Manuela D'Eletto

Transglutaminase 2 is involved in autophagosome maturation

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. “Tissue” transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

The adenine nucleotide translocator 1 acts as a type 2 transglutaminase substrate: implications for mitochondrial-dependent apoptosis.

In this study we provide in vitro and in vivo evidence showing that the protein disulphide isomerase (PDI) activity of type 2 transglutaminase (TG2) regulates the correct assembly and function of the mitochondrial ADP/ATP transporter adenine nucleotide translocator 1 (ANT1). We demonstrate, by means of biochemical and morphological analyses, that ANT1 and TG2 physically interact in the mitochondria. Under physiological conditions, TG2s PDI activity regulates the ADP/ATP transporter function by controlling the oligomerization of ANT1. In fact, mitochondria isolated from hearts of TG2−/− mice exhibit increased polymerization of ANT1, paralleled by an enhanced ADP/ATP carrier activity, as compared to mitochondria belonging to TG2+/+ mice. Interestingly, upon cell-death induction, ANT1 becomes a substrate for TG2s cross-linking activity and the lack of TG2 results in a reduction of apoptosis as well as in a marked sensitivity to the ADP/ATP exchange inhibition by atractyloside. These findings suggest a complex TG2-dependent regulation of the ADP/ATP transporter and reveal new important avenues for its potential applications in the treatment of some mitochondrial-dependent diseases, including cardiovascular and neurodegenerative diseases.

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called ‘aggresomes’, where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzymes crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.

TG2 transamidating activity acts as a reostat controlling the interplay between apoptosis and autophagy

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.

Transglutaminase 2 ablation leads to mitophagy impairment associated with a metabolic shift towards aerobic glycolysis

Macroautophagy selectively degrades dysfunctional mitochondria by a process known as mitophagy. Here we demonstrate the involvement of transglutaminase 2 (TG2) in the turnover and degradation of damaged mitochondria. In TG2-ablated cells we observed the presence of a large number of fragmented mitochondria that display decreased membrane potential, downregulation of IF1 along with increased Drp1 and PINK1 levels, two key proteins regulating the mitochondrial fission. Of note, we demonstrate that in healthy mitochondria, TG2 interacts with the dynamic proteins Drp1 and Fis1; interestingly, their interaction is largely reduced upon induction of the fission process by carbonyl cyanide m-chlorophenyl hydrazine (CCCP). In keeping with these findings, mitochondria lacking TG2 are more susceptible to CCCP treatment. As a consequence of accumulation of damaged mitochondria, cells lacking TG2 increased their aerobic glycolysis and became sensitive to the glycolytic inhibitor 2-deoxy-D-glucose (2-DG). In contrast, TG2-proficient cells are more resistant to 2-DG-induced apoptosis as the caspase 3 is inactivated through the enzyme’s crosslinking activity. The data presented in this study show that TG2 plays a key role in cellular dynamics and consequently influences the energetic metabolism.

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.

The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation

Autophagy is a self-degradative physiological process by which the cell removes worn-out or damaged components. Constant at basal level it may become highly active in response to cellular stress. The type 2 transglutaminase (TG2), which accumulates under stressful cell conditions, plays an important role in the regulation of autophagy and cells lacking this enzyme display impaired autophagy/mitophagy and a consequent shift their metabolism to glycolysis. To further define the molecular partners of TG2 involved in these cellular processes, we analysed the TG2 interactome under normal and starved conditions discovering that TG2 interacts with various proteins belonging to different functional categories. Herein we show that TG2 interacts with pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis which is responsible for maintaining a glycolytic phenotype in malignant cells and displays non metabolic functions, including transcriptional co-activation and protein kinase activity. Interestingly, the ablation of PKM2 led to the decrease of intracellular TG2s transamidating activity paralleled by an increase of its tyrosine phosphorylation. Along with this, a significant decrease of ULK1 and Beclin1 was also recorded, thus suggesting a block in the upstream regulation of autophagosome formation. These data suggest that the PKM2/TG2 interplay plays an important role in the regulation of autophagy in particular under cellular stressful conditions such as those displayed by cancer cells.

Transglutaminase type 2: A multifunctional protein chaperone?

Macroautophagy selectively degrades dysfunctional mitochondria by a process known as mitophagy. The purpose of the study published in Cell Death and Differentiation was to investigate the involvement of transglutaminase 2 (TG2) in the turnover and degradation of damaged mitochondria and its effects on cell metabolism.

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.