Random mutagenesis and disulfide bond formation improved thermostability in microbial transglutaminase.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T50 of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (kcat/Km) at 37.0 °C was increased from 3.3 × 102 M-1 s-1 for the wild type to 5.9 × 102 M-1 s-1. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency. KEY POINTS: • Improved heat resistance is essential to broaden the application of MTG. • The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability. • X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.

Effect of Ferulic Acid Loaded in Nanoparticle on Tissue Transglutaminase Expression Levels in Human Glioblastoma Cell Line.

Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.

Transglutaminase 2-mediated histone monoaminylation and its role in cancer.

Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.

Identifying polyamine related biomarkers in diagnosis and treatment of ulcerative colitis by integrating bulk and single-cell sequencing data.

Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon, and its pathogenesis remains unclear. Polyamine metabolic enzymes play a crucial role in UC. In this study, we aimed to identify pivotal polyamine-related genes (PRGs) and explore the underlying mechanism between PRGs and the disease status and therapeutic response of UC. We analyzed mRNA-sequencing data and clinical information of UC patients from the GEO database and identified NNMT, PTGS2, TRIM22, TGM2, and PPARG as key PRGs associated with active UC using differential expression analysis and weighted gene co-expression network analysis (WCGNA). Receiver operator characteristic curve (ROC) analysis confirmed the accuracy of these key genes in UC and colitis-associated colon cancer (CAC) diagnosis, and we validated their relationship with therapeutic response in external verification sets. Additionally, single-cell analysis revealed that the key PRGs were specific to certain immune cell types, emphasizing the vital role of intestinal tissue stem cells in active UC. The results were validated in vitro and in vivo experiments, including the colitis mice model and CAC mice model. In conclusion, these key PRGs effectively predict the progression of UC patients and could serve as new pharmacological biomarkers for the therapeutic response of UC.

Transglutaminase 2 Regulates HSF1 Gene Expression in the Acute Phase of Fish Optic Nerve Regeneration.

Fish retinal ganglion cells (RGCs) can regenerate after optic nerve lesions (ONLs). We previously reported that heat shock factor 1 (HSF1) and Yamanaka factors increased in the zebrafish retina 0.5-24 h after ONLs, and they led to cell survival and the transformation of neuro-stem cells. We also showed that retinoic acid (RA) signaling and transglutaminase 2 (TG2) were activated in the fish retina, performing neurite outgrowth 5-30 days after ONLs. In this study, we found that RA signaling and TG2 increased within 0.5 h in the zebrafish retina after ONLs. We examined their interaction with the TG2-specific morpholino and inhibitor due to the significantly close initiation time of TG2 and HSF1. The inhibition of TG2 led to the complete suppression of HSF1 expression. Furthermore, the results of a ChIP assay with an anti-TG2 antibody evidenced significant anti-TG2 immunoprecipitation of HSF1 genome DNA after ONLs. The inhibition of TG2 also suppressed Yamanaka factors' gene expression. This rapid increase in TG2 expression occurred 30 min after the ONLs, and RA signaling occurred 15 min before this change. The present study demonstrates that TG2 regulates Yamanaka factors via HSF1 signals in the acute phase of fish optic nerve regeneration.

Screening of novel disease genes of sepsis-induced myocardial Disfunction by RNA sequencing and bioinformatics analysis.

BACKGROUND: There is still a lack of effective treatment for sepsis-induced myocardial dysfunction (SIMD), while the pathogenesis of SIMD still remains largely unexplained. METHODS: RNA sequencing results (GSE267388 and GSE79962) were used for cross-species integrative analysis. Bioinformatic analyses were used to delve into function, tissue- and cell- specificity, and interactions of genes. External datasets and qRT-PCR experiments were used for validation. L1000 FWD was used to predict targeted drugs, and 3D structure files were used for molecular docking. RESULTS: Based on bioinformatic analyses, ten differentially expressed genes were selected as genes of interest, seven of which were verified to be significantly differential expression. Bucladesine was considered as a potential targeted drug for SIMD, which banded to seven target proteins primarily by forming hydrogen bonds. CONCLUSION: It was considered that Cebpd, Timp1, Pnp, Osmr, Tgm2, Cp, and Asb2 were novel disease genes, while bucladesine was a potential therapeutic drug, of SIMD.

Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death.

Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Transglutaminase 2 knockout mice are protected from bleomycin-induced lung fibrosis with preserved lung function and reduced metabolic derangements.

Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.

TG2 participates in pulmonary vascular remodelling by regulating the senescence of pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is a severe cardiovascular disease characterised by pulmonary vascular remodelling. The pivotal role of cellular senescence in vascular remodelling has been acknowledged. Transglutaminase type 2 (TG2), a calcium-dependent enzyme, is intricately linked to both cellular senescence and PH. However, the precise mechanisms underlying the involvement of TG2 in PH remain unclear. In this study, we explored the expression of TG2 and the cellular senescence marker p16INK4a in the pulmonary vasculature of mice with PH induced by hypoxia combined with SU5416. Our findings revealed upregulation of both TG2 and p16INK4a expression in the pulmonary vasculature of PH mice. Additionally, a notable increase in TG2 expression was observed in senescent pulmonary artery smooth muscle cells (PASMC). To delve deeper, we employed proteomic sequencing to reveal seven genes associated with cellular senescence, with a subsequent focus on MAPK14. Our investigation revealed that TG2 regulates senescence in PASMC by modulating the phosphorylation levels of MAPK14. Additionally, in the context of hypoxia combined with SU5416, our observations revealed a noteworthy reduction in both pulmonary vascular remodelling and senescent manifestations in smooth muscle-specific TG2 knockout mice compared with their wild-type counterparts. In summary, our findings indicate that TG2 deficiency lowers the senescence levels of PASMC by inhibiting the activity of MAPK14. This inhibition of senescence in the pulmonary vasculature of PH mice helps to decelerate the progression of pulmonary vascular remodelling and consequently hinders the onset and development of PH.

Integrated strategies for efficient production of Streptomyces mobaraensis transglutaminase in Komagataella phaffii.

Transglutaminase (TGase) from Streptomyces mobaraensis commonly used to improve protein-based foods due to its unique enzymatic reactions, which imply considerable attention in its production. Recently, TGase exhibit broad market potential in non-food industries. However, achieving efficient synthesis of TGase remains a significant challenge. Herein, we achieved a substantial amount of a fully functional and kinetically stable TGase produced by Komagataella phaffii (Pichia pastoris) using multiple strategies including Geneticin (G418) screening, combinatorial mutations, promoter optimization, and co-expression. The active TGase expression reached a maximum of 10.1 U mL-1 in shake flask upon 96 h of induction, which was 3.8-fold of the wild type. Also, the engineered strain exhibited a 6.4-fold increase in half-life and a 2-fold increase in specific activity, reaching 172.67 min at 60 °C (t1/2(60 °C)) and 65.3 U mg-1, respectively. Moreover, the high-cell density cultivation in 5-L fermenter was also applied to test the productivity at large scale. Following optimization at a fermenter, the secretory yield of TGase reached 47.96 U mL-1 in the culture supernatant. Given the complexity inherent in protein expression and secretion, our research is of great significance and offers a comprehensive guide for improving the production of a wide range of heterologous proteins.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

Sphingolipid-Enriched Porcine Placental Extract Promotes the Expression of Structural Genes and Desquamation Enzyme Genes in Cultured Human Keratinocytes.

Porcine placental extract (PPE) is commonly used in various health foods and cosmetics. PPE use in cosmetics predominantly consist of the water-soluble fraction derived from the entire placenta. In this report, we examined the effect of the hydrophobic constituents of the PPE, specifically the sphingolipid-enriched fraction designated as the sphingolipid-enriched porcine placental extract (SLPPE), on the expression of genes associated with skin function in cultured normal human epidermal keratinocytes. Using quantitative RT-PCR (qRT-PCR) analysis, we found that SLPPE concentrations ranging from 25 to 100 µg/mL upregulated the gene expression of key components associated with the cornified envelope structure (filaggrin (FLG), involucrin (IVL) and loricrin (LOR)), cornification enzymes (transglutaminase 1 (TGM1) and TGM5) and the desquamation enzymes (kallikrein 5 (KLK5) and KLK7). Additionally, KLK5p and FLG protein (FLGp) were detected in the culture supernatants of keratinocytes treated with SLPPE at these concentrations. These findings suggest that SLPPE is possible to promote the cornification and desquamation in epidermal keratinocytes, and it may offer potential benefits in cosmetics.

Transglutaminase-mediated stiffening of the glomerular basement membrane mitigates pressure-induced reductions in molecular sieving coefficient by reducing compression.

Proteinuria, the presence of high molecular weight proteins in the urine, is a primary indicator of chronic kidney disease. Proteinuria results from increased molecular permeability of the glomerular filtration barrier combined with saturation or defects in tubular protein reabsorption. Any solute that passes into the glomerular filtrate traverses the glomerular endothelium, the glomerular basement membrane, and the podocyte slit diaphragm. Damage to any layer of the filter has reciprocal effects on other layers to increase glomerular permeability. The GBM is thought to act as a compressible ultrafilter that has increased molecular selectivity with increased pressure due to compression that reduced the porosity of the GBM with increased pressure. In multiple forms of chronic kidney disease, crosslinking enzymes are upregulated and may act to increase GBM stiffness. Here we show that enzymatically crosslinking porcine GBM with transglutaminase increases the stiffness of the GBM and mitigates pressure-dependent reductions in molecular sieving coefficient. This was modeled mathematically using a modified membrane transport model accounting for GBM compression. Changes in the mechanical properties of the GBM may contribute to proteinuria through pressure-dependent effects on GBM porosity.

The Evolution of Transglutaminases Underlies the Origin and Loss of Cornified Skin Appendages in Vertebrates.

Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes.

Deletion of Tgm2 suppresses BMP-mediated hepatocyte-to-cholangiocyte metaplasia in ductular reaction.

Transglutaminase 2 (Tgm2) plays an essential role in hepatic repair following prolonged toxic injury. During cholestatic liver injury, the intrahepatic cholangiocytes undergo dynamic tissue expansion and remodelling, referred to as ductular reaction (DR), which is crucial for liver regeneration. However, the molecular mechanisms governing the dynamics of active cells in DR are still largely unclear. Here, we generated Tgm2-knockout mice (Tgm2-/-) and Tgm2-CreERT2-Rosa26-mTmG flox/flox (Tgm2CreERT2-R26T/Gf/f) mice and performed a three-dimensional (3D) collagen gel culture of mouse hepatocytes to demonstrate how Tgm2 signalling is involved in DR to remodel intrahepatic cholangiocytes. Our results showed that the deletion of Tgm2 adversely affected the functionality and maturity of the proliferative cholangiocytes in DR, thus leading to more severe cholestasis during DDC-induced liver injury. Additionally, Tgm2 hepatocytes played a crucial role in the regulation of DR through metaplasia. We unveiled that Tgm2 regulated H3K4me3Q5ser via serotonin to promote BMP signalling activation to participate in DR. Besides, we revealed that the activation or inhibition of BMP signalling could promote or suppress the development and maturation of cholangiocytes in DDC-induced DR. Furthermore, our 3D collagen gel culture assay indicated that Tgm2 was vital for the development of cholangiocytes in vitro. Our results uncovered a considerable role of BMP signalling in controlling metaplasia of Tgm2 hepatocytes in DR and revealed the phenotypic plasticity of mature hepatocytes.

Superior COL7A1 and TGM1 gene expression in difficult-to-transfect skin cell mediated by highly branched poly(ß-amino esters) through stepwise fractionation.

Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with Mw ranging from 7.6 to 131.8 kg/mol and D < 2.0 from the one crude HPAE23.7k, and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE20.6k, exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases.

Cross-Sectional Study on Autosomal Recessive Congenital Ichthyoses: Association of Genotype with Disease Severity, Phenotypic, and Ultrastructural Features in 74 Italian Patients.

BACKGROUND: Autosomal recessive congenital ichthyoses (ARCIs) are a clinically heterogeneous group of keratinization disorders characterized by generalized skin scaling due to mutations in at least 12 genes. The aim of our study was to assess disease severity, phenotypic, and ultrastructural features and to evaluate their association with genetic findings in ARCI patients. METHODS: Clinical signs and symptoms, and disease severity were scored in a single-center series of patients with a genetic diagnosis of ARCI. Skin ultrastructural findings were reviewed. RESULTS: Seventy-four consecutive patients (mean age 11.0 years, range 0.1-48.8) affected with lamellar ichthyosis (50/74, 67.5%), congenital ichthyosiform erythroderma (18/74, 24.3%), harlequin ichthyosis (two/74, 2.7%), and other minor ARCI subtypes (four/74, 5.4%) were enrolled. Mutated genes were as follows: TGM1 in 18/74 (24.3%) patients, ALOX12B in 18/74 (24.3%), CYP4F22 in 12/74 (16.2%), ABCA12 in nine/74 (12.2%), ALOXE3 in seven/74 (9.5%), NIPAL4 in seven/74 (9.5%), and CERS3, PNPLA1, and SDR9C7 in 1 patient each (1.4%). Twenty-five previously undescribed mutations in the different ARCI causative genes, as well as two microduplications in TGM1, and two microdeletions in CYP4F22 and NIPAL4 were identified. The mean ichthyosis severity score in TGM1- and ABCA12-mutated patients was significantly higher than in all other mutated genes, while the lowest score was observed in CYP4F22-mutated patients. Alopecia, ectropion, and eclabium were significantly associated with TGM1 and ABCA12 mutations, and large, thick, and brownish scales with TGM1 mutations. Among specific phenotypic features, psoriasis-like lesions as well as a trunk reticulate scale pattern and striated keratoderma were present in NIPAL4-mutated patients. Ultrastructural data available for 56 patients showed a 100% specificity of cholesterol clefts for TGM1-mutated cases and revealed abnormal lamellar bodies in SDR9C7 and CERS3 patients. CONCLUSION: Our study expands the phenotypic and genetic characterization of ARCI by the description of statistically significant associations between disease severity, specific clinical signs, and different mutated genes. Finally, we highlighted the presence of psoriasis-like lesions in NIPAL4-ARCI patients as a novel phenotypic feature with diagnostic and possible therapeutic implications.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Transglutaminase 3 regulates cutaneous squamous carcinoma differentiation and inhibits progression via PI3K-AKT signaling pathway-mediated Keratin 14 degradation.

Cutaneous squamous carcinoma is the second most common epithelial malignancy, associated with significant morbidity, mortality, and economic burden. However, the mechanisms underlying cSCC remain poorly understood. In this study, we identified TGM3 as a novel cSCC tumor suppressor that acts via the PI3K-AKT axis. RT-qPCR, IHC and western blotting were employed to assess TGM3 levels. TGM3-overexpression/knockdown cSCC cell lines were utilized to detect TGM3's impact on epithelial differentiation as well as tumor cell proliferation, migration, and invasion in vitro. Additionally, subcutaneous xenograft tumor models were employed to examine the effect of TGM3 knockdown on tumor growth in vivo. Finally, molecular and biochemical approaches were employed to gain insight into the tumor-suppressing mechanisms of TGM3. TGM3 expression was increased in well-differentiated cSCC tumors, whereas it was decreased in poor-differentiated cSCC tumors. Loss of TGM3 is associated with poor differentiation and a high recurrence rate in patients with cSCC. TGM3 exhibited tumor-suppressing activity by regulating cell proliferation, migration, and invasion both in vitro and in vivo. As a novel cSCC tumor differentiation marker, TGM3 expression was positively correlated with cell differentiation. In addition, our results demonstrated an interaction between TGM3 and KRT14 that aids in the degradation of KRT14. TGM3 deficiency disrupts keratinocytes differentiation, and ultimately leads to tumorigenesis. Furthermore, RNA-sequence analysis revealed that loss of TGM3 enhanced EMT via the PI3K-AKT signaling pathway. Deguelin, a PI3K-AKT inhibitor, blocked cSCC tumor growth induced by TGM3 knockdown in vivo. Taken together, TGM3 inhibits cSCC tumor growth via PI3K-AKT signaling, which could also serve as a tumor differentiation marker and a potential therapeutic target for cSCC. Proposed model depicted the mechanism by which TGM3 suppress cSCC development. TGM3 reduces the phosphorylation level of AKT and degrades KRT14. In the epithelial cell layer, TGM3 exhibits a characteristic pattern of increasing expression from bottom to top, while KRT14 and pAKT are the opposite. Loss of TGM3 leads to reduced degradation of KRT14 and activation of pAKT, disrupting keratinocyte differentiation, and eventually resulting in the occurrence of low-differentiated cSCC.