors, which were differentiated in the presence or absence of irisin. Irisin did not exert an effect on the expression of characteristic thermogenic genes, while upregulated genes belonging to various cytokine signaling pathways. Out of the several upregulated cytokines, CXCL1, the highest upregulated, was released throughout the entire differentiation period, and predominantly by differentiated adipocytes. Deep-neck area tissue biopsies also showed a significant release of CXCL1 during 24 h irisin treatment. Gene expression data indicated upregulation of the NFκB pathway upon irisin treatment, which was validated by an increase of p50 and decrease of IκBα protein level, respectively. Continuous blocking of the NFκB pathway, using a cell permeable inhibitor of NFκB nuclear translocation, significantly reduced CXCL1 release. The released CXCL1 exerted a positive effect on the adhesion of endothelial cells. Together, our findings demonstrate that irisin stimulates the release of a novel adipokine, CXCL1, via upregulation of NFκB pathway in neck area derived adipocytes, which might play an important role in improving tissue vascularization.

14.
FEBS J ; 288(22): 6476-6491, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33899329

RESUMO

Necroptosis is a regulated necrotic-like cell death modality which has come into the focus of attention since it is known to contribute to the pathogenesis of many inflammatory and degenerative diseases as well as to tumor regulation. Based on current data, necroptosis serves as a backup mechanism when death receptor-induced apoptosis is inhibited or absent. However, the necroptotic role of the proteins involved in mitochondrial apoptosis has not been investigated. Here, we demonstrated that the stimulation of several death and pattern recognition receptors induced necroptosis under caspase-compromised conditions in wild-type, but not in caspase-9-negative human Jurkat and murine MEF cells. Cerulein-induced pancreatitis was significantly reduced in mice with acinar cell-restricted caspase-9 gene knockout. The absence of caspase-9 led to impaired association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 and resulted in decreased phosphorylation of RIP kinases, but the overexpression of RIPK1 or RIPK3 rescued the effect of caspase-9 deficiency. Inhibition of either Aurora kinase A (AURKA) or its known substrate, glycogen synthase kinase 3ß (GSK3ß) restored necroptosis sensitivity of caspase-9-deficient cells, indicating an interplay between caspase-9 and AURKA-mediated pathways to regulate necroptosis. Our findings suggest that caspase-9 acts as a newly identified regulator of necroptosis, and thus, caspase-9 provides a promising therapeutic target to manipulate the immunological outcome of cell death.


Assuntos
Caspase 9/metabolismo , Necrose/metabolismo , Animais , Morte Celular , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos , Pancreatite/metabolismo
15.
ACS Omega ; 5(43): 28273-28284, 2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33163811

RESUMO

Tissue transglutaminase (TG2) is a multifunctional protein that can act as a cross-linking enzyme, GTPase/ATPase, protein kinase, and protein disulfide isomerase. TG2 is involved in cell adhesion, migration, invasion, and growth, as well as epithelial-mesenchymal transition (EMT). Our previous findings indicate that the increased expression of TG2 in renal cell carcinoma (RCC) results in tumor metastasis with a significant decrease in disease- and cancer-specific survival outcome. Given the importance of the prometastatic activity of TG2 in RCC, in the present study, we aim to investigate the relative contribution of TG2's transamidase and guanosine triphosphate (GTP)-binding/GTPase activity in the cell migration, invasion, EMT, and cancer stemness of RCC. For this purpose, the mouse RCC cell line RenCa was transduced with wild-type-TG2 (wt-TG2), GTP-binding deficient-form TG2-R580A, transamidase-deficient form with low GTP-binding affinity TG2-C277S, and transamidase-inactive form TG2-W241A. Our results suggested that predominantly, GTP-binding activity of TG2 is responsible for cell migration and invasion. In addition, CD marker analysis and spheroid assay confirmed that GTP binding/GTPase activity of TG2 is important in the maintenance of mesenchymal character and the cancer stem cell profile. These findings support a prometastatic role for TG2 in RCC that is dependent on the GTP binding/GTPase activity of the enzyme.

16.
FEBS Open Bio ; 9(2): 396-404, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30761263

RESUMO

Transglutaminases (TGs) are a family of structurally and functionally related enzymes that catalyse calcium-dependent post-translational modifications of proteins through protein-protein crosslinking, amine incorporation, or deamidation. For many years deamidation mediated by TGs was considered to be a side reaction, but recently substrate-specific deamidations have been reported. Here we describe an optimised SDS/PAGE assay for the easy and rapid monitoring of the TG reaction with small peptides. The relative proportion of deamidation to transamidation was evaluated by densitometric analysis and confirmed by nano-liquid chromatography-nano-electrospray ionisation MS. We further investigated the effect of reaction conditions on transamidation and deamidation of TG1, TG2 and blood coagulation factor XIII A-subunit (FXIII-A) enzymes using a panel of glutamine-containing peptide substrates. The ratio of transamidation to deamidation was enhanced at high excess of the acyl-acceptor substrate and increasing pH. In addition, it was influenced by peptide substrates as well. Whereas deamidation was favoured at low cadaverine concentrations and acidic pH, no significant effect of calcium was observed on the ratio of transamidation/deamidation. Under our experimental conditions, deamidation always occurred in vitro even at high excess of the acyl-acceptor substrate, and the reaction outcome was shifted to deamidation at neutral pH. Our results provide clear evidence of the deamidation in the TG reaction, and may serve as an important approach for in vivo analysis of deamidation to better understand the role of TGs in biological events.


Assuntos
Amidas/metabolismo , Transglutaminases/metabolismo , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas em Tandem
17.
J Pediatr Gastroenterol Nutr ; 46(3): 253-61, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18376241

RESUMO

OBJECTIVE: Deamidated gliadin peptides are efficient antigens in diagnostic tests for celiac disease, and results correlate better with transglutaminase 2-based assays than those with native gliadin. We investigated whether deamidated gliadin antigens are structurally similar to transglutaminase 2 or could mimic transglutaminase epitopes. PATIENTS AND METHODS: Serum samples from 74 celiac and 65 control patients, and 13 different transglutaminase 2-specific monoclonal mouse antibodies were investigated for their binding to commercially available deamidated gliadin peptides using enzyme-linked immunosorbent assay, competition studies, and molecular modelling. RESULTS: The enzyme-linked immunosorbent assay with deamidated gliadin peptides had 100% sensitivity and 98.5% specificity in patients. Deamidated gliadin epitopes also were recognized by 3 transglutaminase-specific monoclonal antibodies, and antibodies affinity-purified with deamidated gliadin peptides from celiac patient sera reacted with transglutaminase but did not show endomysial binding. The binding of the monoclonal antibodies to deamidated gliadin was inhibited dose dependently by full-length recombinant human transglutaminase, its fragments containing the binding sites of these monoclonal antibodies, or by celiac patient antibodies. Deamidated gliadin peptides decreased the binding of transglutaminase-specific monoclonal antibodies to transglutaminase. Three different cross-reacting transglutaminase epitopes were found, of which 2 are located in the C-terminal domain and 1 is conformational. The binding of celiac serum samples to deamidated gliadin peptides could not be abolished by transglutaminase or by any of the transglutaminase-specific monoclonals, indicating that celiac sera also contain additional antibodies to gliadin epitopes different from transglutaminase. CONCLUSIONS: Certain deamidated gliadin-derived peptides and transglutaminase 2 epitopes have similar 3-dimensional appearance. This homology may contribute to the induction of transglutaminase autoantibodies by molecular mimicry.


Assuntos
Doença Celíaca/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Ligação ao GTP/imunologia , Gliadina/imunologia , Fragmentos de Peptídeos/imunologia , Transglutaminases/imunologia , Adolescente , Adulto , Animais , Anticorpos Monoclonais , Estudos de Casos e Controles , Criança , Pré-Escolar , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/normas , Epitopos , Feminino , Proteínas de Ligação ao GTP/química , Gliadina/química , Glutens/administração & dosagem , Humanos , Lactente , Masculino , Camundongos , Fragmentos de Peptídeos/química , Proteína 2 Glutamina gama-Glutamiltransferase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transglutaminases/química
18.
Biomedicine (Taipei) ; 7(3): 15, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28840829

RESUMO

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-dependent protein modifications. In addition to being an enzyme, TG2 also serves as a G protein for several seven transmembrane receptors and acts as a co-receptor for integrin ß1 and ß3 integrins distinguishing it from other members of the transglutaminase family. TG2 is ubiquitously expressed in almost all cell types and all cell compartments, and is also present on the cell surface and gets secreted to the extracellular matrix via non-classical mechanisms. TG2 has been associated with various human diseases including inflammation, cancer, fibrosis, cardiovascular disease, neurodegenerative diseases, celiac disease in which it plays either a protective role, or contributes to the pathogenesis. Thus modulating the biological activities of TG2 in these diseases will have a therapeutic value.

19.
PLoS One ; 12(3): e0172189, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28248968

RESUMO

Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with specified roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 has well described enzymatic and non-enzymatic functions but in-spite of numerous studies its physiological function in humans has not been defined. We compared data on non-synonymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 and F13a from the Exome aggregation consortium dataset, and used computational and biochemical analysis to reveal the roles of damaging nsSNVs of TGM2. TGM2 and F13a display rarer damaging nsSNV sites than other TGMs and sequence of TGM2, F13a and TGM1 are evolutionary constrained. TGM2 nsSNVs are predicted to destabilize protein structure, influence Ca2+ and GTP regulation, and non-enzymatic interactions, but none coincide with conserved functional sites. We have experimentally characterized six TGM2 allelic variants detected so far in homozygous form, out of which only one, p.Arg222Gln, has decreased activities. Published exome sequencing data from various populations have not uncovered individuals with homozygous loss-of-function variants for TGM2, TGM3 and TGM7. Thus it can be concluded that human transglutaminases differ in harboring damaging variants and TGM2 is under purifying selection suggesting that it may have so far not revealed physiological functions.


Assuntos
Alelos , Bases de Dados de Proteínas , Evolução Molecular , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Transglutaminases/química , Transglutaminases/genética , Substituição de Aminoácidos , Cálcio/química , Cálcio/metabolismo , Estabilidade Enzimática/genética , Fator XIIIa/química , Fator XIIIa/genética , Fator XIIIa/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/metabolismo
20.
J Inorg Biochem ; 91(1): 320-6, 2002 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-12121791

RESUMO

High kinetic stability is an important requirement for the Gd(3+) complexes used as contrast enhancement agents in magnetic resonance imaging. The kinetic stabilities of the Gd(3+) complexes formed with DTPA-N-mono(methylamide) (L(3)), DTPA-N'-mono(methylamide) (L(2)) and DTPA-bis(methylamide) (L(1)) are characterized by the rates of the exchange reactions with Eu(3+) and the endogenous Cu(2+) and Zn(2+). The exchange reactions occur via the proton-assisted dissociation of the complexes and direct attack of the exchanging metal ions on the complex. On the basis of the line-shape analysis of the 1H NMR spectra of the LaL(2), obtained in the pH range 2.5-3.5, we assume that for the proton-assisted dissociation of the complexes the formation of an intermediate containing a free iminodiacetate group must be followed with the rupture of the metal-central nitrogen bond. At about pH > or = 5, the reactions between GdL(2) or GdL(3) and Cu(2+) or Zn(2+) proceed predominantly by direct reaction of the reactants, through the formation of dinuclear intermediates. The contribution of the proton-assisted dissociation is highly important for GdL(1), but its reaction with Zn(2+) is significantly slower than the reactions of GdL(2) and GdL(3). The overall rates of dissociation of GdL(1), GdL(2), GdL(3) and Gd(DTPA)(2-) through H(+) (pH 7.4), Cu(2+) (1 x 10(-6) M) and Zn(2+) (1 x 10(-5) M)-assisted reactions are surprisingly very similar. Replacement of one or two carboxylates with amide groups results in significantly decreased stability constants, but has practically no effect on the kinetic stability of the Gd(3+) complexes, indicating the lower reactivity of the amide groups with Cu(2+) and Zn(2+).


Assuntos
Gadolínio/química , Ácido Pentético/química , Quelantes/química , Meios de Contraste/química , Cobre/química , Európio/química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Zinco/química
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