Lysine Isopeptide Research Articles

Association between high mobility group box‑1 protein expression and cell death in acute pancreatitis.

The present study used caerulein stimulation of AR42J rat pancreatic cells as an in vitro acute pancreatitis (AP) model to investigate proteins differentially expressed in apoptosis and necrosis. AR42J cells were stimulated with 10-8mol/l caerulein and incubated for 24 h. Apoptosis and necrosis were detected using flow cytometry. The sorted Annexin V-positive cells (apoptotic) and the Annexin V/propidium iodide double-positive cells (necrotic) were analysed using proteomics. Results showed that numerous proteins were differentially expressed in these 2 groups. Functional enrichment analysis was performed on the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery. High mobility group box-1 protein (HMGB1) was specifically expressed in the necrosis group. Models of varying degrees of AP were established using caerulein at concentrations of 10-9, 10-8, 10-7, 10-6 and 10-5 mol/l. The percentage of apoptotic and necrotic cells in each group was determined using flow cytometry. Protein expression levels of HMGB1 were detected by western blot analysis. The present study showed that as the concentration of caerulein increased, the percentage of necrotic cells and the protein expression levels of HMGB1 increased. HMGB1 is involved in many biological processes, including the chromosomal protein glycyl lysine isopeptide cross-link. HMGB1 may be involved in the early stage of pancreatitis, potentially by inducing the development of cell death by necrosis. These results provide an experimental basis for clinical intervention in AP.

Identification of brain substrates of transglutaminase by functional proteomics supports its role in neurodegenerative diseases

Transglutaminases are calcium-dependent enzymes that catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific glutamine and lysine residues. Some transglutaminase isoforms are present in the brain and are thought to participate in the protein aggregation characteristic of neurological diseases such as Huntington, Alzheimer's and Parkinson's disease. We have developed a functional proteomics strategy in which biotinylated amine-donor and amine-acceptor probes were used to identify the transglutaminase substrates present in brain. Bioinformatics analyses revealed that most of the 166 brain substrates identified interacted with huntingtin, the amyloid precursor protein or α-synuclein and that neurological disease was the most significant canonical pathway associated with the substrates. The physiological relevance of the substrates identified by mass spectrometry was confirmed by the fact that three of them (actin, β-tubulin and a neurofilament subunit) were polymerized in neuronal cells when cytosolic calcium concentration was raised. We also showed by in-situ immunolabeling that some of the substrates were part of the protein aggregates found in neurological diseases. These results strongly support the idea that the crosslinking activity of brain transglutaminase participates in the formation of the protein aggregates found in diseases of the central nervous system.

Cross-linking with microbial transglutaminase: Isopeptide bonds and polymer size as drivers for acid casein gel stiffness

Acid casein, cross-linked with microbial transglutaminase for different times, was mixed with uncross-linked reference to obtain samples with identical numbers of cross-links (expressed as N-ε-(γ-glutamyl)-lysine isopeptide content, IC). Mixtures from longer incubated casein solutions, where cross-links were also formed within and between already present polymers, resulted in a lower polymerisation degree because a higher proportion of the reference was necessary to achieve the target IC. For similar gel stiffness at the same isopeptide content, a particular size of the polymers was crucial; high dimer content resulted in lower gel stiffness at a given IC. Gels with high stiffness formed at high amounts of larger polymers. Stiffness at 30 °C was lower at identical IC when casein solutions cross-linked for more than 5 h were used for blending; this was not observed for gelation at 20 or 40 °C. Large deformation properties of the casein gels were clearly affected by cross-linking.

Expressions of isopeptide bonds and corneodesmosin in middle ear cholesteatoma.

Isopeptide bonds form cross-links between constituent proteins in the horny layer of the epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes, which bind corneocytes together. Both play important roles in maintaining epidermal barrier functions. In the present study, we investigated the expressions of isopeptide bonds, CDSN, and related enzymes in middle ear cholesteatoma in comparison with the skin. Prospective case series of patients with middle ear cholesteatoma. Tertiary medical institute. Cholesteatoma and normal postauricular skin were collected from patients with acquired middle ear cholesteatoma during tympanomastoidectomy. Expression of e-(g-glutamyl)lysine isopeptide bonds was examined by immunohistochemistry; Expressions of transglutaminase (TGase)1, TGase2, TGase3, and TGase5 by immunohistochemistry and quantitative RT-PCR (qRT-PCR); expression of CDSN by immunohistochemistry, qRT-PCR, and Western blot; and expressions of tissue kallikrein-related peptidase (KLK)5, KLK7, KLK14, and serine peptidase inhibitor Kazal type 5 (SPINK5) by qRT-PCR. TGase2 was higher (P=0.0046) and TGase5 was lower (P=0.0008) in cholesteatoma than in the postauricular skin. Immunoreactivity for isopeptide bonds was localized in the granular and horny layers, and was not different between the two tissues. Immunoreactivity for CDSN was localized in the granular layer, and was lower in cholesteatoma than in the skin (P=0.0090). Western blot and qRT-PCR confirmed that the expression of CDSN was lower in cholesteatoma than in the skin. Expressions of KLK5, KLK7, KLK14, or SPINK5 were not different between the two tissues. These results indicate that the production of CDSN is likely to be suppressed in cholesteatoma, which would account, at least in part, for the mechanical fragility and increased permeability of the cholesteatoma epithelium.

Hydrogelation of the Short Self-Assembling Peptide I3QGK Regulated by Transglutaminase and Use for Rapid Hemostasis.

The self-assembly of short peptides is a promising route to the creation of smart biomaterials. To combine peptide self-assembly with enzymatic catalysis, we design an amphiphilic short peptide I3QGK that can self-assemble into long nanoribbons in aqueous solution. Upon addition of transglutaminase (TGase), the peptide solution undergoes a distinct sol-gel transition to form a rigid hydrogel, which shows strong shear-thinning and immediate recovery properties. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) measurements indicate the occurrence of considerable nanofibers in addition to the original nanoribbons. Liquid chromatography and mass spectrometry analyses reveal the enzymatic formation of peptide dimers from monomers through intermolecular ε-(γ-glutamyl)lysine isopeptide bonding. The dimers rapidly self-assemble into flexible and entangled nanofibers, and the coexistence of the original nanoribbons and the newly created nanofibers is responsible for hydrogelation. Factor XIII in blood is converted by thrombin to an active TGase (Factor XIIIa) during bleeding, so the peptide solution shows a more rapid and effective hemostasis via a combination of gelling blood and promoting platelet adhesion, relative to other hemostasis methods or materials. These features of I3QGK, together with its low cytotoxicity against normal mammalian cells and noninduction of nonspecific immunogenic responses, endow it with great potential for future clinical hemostasis applications.

Synthesis of a Bis-thio-acetone (BTA) Analogue of the Lysine Isopeptide Bond and its Application to Investigate the Effects of Ubiquitination and SUMOylation on α-Synuclein Aggregation and Toxicity.

The reversible modification of protein by the small protein ubiquitin and other ubiquitin-like modifiers plays important roles in virtually every key biological process in eukaryotic cells. The establishment of a range of chemical methods for the preparation of ubiquitinated proteins has enabled the site-specific interrogation of the consequences of these modifications. However, many of these techniques require significant levels of synthetic expertise, somewhat limiting their widespread application by the biological community. To overcome this issue, the creation of structural analogues of the ubiquitin-protein linkage that can be readily prepared with commercially available reagents and buffers is an important goal. Here we present the development of conditions for the facile synthesis of bis-thio-acetone (BTA) linkages of ubiquitinated proteins in high yields. Additionally, we apply this technique to the preparation of the aggregation prone protein α-synuclein bearing either ubiquitin or the small ubiquitin-like modifier (SUMO). With these proteins, we demonstrate that the BTA linkage recapitulates the previously published effects of either of these proteins on α-synuclein, suggesting that it is a good structural mimic. Notably, the BTA linkage is chemically and enzymatically stable, enabling us to study the consequences of site-specific ubiquitination and SUMOylation on the toxicity of α-synuclein in cell culture, which revealed modification and site-specific differences.

Hyaluronan-based multifunctional hydrogels formed via enzyme-mediated crosslinking

Event Abstract Back to Event Hyaluronan-based multifunctional hydrogels formed via enzyme-mediated crosslinking Daniela S. Ferreira1, 2, Dominic W. Collis1, 2 and Helena S. Azevedo1, 2 1 Queen Mary, University of London, School of Engineering and Materials Science, United Kingdom 2 Queen Mary University of London, Institute of Bioengineering, United Kingdom Introduction: Hydrogels have generated great interest to develop 3D artificial matrices that recapitulate features of the natural extracellular matrix (ECM) for applications in tissue regeneration or cell expansion[1],[2]. Covalent crosslinking is a common method to obtain stable hydrogels with precise control over crosslinking density. However, many of the crosslinking reagents used are toxic, precluding their use for cell encapsulation applications. Enzyme-mediated crosslinking reactions are finding increasing applications for developing hydrogels due to their mild conditions and enzyme specificity[3]. FXIIIa is a transglutaminase that catalyses the reaction between an ε-amine of lysine (K) onto a γ-carboxamide residue of glutamine (Q), resulting in the covalent bond formation of an ε-(γ-glutamyl)lysine isopeptide which is highly resistant to proteolytic degradation[4]. This crosslinking mechanism naturally occurs in vivo during the processes of wound healing. Hyaluronan (HA), a linear polysaccharide present in the ECM of many tissues, has been chemically modified to develop hydrogels[5]-[7]. In this work, HA was functionalized with two different transglutaminase peptide substrates to obtain HA hydrogels by enzyme-mediated crosslinking. Materials and Methods: Peptides were synthesized manually using standard Fmoc based solid phase chemistry and purified, as described elsewhere[8]. Hyaluronan (60 kDa) was functionalized with acrylate groups using a two-step synthesis (Fig 1-A, as described elsewhere[9]. Peptides were coupled to acrylated HA via Michael-type addition by reacting 1.2-fold molar excess of peptide over acrylate groups in 0.3 M triethanolamine (pH 8.0) overnight at 37°C. Results and Discussion: Peptides were designed as factor XIIIa substrates, containing either a lysine donor peptide CH3CONH-GFKGG-ERCG-CONH2 (TG-K, Mw = 950.08 g/mol) or a glutamine acceptor peptide - CH3CONH-GNQEQVSPL-ERCG-CONH2 (TG-Q, Mw = 1457.58 g/mol). In addition, a glutamine acceptor containing a cell adhesion ligand RGDS, which is recognized by several integrin receptors, was also synthesized, CH3CONH--GNQEQVSPLRGDSPG-CONH2 (TG-Q-RGDSPG, Mw = 1581.67 g/mol). To render hydrogels susceptible to degradation by proteolytic enzymes, like natural fibrin, a lysine donor peptide was synthesized bearing a substrate for matrix metalloproteases (MMPs) Ac-GFKGGGPQGIWGQ-ERCG-CONH2 (TG-MMP-K, Mw = 1581.67 g/mol). Acrylated HA was obtained by a two-step reaction and the degree of acrylation was found to be 16%, as determined by NMR (Fig. 1-B). Transglutaminase peptide substrates were grafted onto acrylate groups of HA backbone via Michael-type addition as confirmed by NMR analysis (Fig. 2). Conclusion: HA macromers functionalized with transglutaminase substrate peptides were successfully obtained. These HA-peptide macromers will be used as precursors to develop hydrogels that can be formed in situ (e.g. wound site), where FXIIIa is produced. The incorporation of integrin-specific adhesive ligands and cell-sensitive proteolytic domains in these hydrogels will aid in guiding and enhancing local tissue regeneration. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007‐2013) under REA grant agreement n° 608765.

Effect of high intensity ultrasound on transglutaminase-catalyzed soy protein isolate cold set gel

The effects of high intensity ultrasound (HIU, 105–110W/cm2 for 5 or 40min) pre-treatment of soy protein isolate (SPI) on the physicochemical properties of ensuing transglutaminase-catalyzed soy protein isolate cold set gel (TSCG) were investigated in this study. The gel strength of TSCG increased remarkably from 34.5 to 207.1g for TSCG produced from SPI with 40min HIU pre-treatment. Moreover, gel yield and water holding capacity also increased after HIU pre-treatments. Scanning electron microscopy showed that HIU of SPI resulted in a more uniform and denser microstructure of TSCG. The content of free sulfhydryl (SH) groups was higher in HIU TSCG than non-HIU TSG, even though greater decrease of the SH groups present in HIU treated SPI was observed when the TSCG was formed, suggesting the involvement of disulfide bonds in gel formation. Protein solubility of TSCG in both denaturing and non-denaturing solvents was higher after HIU pretreatment, and changes in hydrophobic amino acid residues as well as in polypeptide backbone conformation and secondary structure of TSCG were demonstrated by Raman spectroscopy. These results suggest that increased inter-molecular ε-(γ-glutamyl) lysine isopeptide bonds, disulfide bonds and hydrophobic interactions might have contributed to the HIU TSCG gel network. In conclusion, HIU changed physicochemical and structural properties of SPI, producing better substrates for TGase. The resulting TSCG network structure was formed with greater involvement of covalent and non-covalent interactions between SPI molecules and aggregates than in the TSCG from non-HIU SPI.

Monitoring of transglutaminase crosslinking reaction by 1H NMR spectroscopy on model substrates

Abstract A new method based on 1H NMR spectroscopy was developed for monitoring transglutaminase crosslinking reaction with model molecules (CBZ-Gln-Gly and N-α-acetyl-lysine). The transglutaminase reaction led to the appearance of new resonances on NMR spectrum as well as significant decrease in others. The new observed resonances, originated from newly formed ɛ-(γ-glutamyl)lysine isopeptide bonds, evidence the enzymatic reaction and allow to quantify the ɛ-(γ-glutamyl)lysine fragment. Moreover, the decrease in resonance intensity, originated from lysine, permit to determine the crosslinking degree. These results obtained by 1H NMR spectroscopy can be used as an alternative method to LC–MS, reducing drastically the analysis time from 7 days to 2 h.

Dark-induced senescence of barley leaves involves activation of plastid transglutaminases.

Transglutaminases (E.C. 2.3.2.13) catalyze the post-translational modification of proteins by establishing ε-(γ-glutamyl) lysine isopeptide bonds and by the covalent conjugation of polyamines to endo-glutamyl residues of proteins. In light of the confirmed role of transglutaminases in animal cell apoptosis and only limited information on the role of these enzymes in plant senescence, we decided to investigate the activity of chloroplast transglutaminases (ChlTGases) and the fate of chloroplast-associated polyamines in Hordeum vulgare L. ‘Nagrad’ leaves, where the senescence process was induced by darkness (day 0) and continued until chloroplast degradation (day 12). Using an anti-TGase antibody, we detected on a subcellular level, the ChlTGases that were associated with destacked/degraded thylakoid membranes, and beginning on day 5, were also found in the stroma. Colorimetric and radiometric assays revealed during senescence an increase in ChlTGases enzymatic activity. The MS/MS identification of plastid proteins conjugated with exogenous polyamines had shown that the ChlTGases are engaged in the post-translational modification of proteins involved in photosystem organization, stress response, and oxidation processes. We also computationally identified the cDNA of Hv-Png1-like, a barley homologue of the Arabidopsis AtPng1 gene. Its mRNA level was raised from days 3 to 10, indicating that transcriptional regulation controls the activity of barley ChlTGases. Together, the presented results deepen our knowledge of the mechanisms of the events happened in dark-induced senescence of barley leaves that might be activation of plastid transglutaminases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-014-1912-y) contains supplementary material, which is available to authorized users.

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Objectives Preeclampsia (PE) is characterized by the presence of autoantibodies that activate the major angiotensin receptor, AT1R (AT1-AA). Little is known about the molecular mechanisms underlying the origination of these pathogenic autoantibodies. We have recently shown that tissue transglutaminase (TG2), modifies AT1R by the introduction of an e -( γ -glutamyl)-lysine isopeptide bond. This finding immediately raises an intriguing hypothesis that the posttranslational modification of AT1Rs by TG2 may generate neoantigen and subsequently trigger AT1-AA production. Methods Pregnant C57Bl/6J mice (Harlan) were anesthetized, and recombinant mouse LIGHT (2 ng) or the same volume of saline was introduced into pregnant mice by retro-orbital sinus injection on embryonic day E13.5 and E14.5. Cystamine dihydrochloride was put in drinking water with the concentration 0.9 g/L. Human NT and PE samples were collected from Memorial Hermann Hospital. Results Here we report that injection of LIGHT, a new TNF- α superfamily member, not only induced hallmark feature of PE including hypertension and proteinuria, but also circulating AT1-AA in the pregnant mice. This finding led us to further discover that circulating and placental TG2 activities were significantly induced in the LIGHT-infused pregnant mice compared to the saline-injected controls. Intriguingly, co-injection of TG2 inhibitor, cystamine, significantly attenuated LIGHT-induced PE features and AT1-AA production in pregnant mice. Mechanistically, we demonstrated that elevated HIF-1 α is a key factor underlying increased TG2 gene expression in the mouse placentas of LIGHT-injected pregnant mice. Finally, we validated our mouse finding in human showing that plasma TG2 activity was positively correlated with blood pressure, urinary protein and circulating AT1-AA. Conclusions Overall, our findings demonstrated for the first time that HIF-1 α -dependent elevated TG2 activity triggers AT1-AA generation in PE likely by posttranslational modification of AT1R. These findings add new chapter for molecular basis underlying production of AT1-AA in PE and highlight the therapeutic possibility for the disease. Disclosures R. Luo: None. C. Liu: None. S. Elliott: None. W. Wang: None. P. Daugherty: None. R. Kellems: None. Y. Xia: None.

Coagulation Factor XIIIa Substrates in Human Plasma: IDENTIFICATION AND INCORPORATION INTO THE CLOT

Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization.

Small Molecule Inhibitors Target the Tissue Transglutaminase and Fibronectin Interaction

Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε−(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination.

Tissue Transglutaminase Contributes to the Pathogenesis of Preeclampsia and Stabilizes Placental Angiotensin Receptor Type 1 by Ubiquitination-Preventing Isopeptide Modification

Preeclampsia is a life-threatening pregnancy disorder that is widely thought to be triggered by impaired placental development. However, the placenta-related pathogenic factors are not fully identified, and their underlying mechanisms in disease development remain unclear. Here, we report that the protein level and enzyme activity of tissue transglutaminase (TG2 or tTG), the most ubiquitous member of a family of enzymes that conducts post-translational modification of proteins by forming ε-(γ-glutamyl)-lysine isopeptide bonds, are significantly elevated in placentas of preeclamptic women. TG2 is localized in the placental syncytiotrophoblasts of patients with preeclampsia where it catalyzes the isopeptide modification of the angiotensin receptor type 1 (AT1). To determine the role of elevated TG2 in preeclampsia, we used a mouse model of preeclampsia based on injection of AT1-agonistic autoantibody. A pathogenic role for TG2 in preeclampsia is suggested by in vivo experiments in which cystamine, a potent transglutaminase inhibitor, or small interfering RNA-mediated TG2 knockdown significantly attenuated autoantibody-induced hypertension and proteinuria in pregnant mice. Cystamine treatment also prevented isopeptide modification of placental AT1 receptors in preeclamptic mice. Mechanistically, we revealed that AT1-agonistic autoantibody stimulation enhances the interaction between AT1 receptor and TG2 and results in increased AT1 receptor stabilization via transglutaminase-mediated isopeptide modification in trophoblasts. Mutagenesis studies further demonstrated that TG2-mediated isopeptide modification of AT1 receptors prevents ubiquitination-dependent receptor degradation. Taken together, our studies not only identify a novel pathogenic involvement of TG2 in preeclampsia but also suggest a previously unrecognized role of TG2 in the regulation of G protein-coupled receptor stabilization by inhibiting ubiquitination-dependent degradation.

Abstract 275: Tissue Transglutaminase Stabilizes Placental AT1 Receptor and Contributes to Pathophysiology in Preeclampsia

Preeclampsia (PE) is a serious hypertensive disease of pregnancy associated with the presence of the major angiotensin receptor AT1-agnostic autoantibodies (AT1-AAs) that cause the defining PE features when introduced into pregnant mice. To understand AT1-AA-mediated receptor activation in PE, we explored the role of tissue transglutaminases (TG2), the most ubiquitous member of a group of enzymes modifying proteins by catalyzing the formation of ε-(γ-glutamyl)-lysine isopeptide bonds. TG2, a versatile protein that can also function as a G protein, is enriched in the syncytiotrophoblasts of placenta, the key organ in PE pathogenesis. Western blot analysis and IHC-IF double staining initially revealed that TG2 and transglutaminase (TG)-mediated isopeptide modification levels are increased approximately 2X in PE vs normotensive (NT) placental tissue (n=4 or 5, p<0.05). Next, we identified a similar increase in PE plasma TG activity (NT=3.1±0.5, mild PE=5.4±0.6, severe PE=9.1±0.7milliunit/ml, n=26-29, p<0.01), and the increased plasma TG activity is positively correlated with the key disease features including blood pressure and proteinuria (r 2 >0.5, p<0.01). We also found a higher level of AT1 receptor with isopeptide modification in PE placentas by immunoprecipitation and IF colocalization. A pathogenic role for TG2 in PE is suggested by in vivo experiments in which TG inhibitor cystamine, or siRNA-mediated TG2 knockdown, significantly attenuated AT1-AA-induced PE features in pregnant mice including hypertension (PE=160±6 vs PE+cystamine=133±3mmHg, p<0.01, n=6; PE+control siRNA=161±3 vs PE+ TG2 siRNA=137±7mmHg, p<0.05, n=5-6), proteinuria (PE=133±21 vs PE+cystamine=58±12ng albumin/mg creatine, p<0.01, n=6; PE+control siRNA=102±51 vs PE+ TG2 siRNA=26±6ng albumin/mg creatine, p<0.05, n=4), and placental isopeptide modification. In trophoblasts, AT1-AA stimulation enhanced the interaction between AT1 receptor and TG2, and resulted in increased AT1 receptor stabilization via isopeptide modification, which is further confirmed by mutagenesis studies. Taken together, our results not only identify a pathogenic role of TG2 in PE, but also shed light on a previously unrecognized mechanism of TG2-mediated GPCR stabilization.

Interaction of the Profilaggrin N-Terminal Domain with Loricrin in Human Cultured Keratinocytes and Epidermis

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

Nɛ–homocysteinyl–lysine Isopeptide is Associated with Progression of Peripheral Artery Disease in Patients Treated with Folic Acid

ObjectiveFolic acid (FA) administration can reduce plasma total homocysteine (tHcy); however, it fails to decrease cardiovascular events and progression of peripheral artery disease (PAD). Nɛ–homocysteinyl–lysine isopeptide (Nɛ–Hcy–Lys) is formed during catabolism of homocysteinylated proteins. We sought to investigate factors that determine the presence of Nɛ–Hcy–Lys in PAD patients with hyperhomocysteinemia receiving FA. Patients and methodsWe studied 131 consecutive PAD patients with tHcy > 15 μmol l−1 taking FA 0.4 mg d−1 for 12 months. Serum Nɛ–Hcy–Lys was determined by high-performance liquid chromatography (HPLC). We also measured interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), asymmetric dimethylarginine (ADMA) and 8-iso-prostaglandin F2α (8-iso-PGF2α). ResultsFA administration resulted in a 70.5% decrease in tHcy (p < 0.0001). However, serum Nɛ–Hcy–Lys was detectable in 28 (21.4%) patients on FA who were more frequently current smokers and survivors of ischaemic stroke (p < 0.001). They had higher tHcy by 46.0%, PAI-1 by 51.7%, 8-iso-PGF2α by 59.1% and ADMA by 26.4% (all, p < 0.0001). The presence of Nɛ–Hcy–Lys was associated with lower ankle-brachial index (ABI) values (p < 0.001) and higher prevalence of cardiovascular events (p < 0.001) following therapy. ConclusionThe presence of Nɛ–Hcy–Lys in one-fifth of hyperhomocysteinemic individuals with PAD despite FA treatment is associated with progression of PAD and with increased ADMA formation, oxidative stress and hypofibrinolysis.

δ-Aminolevulinate synthase is required for apical transcellular barrier formation in the skin of the Drosophila larva

Animals construct a layered skin to prevent dehydration and pathogen entrance. The barrier function of the skin relies on the extensive cross-linking of specialised components. In insects, for instance, epidermal cells produce an apical extracellular cuticle that consists of a network of proteins, chitin and lipids. We have identified mutations in the Drosophila gene coding for the δ-aminolevulinate synthase (Alas) that cause massive water loss. The cuticle of alas mutant larvae detaches from the epidermis and its basal region is frayed suggesting that an Alas dependent pathway is needed to organise the contact between the cuticle and the epidermis and anchor the cuticle to the apical surface of epidermal cells. Concomitantly, reduction of Alas function results in weakening of the extracellular dityrosines network in the cuticle, whereas glutamyl–lysine isopeptide bonds are not affected. The lateral septate junctions of epidermal cells that serve as a paracellular plug are intact, as well. Taken together, we hypothesise that Alas activity, which initiates heme biosynthesis in the mitochondrion, is needed for the formation of a dityrosine-based barrier that confers resistance to the internal hydrostatic pressure protecting both the cuticle from transcellular infiltration of body fluid and the animal from dehydration. We conclude that at least two modules – an apical protein–chitin lattice and the lateral septate junctions, act in parallel to ensure Drosophila skin impermeability.

A Crucial Sequence for Transglutaminase Type 2 Extracellular Trafficking in Renal Tubular Epithelial Cells Lies in Its N-terminal β-Sandwich Domain

Transglutaminase type 2 (TG2) catalyzes the formation of an ε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of β-sandwich domain prevented all TG2 export. Mutations of Asp(94) and Asp(97) within the N-terminal β-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain ((88)WTATVVDQQDCTLSLQLTT(106)). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence (88)WTATVVDQQDCTLSLQLTT(106) within the β-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin.

Targeting Ovarian Tumor Cell Adhesion Mediated by Tissue Transglutaminase

Tissue transglutaminase (TG2) is a transpeptidase involved in protein cross-linking through generation of ε-(γ-glutamyl)lysine isopeptide bonds. It also promotes cell adhesion through interaction with fibronectin and facilitates formation of fibronectin-integrin complexes. This interaction is involved in tumor cell adhesion to the matrix and in the process of tumor dissemination. Its inhibition by small molecules may therefore be useful in blocking metastasis. To that end, we screened more than 800,000 compounds following an in silico docking approach targeting two distinct cavities in the vicinity of the fibronectin-binding site on TG2. A total of 120 compounds were acquired and tested in cell culture-based assays for inhibition of ovarian tumor cell adhesion and proliferation. Seven compounds showed more than 50% inhibition of cell adhesion at a concentration of 25 μmol/L. A follow-up fluorescence polarization study revealed that one compound in particular (ITP-79) inhibited binding of a TG2 peptide to a 42-kDa fragment of fibronectin in a dose-dependent manner. This inhibition was confirmed in cancer cells by coimmunoprecipitation. A competition assay with surface plasmon resonance showed that ITP-79 modulated binding of TG2 to fibronectin. Direct binding of compounds that inhibited adhesion to TG2 were examined with differential scanning fluorimetry, which measures the effect of the compound on the melting temperature of the target. Two compounds, including ITP-79, reduced TG2 stabilization, mimicking the effects of GTP, a known negative allosteric regulator of TG2 enzymatic function. This suggests a potential allosteric mechanism for the compound in light of its distal target site.