Preparation of Transglutaminase-Catalyzed Rice Bran Protein Emulsion Gels as a Curcumin Vehicle.

Protein-based emulsion gels have tunable viscoelasticity that can be applied to improve the stability of bioactive ingredients. As the by-product of rice processing, rice bran protein (RBP) has high nutritional value and good digestibility, exhibiting unique value in the development of hypoallergenic formula. In this study, the effect of transglutaminase (TGase) cross-linking on the physicochemical properties of RBP emulsion gels was investigated. To improve the stability of curcumin against environmental stress, the entrapment efficiency and stability of curcumin in the emulsion gel systems were also evaluated. The results indicated that TGase increased the viscoelastic modulus of RBP emulsion gels, resulting in a solid-like structure. Moreover, the entrapment efficiency of curcumin was increased to 93.73% after adding TGase. The thermal stability and photo-stability of curcumin were enhanced to 79.54% and 85.87%, respectively, compared with the sample without TGase addition. The FTIR results showed that TGase induced the cross-linking of protein molecules and the secondary structure change in RBP. Additionally, SEM observation confirmed that the incorporation of TGase promoted the formation of a compact network structure. This study demonstrated the potential of RBP emulsion gels in protecting curcumin and might provide an alternative strategy to stabilize functional ingredients.

Effects of Laccase and Transglutaminase on the Physicochemical and Functional Properties of Hybrid Lupin and Whey Protein Powder.

Plant-based protein is considered a sustainable protein source and has increased in demand recently. However, products containing plant-based proteins require further modification to achieve the desired functionalities akin to those present in animal protein products. This study aimed to investigate the effects of enzymes as cross-linking reagents on the physicochemical and functional properties of hybrid plant- and animal-based proteins in which lupin and whey proteins were chosen as representatives, respectively. They were hybridised through enzymatic cross-linking using two laccases (laccase R, derived from Rhus vernicifera and laccase T, derived from Trametes versicolor) and transglutaminase (TG). The cross-linking experiments were conducted by mixing aqueous solutions of lupin flour and whey protein concentrate powder in a ratio of 1:1 of protein content under the conditions of pH 7, 40 °C for 20 h and in the presence of laccase T, laccase R, or TG. The cross-linked mixtures were freeze-dried, and the powders obtained were assessed for their cross-linking pattern, colour, charge distribution (ζ-potential), particle size, thermal stability, morphology, solubility, foaming and emulsifying properties, and total amino acid content. The findings showed that cross-linking with laccase R significantly improved the protein solubility, emulsion stability and foaming ability of the mixture, whereas these functionalities were lower in the TG-treated mixture due to extensive cross-linking. Furthermore, the mixture treated with laccase T turned brownish in colour and showed a decrease in total amino acid content which could be due to the enzyme's oxidative cross-linking mechanism. Also, the occurrence of cross-linking in the lupin and whey mixture was indicated by changes in other investigated parameters such as particle size, ζ-potential, etc., as compared to the control samples. The obtained results suggested that enzymatic cross-linking, depending on the type of enzyme used, could impact the physicochemical and functional properties of hybrid plant- and animal-based proteins, potentially influencing their applications in food.

Characteristics of the Mixed Yogurt Fermented from Cow-Soy Milk in the Presence of Transglutaminase.

The mixed yogurt was fermented from Cow-Soy milk and modified by transglutaminase (TG). The effects of mixed milk and TG on the quality characteristics of mixed yogurt were investigated by texture characteristics, rheology (rheometer) and structure (scanning electron microscopy). The findings revealed that the mixed yogurt with 50% cow milk exhibited lower hardness, viscosity and consistency. Furthermore, when TG was added, the yogurt showed better rheological properties, sensory score and a more stable microstructure. Compared with the samples without TG modification, the viscosity and cohesiveness of the modified samples increased by 10% and 100%, respectively. The combination of cow milk and soy milk improved the texture of yogurt, and the TG addition further improved the physicochemical properties of yogurt. This finding provided a meaningful reference for the development of mixed yogurt with a suitable taste from animal and plant milk, and laid a basis for the practical application of mixed yogurt in the dairy industry, which will meet the requirements for dairy products for consumers in future.

The Frequently Used Industrial Food Process Additive, Microbial Transglutaminase: Boon or Bane.

Microbial transglutaminase (mTG) is a frequently consumed processed food additive, and use of its cross-linked complexes is expanding rapidly. It was designated as a processing aid and was granted the generally recognized as safe (GRAS) classification decades ago, thus avoiding thorough assessment according to current criteria of toxicity and public health safety. In contrast to the manufacturer's declarations and claims, mTG and/or its transamidated complexes are proinflammatory, immunogenic, allergenic, pathogenic, and potentially toxic, hence raising concerns for public health. Being a member of the transglutaminase family and functionally imitating the tissue transglutaminase, mTG was recently identified as a potential inducer of celiac disease. Microbial transglutaminase and its docked complexes have numerous detrimental effects. Those harmful aspects are denied by the manufacturers, who claim the enzyme is deactivated when heated or by gastric acidity, and that its covalently linked isopeptide bonds are safe. The present narrative review describes the potential side effects of mTG, highlighting its thermostability and activity over a broad pH range, thus, challenging the manufacturers' and distributers' safety claims. The national food regulatory authorities and the scientific community are urged to reevaluate mTG's GRAS status, prioritizing public health protection against the possible risks associated with this enzyme and its health-damaging consequences.

Characteristics of soy films as affected by transglutaminase cross-linking and inclusions of pectin and protein enhancers.

Previously, we showed that water extract (soymilk, except pH was increased to 8 from 6.5) of whole soybean could be used directly as a raw material for producing edible soy films by deposition of the film-forming solution (soy extract with enhancers). However, the strength of such soy films needed improvement because they were weak. The purpose of this study was to investigate how transglutaminase (TG) cross-linking reactions and film enhancers, including pectin (low- and high-methoxyl pectin), whey protein isolate (WPI), and soy protein isolate (SPI), improve the physical properties of soy films. Soy films prepared with TG had tensile strength (TS) of 3.01 MPa and puncture strength (PS) of 0.78 MPa, which were higher by as much as 51% and 30% than that of soy films without TG treatment, respectively. Pectin showed significant effects on the mechanical properties of TG-added soy films in terms of TS, PS, and % elongation. On the other hand, only TS and PS were increased by the addition of WPI or SPI. Heat curing had a significant effect on soy film's physical properties. TG treatment significantly reduced film solubility when soaked in water and various levels of acid (vinegar) and base (baking soda) solutions. Under the experimental conditions of 35 unit TG and 28 min of reaction, the degrees of cross-linking were evidenced by the disappearance of individual protein subunits, except the basic subunit of glycinin, and the reduction of 21% of lysine residues of the proteins. HIGHLIGHTS: Edible soy films were made with transglutaminase and about 21% lysine cross-linked. The mechanical strength of soy films was increased by incorporating film enhancers. Transglutaminase enhanced the mechanical properties of soy films.

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.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state.

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

An i-type lysozyme from a crustacean, Pacifastacus leniusculus, functions as a clot-destabilizing enzyme.

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bond formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the ß-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate L-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilizing activity in crustacean blood.

Rapid aggregation of amyloid-like protein enhanced by mTGase to prepare functional wool fabrics for efficient and sustainable remove heavy metals from wastewater.

To counteract the increasing severity of water pollution and purify water sources, wastewater treatment materials are essential. In particular, it is necessary to improve the bonding strength between the adsorption material and the substrate in a long-term humid environment, and resist the invasion of microorganisms to prolong the service life. In this study, an amyloid-like aggregation method of lysozyme catalyzed by microbial transglutaminase (mTGase). Lysozyme self-assembles into an amyloid-like phase-transited lysozyme (PTL) in the presence of a reducing agent. Simultaneously, mTGase catalyzes acyl transfer reactions within lysozyme molecules or between lysozyme and keratin molecules, and driving PTL assembly on the wool fiber (TG-PTL@wool). This process enhances the grafting amount and fastness of PTL on the wool. Moreover, the tensile strength of wool fabric increased to 523 N. TG-PTL@wool achieves a 97.32 % removal rate of heavy metals, maintaining a removal rate of over 95 % after 5 cycles. TG-PTL@wool has excellent antibacterial property (99 %), and it remains above 90 % after 50 times of circulating washing. This study proved that mTGase can enhance the amyloid aggregation of lysozyme and enhance the bonding strength between PTL coating and substrate. Moreover, TG-PTL@wool provides a sustainable, efficient and cleaner solution for removing heavy metals from water.

Improvement of Surimi Gel from Frozen-Stored Silver Carp.

Silver Carp (SC) is an under-utilized, invasive species in North American river systems. In this study, the synergistic effects of manufactured Microfiber (MMF), Transglutaminase (TG), and chicken skin collagen (CLG)) to enhance surimi gel quality from frozen SC were studied. The gel strength, textural properties, rheological properties, water-holding capacity (WHC), water mobility, microstructure, and protein composition of the gel samples were determined to assess the impact of the additives individually and synergistically. The results suggested that TG had the most pronounced effect on the surimi gel properties by promoting protein cross-linking. Synergistic effects between TG, MMF, and CLG can bring effective gel property enhancement larger than the individual effect of each additive alone. With the established response-surface models, the combination of CLG and MMF can be optimized to produce surimi gels with less TG but comparable in properties to that of the optimal result with high TG usage. The findings of this study provided a technical foundation for making high-quality surimi gel products out of frozen-stored SC with synergistic utilization of additives, which could serve as guidelines for the industrial development of new surimi products.

3D printing of prawn mimics with faba proteins: The effects of transglutaminase and curdlan gum on texture.

With its capability for automated production of high-resolution structures, 3D printing can develop plant-based seafood mimics with comparable protein content. However, the challenge lies in solidifying 3D printed products to achieve the firmness of seafood. Targeting prawn, texturisation of its 3D printed mimic by curdlan gum was compared against incubation with a protein cross-linking enzyme, microbial transglutaminase. Faba bean protein extract (FBP) was selected for its lightest colour. To confer structural stability to the FBP-based ink without hindering extrudability, adding 1 % xanthan gum was optimal. Printed curdlan-containing mimics were steamed for 9 min, while printed transglutaminase-containing mimics were incubated at 55 °C before steaming. Either adding 0.0625 % or 0.125 % w/w curdlan or, incubating the transglutaminase-containing mimics for an hour achieved chewiness of 818.8-940.6 g, comparable to that of steamed prawn (953.13 g). Curdlan hydrogel penetrated and reinforced the FBP network as observed under confocal imaging. Whereas incubation of transglutaminase-containing mimics enhanced microstructural connectivity, attributable to transglutaminase-catalysed isopeptide cross-linkages, and the consequent increase in disulfide bonding and ß-sheet. Ultimately, transglutaminase treatment appeared more suitable than curdlan, as it yielded mimics with cutting strength comparable to steamed prawn. Both demonstrated promising potential to broaden the variety of 3D printed seafood mimics.

Lactoferrin-Chitosan Composite Hydrogels Induced by Microbial Transglutaminase: Potential Delivery Systems for Thermosensitive Bioactive Substances.

In this work, lactoferrin (LF)-chitosan (CS) composite hydrogels with good loading capacity of thermosensitive bioactive substances were successfully obtained by microbial transglutaminase (MTG)-induced cross-linking. We evaluated the rheological, textural, and microstructural characteristics of the composite hydrogels under different conditions. The results demonstrated that the concentrations of LF and CS as well as the amount of MTG could regulate the textural properties, rheological properties, and water holding capability. The results of FTIR and fluorescence spectroscopy indicated that the main interactions within the composite gel were hydrogen and isopeptide bonds. Additionally, in vitro digestion simulation results verified that riboflavin kept stable in stomach due to the protection of LF-CS composite hydrogels and was released in small intestine. These results suggested that thermosensitive bioactive substance could be encapsulated and delivered by the LF-CS composite hydrogel, which could be applied in lots of potential applications in functional food as a new material.

Potential Celiac Disease in Children: Health Status on A Long-Term Gluten-Containing Diet.

Potential celiac disease (PCD) is a clinical condition characterised by the presence of a positive CD-specific serology and a normal intestinal architecture. Asymptomatic PCD patients are generally advised to continue on a gluten-containing diet (GCD), but long-term risks of this approach have never been explored. In the present study, we aimed to investigate nutritional and autoimmune complications possibly developing overtime in a cohort of asymptomatic PCD children on a GCD. We compared children's parameters of growth, nutritional status, and autoimmunity between the time of diagnosis and on the occasion of their last medical check, after a long-term gluten-containing diet. Altogether, we collected data from 171 PCD children with a mean follow-up time of 3 years (range 0.35-15.3 years). During follow-up, although patients did not reduce their amount of daily gluten intake, their anti-tissue transglutaminase (anti-TG2) antibodies spontaneously and significantly decreased. Most parameters analysed had not changed during follow-up (height centile, ferritin, albumin, cholesterol, calcium, alkaline phosphatase, parathormone, and vitamin D) or even improved significantly (weight and BMI centile, haemoglobin, blood iron, HDL, glycaemia, and HbA1C, p < 0.05), always remaining within the limit of normality. Equally, autoantibodies for other concomitant autoimmune disorders did not increase overtime. Similar results were obtained excluding from analysis patients who had stopped producing anti-TG2 and those with a follow-up time < 3 years. Our pilot study has provided reassuring results regarding the maintenance of a gluten-containing diet in asymptomatic PCD children, even when long-term follow-up was considered.

Production of microbial transglutaminase by Streptoverticillium cinnamoneum KKP 1658.

Transglutaminase finds broad applications in the food industry, influencing texture, shelf life and overall food quality. It can be utilized to create products with enhanced sensory and technological properties and serves as a tool to reduce food waste. The aim of this study was to optimize the production of microbial transglutaminase (MTG) by the genetically unmodified strain of Streptoverticillium cinnamoneum KKP 1658. Tryptone soy broth (TSB) was chosen as the optimal inoculation medium due to its high MTG activity in the cultivation substrate. The optimal inoculum incubation time was determined as 24 hours, with a dosage of 10 %. Various nitrogen sources were investigated while maintaining a consistent nitrogen dosage (0.2 %) (including aminobak, corn steep liquor, ammonium nitrate and ammonium sulphate) to achieve the highest microbiological transglutaminase activity. The combination of aminobak with corn steep liquor and a cultivation period of 72 hours (28 °C; pH 6.0-6.5) yielded the highest MTG activity at 6.59 U/mL.

From waste to strength: Tailor-made enzyme activation design transformation of denatured soy meal into high-performance all-biomass adhesive.

Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance.

Effect of transglutaminase on gelation and functional proteins of mung bean protein isolate.

This study aimed to improve mung bean protein's gelation qualities via microbial transglutaminase (mTGase) cross-linking. The mTGase treatment significantly improved gel hardness and storage modulus (G') at higher enzyme levels (2 IU/g), peaking hardness at 3 h. The scanning electron microscopy imaging demonstrated more cross-linked structures at 2 IU/g, evolving into a dense network by 3 h. The water-holding capacity for mTGase-treated samples (2 IU/g, 3 h, 55 °C) tripled to 3.77 ± 0.06 g/g versus control (1.24 ± 0.02 g/g), alongside a 15 % decrease in zeta potential (-30.84 ± 0.901 mV versus control's -26.63 ± 0.497 mV) and an increase in emulsifying activity index to 4.519 ± 0.004 m2/g from 3.79 ± 0.01 m2/g (control). The confocal images showed a more uniform lipid droplet distribution in mTGase-treated samples, suggesting enhanced emulsifying activity. Thus, mTGase treatment significantly improved gel strength and emulsifying properties, making it ideal for plant-based seafood products.

Outcome in pediatric celiac disease is independent of the diagnostic approach in patients with high antibody levels.

OBJECTIVES: European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines enable the diagnosis of celiac disease (CD) without biopsies in patients with immunoglobulin A (IgA)-antibodies against tissue transglutaminase (TGA-IgA) ≥ 10× the upper limit of normal (ULN) and positivity of endomysial antibodies in a second blood sample. Limited data exist comparing the biopsy versus the nonbiopsy diagnostic approach regarding long-term outcomes in CD patients. Our study aimed to investigate the influence of the diagnostic approach on adherence to gluten-free diet (GFD), serological remission (defined as normalization of TGA-IgA during follow-up (FU)) and clinical remission in CD patients with TGA-IgA ≥ 10× ULN. METHODS: Retrospective multicenter study. Patients with CD and TGA-IgA ≥ 10× ULN at diagnosis were included in the study. Patients with confirmed diagnosis by biopsy were compared to patients diagnosed by nonbiopsy approach using univariate analysis, Kaplan-Meier survival curve, and logistic regression models. RESULTS: A total of 282 CD patients (192 [68.1%] in the biopsy group; 90 [31.9%] in the nonbiopsy group) were analyzed. The median time to normalization of TGA-IgA was 16.5 months [interquartile range, IQR: 13, 28] in the biopsy and 15 months [IQR: 12, 26] in the nonbiopsy group; p = 0.14). Rates of normalized TGA-IgA at first to third-year FU were comparable between both groups. Adherence to GFD did not seem to be influenced by the diagnostic approach. CONCLUSIONS: The nonbiopsy approach is not inferior to the biopsy approach in terms of adherence to GFD and serological remission in patients with CD.

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.

Analysis on promotive effect of rocking culture on keratinocyte differentiation in 3-dimensional reconstitution human epidermis.

A 3-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by downregulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study of cultured keratinocytes with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analyses. In addition, transcriptome analysis revealed the expected similar pattern between air-exposed and rocking cultures, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.

Membrane Association of the Short Transglutaminase Type 2 Splice Variant (TG2-S) Modulates Cisplatin Resistance in a Human Hepatocellular Carcinoma (HepG2) Cell Line.

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy with complex carcinogenesis. Although there has been significant progress in the treatment of HCC over the past decades, drug resistance to chemotherapy remains a major obstacle in its successful management. In this study, we were able to reduce chemoresistance in cisplatin-resistant HepG2 cells by either silencing the expression of transglutaminase type 2 (TG2) using siRNA or by the pre-treatment of cells with the TG2 enzyme inhibitor cystamine. Further analysis revealed that, whereas the full-length TG2 isoform (TG2-L) was almost completely cytoplasmic in its distribution, the majority of the short TG2 isoform (TG2-S) was membrane-associated in both parental and chemoresistant HepG2 cells. Following the induction of cisplatin toxicity in non-chemoresistant parental cells, TG2-S, together with cisplatin, quickly relocated to the cytosolic fraction. Conversely, no cytosolic relocalisation of TG2-S or nuclear accumulation cisplatin was observed, following the identical treatment of chemoresistant cells, where TG2-S remained predominantly membrane-associated. This suggests that the deficient subcellular relocalisation of TG2-S from membranous structures into the cytoplasm may limit the apoptic response to cisplatin toxicity in chemoresistant cells. Structural analysis of TG2 revealed the presence of binding motifs for interaction of TG2-S with the membrane scaffold protein LC3/LC3 homologue that could contribute to a novel mechanism of chemotherapeutic resistance in HepG2 cells.