Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes:a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Tissue transglutaminase and the stress response

The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions. Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines. These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions. © 2007 Springer-Verlag.

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts:the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

Nonlinear dynamics of cilia and flagella

Cilia and flagella are hairlike extensions of eukaryotic cells which generate oscillatory beat patterns that can propel micro-organisms and create fluid flows near cellular surfaces. The evolutionary highly conserved core of cilia and flagella consists of a cylindrical arrangement of nine microtubule doublets, called the axoneme. The axoneme is an actively bending structure whose motility results from the action of dynein motor proteins cross-linking microtubule doublets and generating stresses that induce bending deformations. The periodic beat patterns are the result of a mechanical feedback that leads to self-organized bending waves along the axoneme. Using a theoretical framework to describe planar beating motion, we derive a nonlinear wave equation that describes the fundamental Fourier mode of the axonemal beat. We study the role of nonlinearities and investigate how the amplitude of oscillations increases in the vicinity of an oscillatory instability. We furthermore present numerical solutions of the nonlinear wave equation for different boundary conditions. We find that the nonlinear waves are well approximated by the linearly unstable modes for amplitudes of beat patterns similar to those observed experimentally.

Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.

The role of tissue transglutaminase in 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells

Tissue transglutaminase (TG2) can induce post-translational modification of proteins, resulting in protein cross-linking or incorporation of polyamines into substrates, and can also function as a signal transducing G protein. The role of TG2 in the formation of insoluble cross-links has led to its implication in some neurodegenerative conditions. Exposure of pre-differentiated SH-SY5Y cells to the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) resulted in significant dose-dependent reductions in TG2 protein levels, measured by probing Western blots with a TG2-specific antibody. Transglutaminase (TG) transamidating activity, on the other hand, monitored by incorporation of a polyamine pseudo-substrate into cellular proteins, was increased. Inhibitors of TG (putrescine) and TG2 (R283) exacerbated MPP+ toxicity, suggesting that activation of TG2 may promote a survival response in this toxicity paradigm.

Transglutaminase treatment of wool fabrics leads to resistance to detergent damage

Biological detergents are now routinely used in domestic laundry because the enzymes they contain provide the added benefit of low temperature washes with improved cleaning performance. One of the key enzymes found in these detergents are proteases, which if exposed to natural protein fibres such as wool or silk can cause irreversible damage, leading to loss of fabric strength, shape and poor colour fastness. Transglutaminases (TGases) are protein cross-linking enzymes capable of adding tensile strength to wool proteins, and as a consequence are capable of remediating the damage caused by previous chemical treatments, and more importantly, by proteases. In this paper we treated dyed wool fabric with TGase and then washed the fabric with biological and non-biological detergents to investigate whether TGases would protect wool garments from damage by the undue use of biological detergents in domestic laundry. We demonstrate using different cycles of detergent washes containing biological and non-biological detergents and different TGase treatments, that wool fabric treated previously with TGase release less dye into the washing liquor and in addition maintain fabric strength at levels greater than the washed controls. As a consequence, wool garments previously treated with TGase are likely to have increased resistance to domestic washing and thus provide increased longevity. © 2005 Elsevier B.V. All rights reserved.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Enzymatic stabilization of gelatin-based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

Inhibition of transglutaminase activity reduces extracellular matrix accumulation induced by high glucose levels in proximal tubular epithelial cells

Diabetic nephropathy affects 30-40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product epsilon(gamma-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mm glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM epsilon(gamma-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECM-associated epsilon(gamma-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor beta1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor beta1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated.

Characterization of tissue transglutaminase 2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in a number of cell functions. Up-regulation of TG2 is thought to be involved in monocyte to macrophage differentiation and defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be fully elucidated. Cell surface-associated TG2 is now recognized as being important in regulating cell adhesion and migration, via its association with cell surface receptors such as syndecan-4, ß1 and ß3 integrin, but its extracellular role in the clearance of apoptotic cells is still not fully explored. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function within the framework of apoptotic cell clearance. Both THP-1 cell-derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors (both cell permeable and impermeable, irreversible and active site directed) resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. Macrophage cell surface TG2 and, in particular, its cell surface crosslinking activity was found to be crucial in dictating apoptotic cell clearance. Our further studies demonstrate syndecan-4 association with TG2 and imply possible cooperation of these proteins in apoptotic cell clearance. Knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance which seems to involve protein cross linking and interaction with other cell surface receptors.

Towards development of a dermal rudiment for enhanced wound healing response

Enhancement of collagen's physical characteristics has been traditionally approached using various physico-chemical methods frequently compromising cell viability. Microbial transglutaminase (mTGase), a transamidating enzyme obtained from Streptomyces mobaraensis, was used in the cross-linking of collagen-based scaffolds. The introduction of these covalent bonds has previously indicated increased proteolytic and mechanical stability and the promotion of cell colonisation. The hypothesis behind this research is that an enzymatically stabilised collagen scaffold will provide a dermal precursor with enhanced wound healing properties. Freeze-dried scaffolds, with and without the loading of a site-directed mammalian transglutaminase inhibitor to modulate matrix deposition, were applied to full thickness wounds surgically performed on rats’ dorsum and explanted at three different time points (3, 7 and 21 days). Wound healing parameters such as wound closure, epithelialisation, angiogenesis, inflammatory and fibroblastic cellular infiltration and scarring were analysed and quantified using stereological methods. The introduction of this enzymatic cross-linking agent stimulated neovascularisation and epithelialisation resisting wound contraction. Hence, these characteristics make this scaffold a potential candidate to be considered as a dermal precursor.

Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy?

Background. Diabetic nephropathy is the leading cause of end-stage kidney failure worldwide. It is characterized by excessive extracellular matrix accumulation. Transforming growth factor beta 1 (TGF-ß1) is a fibrogenic cytokine playing a major role in the healing process and scarring by regulating extracellular matrix turnover, cell proliferation and epithelial mesanchymal transdifferentiation. Newly synthesized TGF-ß is released as a latent, biologically inactive complex. The cross-linking of the large latent TGF-ß to the extracellular matrix by transglutaminase 2 (TG2) is one of the key mechanisms of recruitment and activation of this cytokine. TG2 is an enzyme catalyzing an acyl transfer reaction leading to the formation of a stable e(?-glutamyl)-lysine cross-link between peptides.Methods. To investigate if changes in TG activity can modulate TGF-ß1 activation, we used the mink lung cell bioassay to assess TGF-ß activity in the streptozotocin model of diabetic nephropathy treated with TG inhibitor NTU281 and in TG2 overexpressing opossum kidney (OK) proximal tubular epithelial cells.Results. Application of the site-directed TG inhibitor NTU281 caused a 25% reduction in kidney levels of active TGF-ß1. Specific upregulation of TG2 in OK proximal tubular epithelial cells increased latent TGF-ß recruitment and activation by 20.7% and 19.7%, respectively, in co-cultures with latent TGF-ß binding protein producing fibroblasts.Conclusions. Regulation of TG2 directly influences the level of active TGF-ß1, and thus, TG inhibition may exert a renoprotective effect by targeting not only a direct extracellular matrix deposition but also TGF-ß1 activation and recruitment.