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Appelqvist H.,Linkoping University | Johansson A.-C.,Linkoping University | Linderoth E.,Linkoping University | Linderoth E.,University College London | And 5 more authors.
Annals of Clinical and Laboratory Science | Year: 2012

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin Dmediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid. © 2012 by the Association of Clinical Scientists, Inc.

Li T.,Indiana University | Brustovetsky T.,Indiana University | Antonsson B.,Geneva Research Center | Brustovetsky N.,Indiana University
Biochimica et Biophysica Acta - Bioenergetics | Year: 2010

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAXoligo) and by a combination of monomeric BAX (BAXmono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, tcBID). We also examined whether the effects induced by BAXoligo or by BAXmono activated with tcBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that tcBID plus BAXmono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAXoligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAXoligo could be at least partially suppressed by mitochondrial depolarization. The effects of tcBID plus BAXmono were insensitive to depolarization. BAXoligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K+ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAXoligo, but failed to influence the effects produced by tcBID plus BAXmono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAXoligo and by BAX activated with tcBID. © 2009 Elsevier B.V. All rights reserved.

Brustovetsky T.,Indiana University | Li T.,Indiana University | Yang Y.,Indiana University | Zhang J.-T.,Indiana University | And 2 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2010

BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/ oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state. © 2010 Elsevier B.V.

Nellen A.,RWTH Aachen | Heinrichs D.,RWTH Aachen | Berres M.-L.,RWTH Aachen | Sahin H.,RWTH Aachen | And 4 more authors.
PLoS ONE | Year: 2012

Background: The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein (44AANA47-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo. Methodology: Liver injury was induced in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride (CCl4) in an acute and a chronic liver injury model. Simultaneously, mice received either 44AANA47-CCL5 or vehicle. Liver cell necrosis and fibrosis was analyzed by histology, and measurement of serum transaminases and hydroxyproline. Intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes. Principal Findings: 44AANA47-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cell necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline content) compared to vehicle treated mice. Ameliorated fibrosis by 44AANA47-CCL5 was associated with a decreased expression of fibrosis related genes, decreased α-smoth muscle antigen (αSMA) and a reduction of infiltrating immune cells. In the acute model, 44AANA47-CCL5 treated mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL-1 and CCL3 compared to vehicle treated mice. In vitro, conditioned medium of T-cell enriched splenocytes of 44AANA47-CCL5 treated mice inhibited the chemotaxis and proliferation of HSC. Conclusions: The results provide evidence that inhibition of oligomerization and glycosaminoglycan binding of the chemokine CCL5 is a new therapeutic strategy for the treatment of acute and chronic liver injuries and represents an alternative to chemokine receptor antagonism. © 2012 Nellen et al.

Grouls C.,RWTH Aachen | Hatting M.,RWTH Aachen | Rix A.,RWTH Aachen | Pochon S.,Geneva Research Center | And 6 more authors.
Radiology | Year: 2013

Purpose: To investigate the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted ultrasonographic (US) microbubbles for the assessment of liver dysplasia in transgenic mice. Materials and Methods: Animal experiments were approved by the governmental review committee. Nuclear factor-kB essential modulator knock-out mice with liver dysplasia and wild-type mice underwent liver imaging by using a clinical US system. Two types of contrast agents were investigated: nontargeted, commercially available, second-generation microbubbles (SonoVue) and clinically translatable PEGylated VEGFR2-targeted microbubbles (BR55). Microbubble kinetics was investigated over the course of 4 minutes. Targeted contrast material-enhanced US signal was quantified 5 minutes after injection. Competitive in vivo binding experiments with BR55 were performed in knock-out mice. Immunohistochemical and hematoxylin-eosin staining of liver sections was performed to validate the in vivo US results. Groups were compared by using the Mann-Whitney test. Results: Peak enhancement after injection of SonoVue and BR55 did not differ in healthy and dysplastic livers (SonoVue, P = .46; BR55, P = .43). Accordingly, immunohistochemical findings revealed comparable vessel densities in both groups. The specificity of BR55 to VEGFR2 was proved by in vivo competition (P = .0262). While the SonoVue signal decreased similarly in healthy and dysplastic livers during the 4 minutes, there was an accumulation of BR55 in dysplastic livers compared with healthy ones. Furthermore, targeted contrast-enhanced US signal indicated a significantly higher site-specific binding of BR55 in dysplastic than healthy livers (P = .005). Quantitative immunohistologic findings confirmed significantly higher VEGFR2 levels in dysplastic livers (P = .02). Conclusion: BR55 enables the distinction of early stages of liver dysplasia from normal liver. © RSNA, 2013.

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