RESUMO
One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.
Assuntos
Montagem e Desmontagem da Cromatina , Citoplasma , Epigênese Genética , Histonas , Transglutaminases , Humanos , Acetilação , Cromatina , DNA/genética , Metilação de DNA , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Transglutaminases/genética , Transglutaminases/metabolismo , Citoplasma/enzimologia , Citoplasma/genética , Citoplasma/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologiaRESUMO
Transglutaminase type 2 (TG2) is the most ubiquitously expressed and well characterized member of the transglutaminase family. It is a ubiquitous multifunctional enzyme implicated in the regulation of several cellular pathways that support the survival, death, and general homeostasis of eukaryotic cells. Due to its multiple localizations both inside and outside the cell, TG2 participates in the regulation of many crucial intracellular signaling cascades in a tissue- and cell-specific manner, making this enzyme an important player in disease development and progression. Moreover, TG2 is capable of modulating the tumor microenvironment, a process of dynamic tissue remodeling and biomechanical events, resulting in changes which influence tumor initiation, growth, and metastasis. Even if generally related to the Ca2+-dependent post-translational modification of proteins, a number of different biological functions have been ascribed to TG2, like those of a peptide isomerase, protein kinase, guanine nucleotide binder, and cytosolic-nuclear translocator. With respect to cancer, TG2's role is controversial and highly debated; it has been described both as an anti- and pro-apoptotic factor and is linked to all the processes of tumorigenesis. However, numerous pieces of evidence support a tissue-specific role of TG2 so that it can assume both oncogenic and tumor-suppressive roles.
Assuntos
Neoplasias , Proteína 2 Glutamina gama-Glutamiltransferase , Humanos , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.
Assuntos
Doença Celíaca/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Gliadina/farmacologia , Fragmentos de Peptídeos/farmacologia , Adolescente , Aminofenóis/administração & dosagem , Aminofenóis/farmacologia , Animais , Células CACO-2 , Doença Celíaca/tratamento farmacológico , Doença Celíaca/genética , Linhagem Celular , Criança , Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios Proteicos , Quinolonas/administração & dosagem , Quinolonas/farmacologia , Adulto JovemRESUMO
We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.
Assuntos
COVID-19/imunologia , Proteínas de Ligação ao GTP/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Transglutaminases/imunologia , Animais , COVID-19/genética , COVID-19/patologia , Proteínas de Ligação ao GTP/genética , Humanos , Fator Regulador 3 de Interferon/genética , Interferon beta/genética , Interferon beta/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transglutaminases/genéticaRESUMO
Host-directed therapies are emerging as a promising tool in the curing of difficult-to-treat infections, such as those caused by drug-resistant bacteria. In this study, we aim to test the potential activity of the FDA- and EMA-approved drugs cysteamine and cystamine against Mycobacterium abscessus. In human macrophages (differentiated THP-1 cells), these drugs restricted M. abscessus growth similar to that achieved by amikacin. Here, we use the human ex vivo granuloma-like structures (GLS) model of infection with the M. abscessus rough (MAB-R) and smooth (MAB-S) variants to study the activity of new therapies against M. abscessus. We demonstrate that cysteamine and cystamine show a decrease in the number of total GLSs per well in the MAB-S and MAB-R infected human peripheral blood mononuclear cells (PBMCs). Furthermore, combined administration of cysteamine or cystamine with amikacin resulted in enhanced activity against the two M. abscessus morpho variants compared to treatment with amikacin only. Treatment with cysteamine and cystamine was more effective in reducing GLS size and bacterial load during MAB-S infection compared with MAB-R infection. Moreover, treatment with these two drugs drastically quenched the exuberant proinflammatory response triggered by the MAB-R variant. These findings showing the activity of cysteamine and cystamine against the R and S M. abscessus morphotypes support the use of these drugs as novel host-directed therapies against M. abscessus infections.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Humanos , Amicacina/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cisteamina/farmacologia , Cisteamina/uso terapêutico , Cistamina/farmacologia , Cistamina/uso terapêutico , Leucócitos Mononucleares , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
The cellular environment needs to be strongly regulated and the maintenance of protein homeostasis is crucial for cell function and survival. HSF1 is the main regulator of the heat shock response (HSR), the master pathway required to maintain proteostasis, as involved in the expression of the heat shock proteins (HSPs). HSF1 plays numerous physiological functions; however, the main role concerns the modulation of HSPs synthesis in response to stress. Alterations in HSF1 function impact protein homeostasis and are strongly linked to diseases, such as neurodegenerative disorders, metabolic diseases, and different types of cancers. In this context, type 2 Transglutaminase (TG2), a ubiquitous enzyme activated during stress condition has been shown to promote HSF1 activation. HSF1-TG2 axis regulates the HSR and its function is evolutionary conserved and implicated in pathological conditions. In this review, we discuss the role of HSF1 in the maintenance of proteostasis with regard to the HSF1-TG2 axis and we dissect the stress response pathways implicated in physiological and pathological conditions.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Transglutaminases/metabolismo , Animais , Desenvolvimento Embrionário , Resposta ao Choque Térmico , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response.
Assuntos
Fibrose Cística/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação ao GTP/genética , Fatores de Transcrição de Choque Térmico/genética , Transglutaminases/genética , Animais , Fibrose Cística/patologia , Regulação da Expressão Gênica , Resposta ao Choque Térmico/genética , Humanos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/genética , Proteína 2 Glutamina gama-Glutamiltransferase , Processamento de Proteína Pós-Traducional/genética , Proteostase/genética , Transdução de SinaisRESUMO
BACKGROUND: Clinical scores have been proposed to stratify the risk of pulmonary thromboembolism (PTE), although this approach suffers a low specificity and the unavoidable need for computed tomography pulmonary angiography (CTPA) scans. OBJECTIVE: Our study aimed to investigate a simple modification to the already validated Wells' score to improve its diagnostic accuracy in the emergency department (ED). METHODS: We retrospectively reviewed all CTPA scans performed in the ED setting to rule out PTE over a 1-year (2017) period. Clinical variables potentially associated with PTE were assessed to improve diagnostic accuracy of the Wells' score, thus introducing a modified Wells' score (mWells). RESULTS: Four thousand four hundred thirteen CTPAs were identified, of which 504 were for suspected PTE. The prevalence of PTE was 23.9%. Among clinical data, only peripheral capillary oxygen saturation was consistently correlated with PTE at univariate (odds ratio 2.75 [95% confidence interval 1.61-4.73]) and multivariate (odds ratio 3.78 [95% confidence interval 2.13-6.72]) logistic regression analysis. The mWells' score had a higher area under the receiver operating characteristic curve compared with the original Wells' score: 0.71 (95% confidence interval 0.67-0.75) vs. 0.65 (95% confidence interval 0.61-0.69) (p < .01) and improved diagnostic accuracy. CONCLUSIONS: Current clinical stratification tools for PTE are characterized by low specificity, leading to an overuse of CTPA. mWells', rather than Wells', score showed a better predictive performance of PTE detection. Our results suggest that current diagnostic pathway for PTE may be improved by simple adjustments (i.e., mWells') of clinical prediction scores.
Assuntos
Embolia Pulmonar , Angiografia , Angiografia por Tomografia Computadorizada , Serviço Hospitalar de Emergência , Humanos , Embolia Pulmonar/diagnóstico , Estudos RetrospectivosRESUMO
The maintenance of protein homeostasis (proteostasis) is a fundamental aspect of cell physiology that is essential for the survival of organisms under a variety of environmental and/or intracellular stress conditions. Acute and/or persistent stress exceeding the capacity of the intracellular homeostatic systems results in protein aggregation and/or damaged organelles that leads to pathological cellular states often resulting in cell death. These events are continuously suppressed by a complex macromolecular machinery that uses different intracellular pathways to maintain the proteome integrity in the various subcellular compartments ensuring a healthy cellular life span. Recent findings have highlighted the role of the multifunctional enzyme type 2 transglutaminase (TG2) as a key player in the regulation of intracellular pathways, such as autophagy/mitophagy, exosomes formation and chaperones function, which form the basis of proteostasis regulation under conditions of cellular stress. Here, we review the role of TG2 in these stress response pathways and how its various enzymatic activities might contributes to the proteostasis control.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteostase , Transglutaminases/metabolismo , Animais , Autofagia , Doença , Exossomos/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Estresse FisiológicoRESUMO
Numerous studies are revealing a role of exosomes in intercellular communication, and growing evidence indicates an important function for these vesicles in the progression and pathogenesis of cancer and neurodegenerative diseases. However, the biogenesis process of exosomes is still unclear. Tissue transglutaminase (TG2) is a multifunctional enzyme with different subcellular localizations. Particularly, under stressful conditions, the enzyme has been also detected in the extracellular matrix, but the mechanism(s) by which TG2 is released outside the cells requires further investigation. Therefore, the goal of the present study was to determine whether exosomes might be a vehicle for TG2 to reach the extracellular space, and whether TG2 could be involved in exosomes biogenesis. To address this issue, we isolated and characterized exosomes derived from cells either expressing or not TG2, under stressful conditions (i.e. proteasome impairment or expressing a mutated form of huntingtin (mHtt) containing 84 polyglutamine repeats). Our results show that TG2 is present in the exosomes only upon proteasome blockade, a condition in which TG2 interacts with TSG101 and ALIX, two key proteins involved in exosome biogenesis. Interestingly, we found that TG2 favours the assembly of a protein complex including mHtt, ALIX, TSG101 and BAG3, a co-chaperone involved in the clearance of mHtt. The formation of this complex is paralleled by the selective recruitment of mHtt and BAG3 in the exosomes derived from TG2 proficient cells only. Overall, our data indicate that TG2 is an important player in the biogenesis of exosomes controlling the selectivity of their cargo under stressful cellular conditions. In addition, these vesicles represent the way by which cells can release TG2 into the extracellular space under proteostasis impairment.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Exossomos/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Leupeptinas/farmacologia , Transporte Proteico/fisiologia , Estresse Fisiológico/fisiologia , Transglutaminases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fibroblastos , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Mapeamento de Interação de Proteínas , Fatores de Transcrição/metabolismo , Transglutaminases/deficiência , Transglutaminases/genética , Repetições de TrinucleotídeosRESUMO
The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and the determination of cell fate under stress conditions. Reticulon-1C (RTN-1C) is a member of the reticulon family proteins localized primarily on the ER membrane and known to regulate ER structure and function. Several cellular processes depend on the structural and functional crosstalk between different organelles, particularly on the endoplasmic reticulum and mitochondria. These dynamic contacts, called mitochondria-associated ER membranes (MAMs), are essential for the maintenance of mitochondrial structure and participate in lipid and calcium exchanges between the two organelles. In this study we investigated the impact of RTN-1C modulation on mitochondrial dynamics. We demonstrate that RTN-1C controls mitochondrial structure and function affecting intracellular Ca2+ homeostasis and lipid exchange between ER and mitochondria. We propose that these events depend on RTN-1C involvement in the regulation of ER-mitochondria cross-talk and define a role for RTN-1C in maintaining the function of contacts between the two organelles.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Membranas Mitocondriais/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Ligação Proteica , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
Cystic fibrosis (CF) is a rare autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is F508del-CFTR (ΔF) which leads the encoded ion channel towards misfolding and premature degradation. The disease is characterized by chronic bronchopulmonary obstruction, inflammation and airways colonization by bacteria, which are the major cause of morbidity and mortality. The STING pathway is the main signaling route activated in the presence of both self and pathogen DNA, leading to Type I Interferon (IFN I) production and the innate immune response. In this study, we show for the first time the relationship existing in CF between resistant and recurrent opportunistic infections by Pseudomonas aeruginosa and the innate immunity impairment. We demonstrate through ex vivo and in vivo experiments that the pathway is inadequately activated in ΔF condition and the use of direct STING agonists, as 2',3'-cyclic GMP-AMP (2', 3' cGAMP), is able to restore the immune response against bacterial colonization. Indeed, upon treatment with the STING pathway agonists, we found a reduction of colony forming units (CFUs) consequent to IFN-ß enhanced production in Pseudomonas aeruginosa infected bone marrow derived macrophages and lung tissues from mice affected by Cystic Fibrosis. Importantly, we also verified that the impairment detected in the primary PBMCs obtained from ΔF patients can be corrected by 2', 3' cGAMP. Our work indicates that the cGAS/STING pathway integrity is crucial in the Cystic Fibrosis response against pathogens and that the restoration of the pathway by 2', 3' cGAMP could be exploited as a possible new target for the symptomatic treatment of the disease.
Assuntos
Fibrose Cística , Interferon Tipo I , Camundongos , Animais , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística , Imunidade Inata/genética , Interferon Tipo I/metabolismo , Macrófagos , Proteínas Serina-Treonina Quinases/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismoRESUMO
BACKGROUND: Renowned since ancient times for its medical properties, liquorice is nowadays mainly used for flavoring candies or soft drinks. Continuous intake of large amounts of liquorice is a widely known cause of pseudo-hyperaldosteronism leading to hypertension and hypokalemia. These manifestations are usually mild, although in some cases may generate life-threatening complications, i.e., arrhythmias, muscle paralysis, rhabdomyolysis, and coma. In addition, liquorice has an important estrogenic-like activity. METHODS: We summarized the current knowledge about liquorice and reviewed 104 case reports in both the English and Italian languages from inception to June 2023 concerning complications due to an excess of liquorice intake. RESULTS: In contrast to most published data, female sex and old age do not appear to be risk factors. However, hypertension and electrolyte imbalance (mainly hypokalemia) are prevalent features. The detection of glycyrrhetinic acid in blood is very uncommon, and the diagnosis is essentially based on an accurate history taking. CONCLUSIONS: Although there is not a significant mortality rate, liquorice toxicity often requires hospitalization and therefore represents a significant health concern. Major pharmaceutical drug regulatory authorities should solicit public awareness about the potentially dangerous effects caused by excessive use of liquorice.
Assuntos
Glycyrrhiza , Hipertensão , Hipopotassemia , Glycyrrhiza/efeitos adversos , Hipopotassemia/induzido quimicamente , Doces , Bebidas Gaseificadas , Hipertensão/induzido quimicamenteRESUMO
Skin cutaneous melanoma (SKCM) is the deadliest form of skin cancer due to its high heterogeneity that drives tumor aggressiveness. Melanoma plasticity consists of two distinct phenotypic states that co-exist in the tumor niche, the proliferative and the invasive, respectively associated with a high and low expression of MITF, the master regulator of melanocyte lineage. However, despite efforts, melanoma research is still far from exhaustively dissecting this phenomenon. Here, we discovered a key function of Transglutaminase Type-2 (TG2) in regulating melanogenesis by modulating MITF transcription factor expression and its transcriptional activity. Importantly, we demonstrated that TG2 expression affects melanoma invasiveness, highlighting its positive value in SKCM. These results suggest that TG2 may have implications in the regulation of the phenotype switching by promoting melanoma differentiation and impairing its metastatic potential. Our findings offer potential perspectives to unravel melanoma vulnerabilities via tuning intra-tumor heterogeneity.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanócitos/metabolismo , Fenótipo , Fator de Transcrição Associado à Microftalmia/genética , Linhagem Celular Tumoral , Melanoma Maligno CutâneoRESUMO
Tissue transglutaminase (TG2) activity has been implicated in inflammatory disease processes such as Celiac disease, infectious diseases, cancer, and neurodegenerative diseases, such as Huntington's disease. Furthermore, four distinct biochemical activities have been described for TG2 including protein crosslinking via transamidation, GTPase, kinase and protein disulfide isomerase activities. Although the enzyme plays a complex role in the regulation of cell death and autophagy, the molecular mechanisms and the putative biochemical activity involved in each is unclear. Therefore, the goal of the present study was to determine how TG2 modulates autophagy and/or apoptosis and which of its biochemical activities is involved in those processes. To address this question, immortalized embryonic fibroblasts obtained from TG2 knock-out mice were reconstituted with either wild-type TG2 or TG2 lacking its transamidating activity and these were subjected to different treatments to induce autophagy or apoptosis. We found that knock out of the endogenous TG2 resulted in a significant exacerbation of caspase 3 activity and PARP cleavage in MEF cells subjected to apoptotic stimuli. Interestingly, the same cells showed the accumulation of LC3 II isoform following autophagy induction. These findings strongly suggest that TG2 transamidating activity plays a protective role in the response of MEF cells to death stimuli, because the expression of the wild-type TG2, but not its transamidation inactive C277S mutant, resulted in a suppression of caspase 3 as well as PARP cleavage upon apoptosis induction. Additionally, the same mutant was unable to catalyze the final steps in autophagosome formation during autophagy. Our findings clearly indicate that the TG2 transamidating activity is the primary biochemical function involved in the physiological regulation of both apoptosis and autophagy. These data also indicate that TG2 is a key regulator of cross-talk between autophagy and apoptosis.
Assuntos
Apoptose , Autofagia , Transglutaminases/genética , Transglutaminases/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Caspase 3/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mutantes/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , TransfecçãoRESUMO
cGAS/STING axis is the major executor of cytosolic dsDNA sensing that leads to the production of type I interferon (IFNI) not only upon bacterial infection, but also in cancer cells, upon DNA damage. In fact, DNA damage caused by ionizing radiations and/or topoisomerase inhibitors leads to a release of free DNA into the cytosol, which activates the cGAS/STING pathway and the induction of IFNI expression. Doxorubicin-induced apoptotic cancer cells release damage-associated molecular patterns (DAMPs), including IFNI, which are able to stimulate the immune system. Our results indicate that Transglutaminase type 2 (TG2) is directly involved in the formation of a covalent cross-linked IRF3 (Interferon regulatory factor 3) dimers, thereby limiting the production of IFNI. Indeed, we demonstrated that upon doxorubicin treatment TG2 translocates into the nucleus of apoptotic melanoma cells interacting with IRF3 dimers. Interestingly, we show that both the knockdown of the enzyme as well as the inhibition of its transamidating activity lead to a decrease in the dimerization of IRF3 correlated with an increase in the IFNI mRNA levels. Taken together, these data demonstrate that TG2 negatively regulates the IRF3 pathway in human melanoma cells suggesting a so far unknown TG2-dependent mechanism by which cancer cells reduce the IFNI production after DNA damage to limit the immune system response.
RESUMO
Skeletal muscle is a very dynamic and plastic tissue, being essential for posture, locomotion and respiratory movement. Muscle atrophy or genetic muscle disorders, such as muscular dystrophies, are characterized by myofiber degeneration and replacement with fibrotic tissue. Recent studies suggest that changes in muscle metabolism such as mitochondrial dysfunction and dysregulation of intracellular Ca2+ homeostasis are implicated in many adverse conditions affecting skeletal muscle. Accumulating evidence also suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders. Among the different known proteins regulating ER structure and function, we focused on RTN-1C, a member of the reticulon proteins family localized on the ER membrane. We previously demonstrated that RTN-1C expression modulates cytosolic calcium concentration and ER stress pathway. Moreover, we recently reported a role for the reticulon protein in autophagy regulation. In this study, we found that muscle differentiation process positively correlates with RTN-1C expression and UPR pathway up-regulation during myogenesis. To better characterize the role of the reticulon protein alongside myogenic and muscle regenerative processes, we performed in vivo experiments using either a model of muscle injury or a photogenic model for Duchenne muscular dystrophy. The obtained results revealed RTN-1C up-regulation in mice undergoing active regeneration and localization in the injured myofibers. The presented results strongly suggested that RTN-1C, as a protein involved in key aspects of muscle metabolism, may represent a new target to promote muscle regeneration and repair upon injury.
RESUMO
The ongoing pandemic of coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), needs better treatment options both at antiviral and anti-inflammatory levels. It has been demonstrated that the aminothiol cysteamine, an already human applied drug, and its disulfide product of oxidation, cystamine, have anti-infective properties targeting viruses, bacteria, and parasites. To determine whether these compounds exert antiviral effects against SARS-CoV-2, we used different in vitro viral infected cell-based assays. Moreover, since cysteamine has also immune-modulatory activity, we investigated its ability to modulate SARS-CoV-2-specific immune response in vitro in blood samples from COVID-19 patients. We found that cysteamine and cystamine decreased SARS-CoV-2-induced cytopathic effects (CPE) in Vero E6 cells. Interestingly, the antiviral action was independent of the treatment time respect to SARS-CoV-2 infection. Moreover, cysteamine and cystamine significantly decreased viral production in Vero E6 and Calu-3 cells. Finally, cysteamine and cystamine have an anti-inflammatory effect, as they significantly decrease the SARS-CoV-2 specific IFN-γ production in vitro in blood samples from COVID-19 patients. Overall, our findings suggest that cysteamine and cystamine exert direct antiviral actions against SARS-CoV-2 and have in vitro immunomodulatory effects, thus providing a rational to test these compounds as a novel therapy for COVID-19.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Cisteamina/farmacologia , Reposicionamento de Medicamentos/métodos , Agentes de Imunomodulação/farmacologia , SARS-CoV-2/efeitos dos fármacos , Idoso , Animais , COVID-19/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Cistamina/farmacologia , Eliminadores de Cistina/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
TG2 is a multifunctional enzyme involved in several cellular processes and has emerging as a potential regulator of gene expression. In this regard, we have recently shown that TG2 is able to activate HSF1, the master transcriptional regulator of the stress-responsive genes; however, its effect on the overall gene expression remains unclear. To address this point, we analyzed, by RNA-seq, the effect of TG2 on the overall transcriptome as well as we characterized the TG2 interactome in the nucleus. The data obtained from these omics approaches reveal that TG2 markedly influences the overall cellular transcriptome profile and specifically the Wnt and HSF1 pathways. In particular, its ablation leads to a drastic downregulation of many key members of these pathways. Interestingly, we found that key components of the Wnt/ß-catenin pathway are also downregulated in cells lacking HSF1, thus confirming that TG2 regulates the HSF1 and this axis controls the Wnt signaling. Mechanistic studies revealed that TG2 can regulate the Wnt pathway by physically interacts with ß-catenin and its nuclear interactome includes several proteins known to be involved in the regulation of the Wnt signaling. In order to verify whether this effect is playing a role in vivo, we ablated TG2 in Danio rerio. Our data show that the zebrafish lacking TG2 cannot complete the development and their death is associated with an evident downregulation of the Wnt pathway and a defective heat-shock response. Our findings show for the first time that TG2 is essential for the correct embryonal development of lower vertebrates, and its action is mediated by the Wnt/HSF1 axis.
Assuntos
Fibroblastos/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Transglutaminases/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Transcrição Gênica , Transcriptoma , Transglutaminases/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Under physiological conditions, a finely tuned system of cellular adaptation allows the intestinal mucosa to maintain the gut barrier function while avoiding excessive immune responses to non-self-antigens from dietary origin or from commensal microbes. This homeostatic function is compromised in cystic fibrosis (CF) due to loss-of-function mutations in the CF transmembrane conductance regulator (CFTR). Recently, we reported that mice bearing defective CFTR are abnormally susceptible to a celiac disease-like enteropathy, in thus far that oral challenge with the gluten derivative gliadin elicits an inflammatory response. However, the mechanisms through which CFTR malfunction drives such an exaggerated response to dietary protein remains elusive. Here we demonstrate that the proteostasis regulator/transglutaminase 2 (TGM2) inhibitor cysteamine restores reduced Beclin 1 (BECN1) protein levels in mice bearing cysteamine-rescuable F508del-CFTR mutant, either in homozygosis or in compound heterozygosis with a null allele, but not in knock-out CFTR mice. When cysteamine restored BECN1 expression, autophagy was increased and gliadin-induced inflammation was reduced. The beneficial effects of cysteamine on F508del-CFTR mice were lost when these mice were backcrossed into a Becn1 haploinsufficient/autophagy-deficient background. Conversely, the transfection-enforced expression of BECN1 in human intestinal epithelial Caco-2 cells mitigated the pro-inflammatory cellular stress response elicited by the gliadin-derived P31-43 peptide. In conclusion, our data provide the proof-of-concept that autophagy stimulation may mitigate the intestinal malfunction of CF patients.