Highlights d TG2 interacts with GRP75 in mitochondria-associated membranes (MAMs) d TG2 influences the number of ER-mitochondria contact sites d TG2 regulates the interaction between IP3R3 and GRP75 d TG2 controls ER-mitochondrial Ca 2+ flux and protein expression in MAMs

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.

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/β-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 β-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.

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-β 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 IFNβ 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-β production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

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.