Transglutaminase Type 2-MITF axis regulates phenotype switching in skin cutaneous melanoma.

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.

The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis.

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.

Type 2 transglutaminase in the nucleus: the new epigenetic face of a cytoplasmic enzyme.

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.

Cysteamine/Cystamine Exert Anti-Mycobacterium abscessus Activity Alone or in Combination with Amikacin.

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.

Transglutaminase type 2-dependent crosslinking of IRF3 in dying melanoma cells.

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.

Overexpression of α-synuclein inhibits mitochondrial Ca2+ trafficking between the endoplasmic reticulum and mitochondria through MAMs by altering the GRP75-IP3R interaction.

Mitochondria-associated ER membranes (MAMs) are formed by close and specific components in the contact sites between the endoplasmic reticulum (ER) and mitochondria, which participate in several cell functions, including lipid metabolism, autophagy, and Ca2+ signaling. Particularly, the presence of α-synuclein (α-syn) in MAMs was previously demonstrated, indicating a physical interaction among some proteins in this region and a potential involvement in cell dysfunctions. MAMs alterations are associated with neurodegenerative diseases such as Parkinson's disease (PD) and contribute to the pathogenesis features. Here, we investigated the effects of α-syn on MAMs and Ca2+ transfer from the ER to mitochondria in WT- and A30P α-syn-overexpressing SH-SY5Y or HEK293 cells. We observed that α-syn potentiates the mitochondrial membrane potential (Δψm ) loss induced by rotenone, increases mitophagy and mitochondrial Ca2+ overload. Additionally, in α-syn-overexpressing cells, we found a reduction in ER-mitochondria contact sites through the impairment of the GRP75-IP3R interaction, however, with no alteration in VDAC1-GRP75 interaction. Consequently, after Ca2+ release from the ER, α-syn-overexpressing cells demonstrated a reduction in Ca2+ buffering by mitochondria, suggesting a deregulation in MAM activity. Taken together, our data highlight the importance of the α-syn/MAMs/Ca2+ axis that potentially affects cell functions in PD.

The Multifaceted Role of HSF1 in Pathophysiology: Focus on Its Interplay with TG2.

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.

Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

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.

Transglutaminase Type 2 regulates the Wnt/ß-catenin pathway in vertebrates.

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.

Cysteamine with In Vitro Antiviral Activity and Immunomodulatory Effects Has the Potential to Be a Repurposing Drug Candidate for COVID-19 Therapy.

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.

Reticulon Homology Domain-Containing Proteins and ER-Phagy.

The endoplasmic reticulum (ER) is a dynamic membrane system comprising different and interconnected subdomains. The ER structure changes in response to different stress conditions through the activation of a selective autophagic pathway called ER-phagy. This represents a quality control mechanism for ER turnover and component recycling. Several ER-resident proteins have been indicated as receptors for ER-phagy; among these, there are proteins characterized by the presence of a reticulon homology domain (RHD). RHD-containing proteins promote ER fragmentation by a mechanism that involves LC3 binding and lysosome delivery. Moreover, the presence of a correct RHD structure is closely related to their capability to regulate ER shape and morphology by curvature induction and membrane remodeling. Deregulation of the ER-selective autophagic pathway due to defects in proteins with RHD has been implicated in several human diseases, infectious and neurodegenerative diseases in particular, as well as in cancer development. While the molecular mechanisms and the physiological role of ER-phagy are not yet fully understood, it is quite clear that this process is involved in different cellular signaling pathways and has an impact in several human pathologies.

Modulation of autophagy by RTN-1C: role in autophagosome biogenesis.

The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and to determine the cell's fate under stress conditions. Among the known proteins that regulate ER structure and function there is Reticulon-1C (RTN-1C), a member of the reticulon family localized primarily on the ER membrane. We previously demonstrated that RTN-1C expression affects ER function and stress condition. ER is an essential site for the regulation of apoptotic pathways and it has also been recently recognized as an important component of autophagic signaling. Based on these evidences, we have investigated the impact of RTN-1C modulation on autophagy induction. Interestingly we found that reticulon overexpression is able to activate autophagic machinery and its silencing results in a significative inhibition of both basal and induced autophagic response. Using different experimental approaches we demonstrated that RTN-1C colocalizes with ATG16L and LC3II on the autophagosomes. Considering the key role of reticulon proteins in the control of ER membrane shaping and homeostasis, our data suggest the participation of RTN-1C in the autophagic vesicle biogenesis at the level of the ER compartment. Our data indicate a new mechanism by which this structural ER protein modulates cellular stress, that is at the basis of different autophagy-related pathologies.

Genistein antagonizes gliadin-induced CFTR malfunction in models of celiac disease.

In celiac disease (CD), an intolerance to dietary gluten/gliadin, antigenic gliadin peptides trigger an HLA-DQ2/DQ8-restricted adaptive Th1 immune response. Epithelial stress, induced by other non-antigenic gliadin peptides, is required for gliadin to become fully immunogenic. We found that cystic-fibrosis-transmembrane-conductance-regulator (CFTR) acts as membrane receptor for gliadin-derived peptide P31-43, as it binds to CFTR and impairs its channel function. P31-43-induced CFTR malfunction generates epithelial stress and intestinal inflammation. Maintaining CFTR in an active open conformation by the CFTR potentiators VX-770 (Ivacaftor) or Vrx-532, prevents P31-43 binding to CFTR and controls gliadin-induced manifestations. Here, we evaluated the possibility that the over-the-counter nutraceutical genistein, known to potentiate CFTR function, would allow to control gliadin-induced alterations. We demonstrated that pre-treatment with genistein prevented P31-43-induced CFTR malfunction and an epithelial stress response in Caco-2 cells. These effects were abrogated when the CFTR gene was knocked out by CRISP/Cas9 technology, indicating that genistein protects intestinal epithelial cells by potentiating CFTR function. Notably, genistein protected gliadin-sensitive mice from intestinal CFTR malfunction and gliadin-induced inflammation as it prevented gliadin-induced IFN-γ production by celiac peripheral-blood-mononuclear-cells (PBMC) cultured ex-vivo in the presence of P31-43-challenged Caco-2 cells. Our results indicate that natural compounds capable to increase CFTR channel gating might be used for the treatment of CD.

Transglutaminase type 2 in the regulation of proteostasis.

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.

Transglutaminase Type 2 Regulates ER-Mitochondria Contact Sites by Interacting with GRP75.

Transglutaminase type 2 (TG2) is a multifunctional enzyme that plays a key role in mitochondria homeostasis under stressful cellular conditions. TG2 interactome analysis reveals an enzyme interaction with GRP75 (glucose-regulated protein 75). GRP75 localizes in mitochondria-associated membranes (MAMs) and acts as a bridging molecule between the two organelles by assembling the IP3R-GRP75-VDAC complex, which is involved in the transport of Ca2+ from the endoplasmic reticulum (ER) to mitochondria. We demonstrate that the TG2 and GRP75 interaction occurs in MAMs. The absence of the TG2-GRP75 interaction leads to an increase of the interaction between IP3R-3 and GRP75; a decrease of the number of ER-mitochondria contact sites; an impairment of the ER-mitochondrial Ca2+ flux; and an altered profile of the MAM proteome. These findings indicate TG2 is a key regulatory element of the MAMs.

TG2 regulates the heat-shock response by the post-translational modification of HSF1.

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.

Non-alcoholic fatty liver disease severity is modulated by transglutaminase type 2.

Non-alcoholic fatty liver disease (NAFLD) is one of the most important liver diseases worldwide. Currently, no effective treatment is available, and NAFLD pathogenesis is incompletely understood. Transglutaminase type 2 (TG2) is a ubiquitous enzyme whose dysregulation is implicated in the pathogenesis of various human diseases. Here we examined the impact of TG2 on NAFLD progression using the high-fat-diet-induced model in both wild-type and TG2-deficient mice. Animals were fed with a standard chow diet or a high-fat diet (42% of the energy from fat) for 16 weeks. Results demonstrated that the absence of a functional enzyme, which causes the impairment of autophagy/mitophagy, leads to worsening of disease progression. Data were confirmed by pharmacological inhibition of TG2 in WT animals. In addition, the analysis of human liver samples from NAFLD patients validated the enzyme's involvement in the liver fat disease pathogenesis. Our findings strongly suggest that TG2 activation may offer protection in the context of NAFLD, thus representing a novel therapeutic target for tackling the NAFLD progression.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation.

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Transglutaminase type 2-dependent selective recruitment of proteins into exosomes under stressful cellular conditions.

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.