Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of themitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization.Peer reviewe

Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA

Purpose: To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation. Methods: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. Results: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. Conclusions: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX

Cardiac fibrosis can be attenuated by blocking the activity of transglutaminase 2 using a selective small-molecule inhibitor

Cardiac fibrosis is implicit in all forms of heart disease but there are no effective treatments. In this report, we investigate the role of the multi-functional enzyme Transglutaminase 2 (TG2) in cardiac fibrosis and assess its potential as a therapeutic target. Here we describe the use a highly selective TG2 small-molecule inhibitor to test the efficacy of TG2 inhibition as an anti-fibrotic therapy for heart failure employing two different in vivo models of cardiac fibrosis: Progressively induced interstitial cardiac fibrosis by pressure overload using angiotensin II infusion: Acutely induced focal cardiac fibrosis through myocardial infarction by ligation of the left anterior descending coronary artery (AMI model). In the AMI model, in vivo MRI showed that the TG2 inhibitor 1–155 significantly reduced infarct size by over 50% and reduced post-infarct remodelling at 20 days post insult. In both models, Sirius red staining for collagen deposition and levels of the TG2-mediated protein crosslink ε(γ-glutamyl)lysine were significantly reduced. No cardiac rupture or obvious signs of toxicity were observed. To provide a molecular mechanism for TG2 involvement in cardiac fibrosis, we show that both TGFβ1-induced transition of cardiofibroblasts into myofibroblast-like cells and TGFβ1- induced EndMT, together with matrix deposition, can be attenuated by the TG2 selective inhibitor 1–155, suggesting a new role for TG2 in regulating TGFβ1 signalling in addition to its role in latent TGFβ1 activation. In conclusion, TG2 has a role in cardiac fibrosis through activation of myofibroblasts and matrix deposition. TG2 inhibition using a selective small-molecule inhibitor can attenuate cardiac fibrosis

Transglutaminase 2 in human diseases

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Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature

Transglutaminaasi 2 proteiinin toiminnan vaikutukset endoteelisolujen biologiaan

Transglutaminaasi 2 proteiinin toiminnan vaikutukset endoteelisolujen biologiaan Ohutsuolen limakalvon nukkalisäkkeet ovat keskeisessä merkityksessä ravinteiden imeyttämisessä. Nukkalisäkkeiden rakenteen ylläpitämiseen liittyy keskeisesti niiden keskellä oleva verisuonitus. Keliakia on ravinnon gluteenin laukaisema sairaus, jolle on tyypillistä ohutsuolen limakalvovaurio, ja tämän yhteydessä nähtävä verisuonituksen poikkeava rakenne. Lisäksi keliakiapotilaille kehittyy kehon omaa proteiinin, transglutaminaasi 2:ta (TG2) tunnistavia vasta-aineita. TG2-proteiinilla on useita toimintoja ja se osallistuu muun muassa verisuonten muodostumiseen. Aiemmat tutkimukset viittaavat siihen, että keliakiapotilaiden TG2:ta tunnistavat vasta-aineet estävät verisuonten muodostumista. Tutkimuksen tarkoituksena oli selvittää TG2n roolia verisuonten muodostuksessa sekä mekanismia, jolla keliakiapotilaiden vasta-aineet estävät verisuoniston kehittymistä. Tutkimuksessa selvisi, että verisuonen seinämän endoteelisolujen ulkopuolinen TG2 osallistuu solujen kiinnittymistä alustaansa, kun solun sisäinen TG2 puolestaan vaikuttaa solusykliin ja apoptoosiin. Tulosten mukaan keliaakikon vasta-aineet häiritsevät solun ulkopuolisen TG2n toimintaa, ja täten vaikuttaa endoteelisolujen kiinnittymiseeen alustaansa, solujen polarisoitumiseen ja liikkumiseen. Keliakiapotilaiden vasta-aineet myös lisäsivät thioredoksiini -nimisen proteiinin erittymistä verisuonen seinämän soluista. Väitöskirjan tulokset viittaavat siihen, että thioredoksiini-proteiinin eritys luultavasti vaikuttaa TG2n kolmiulotteiseen rakenteeseen siten, että keliakiapotilaiden vasta-aineet sitoutuvat siihen paremmin. Kaikkineen väitöskirjan tulokset siis selittävät osaltaan millä mekanismilla keliaakikon vasta-aineet estävät verisuonten muodostumista ja mistä keliaakikon ohutsuolen limakalvon verisuonituksen epänormaalius voisi johtua.In the small intestine villi offers an extensive epithelial surface area for absorptive function and their integrity relies on an appropriate vascular network. In celiac disease (CD), an autoimmune disorder triggered by ingested dietary gluten in susceptible individuals with HLA-DQ2 or DQ8 genotype, the overall mucosal vascular architecture is completely altered. CD is predominately characterized by a strong antibody response targeted against dietary gluten-derived deamidated gliadin peptides and the self-antigen, transglutaminase 2 (TG2). TG2 is a complex multifunctional protein involved in a variety of basic biological processes, including angiogenesis, where its role is still not fully understood. Since our group has previously shown that CD patient derived autoantibodies disturb several steps of angiogenesis, the aim of this thesis project was to deepen our knowledge of TG2 in the endothelium and to clarify the molecular mechanism behind the anti-angiogenic effects exerted by CD antibodies. This work shed new light on the role of TG2 in the endothelium where the extracellular pool of TG2 is involved in regulating cell-matrix adhesion while the cytoplasmic TG2 is important for cell cycle progression and survival. Furthermore, it was shown that CD IgA antibodies disturb the extracellular function of TG2, thus altering endothelial cell-extracellular matrix (ECM) interactions and thereby affecting endothelial cell adhesion, polarization and motility. Intriguingly, CD IgA promoted the secretion of the redox sensor protein TRX, which probably is needed to keep TG2 in a conformation suitable for the constitutive antibody binding and activation. Thus, this study set up a possible molecular mechanism by which CD antibodies exert their anti-angiogenic functions, which in turn might help to explain the altered small-bowel mucosal microvasculature observed in untreated CD patients

L'immagine dell'universita' fornita dai quotidiani

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Does the 'European grouping of territorial co-operation' promote multi-level governance within the european union?

Through the analysis of Regulation 1082/2006 (also known as 'The European Grouping of Territorial Co-operation'), which enables regional and local authorities from different European Union (EU) countries to set up co-operation groupings as legal entitie