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Casanovas G.,University of Heidelberg | Casanovas G.,Molecular Medicine Partnership Unit | Casanovas G.,European Molecular Biology Laboratory | Spasic M.V.,University of Heidelberg | And 7 more authors.
Haematologica | Year: 2013

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss. © 2013 Ferrata Storti Foundation.


Konvalinka J.,Gilead Sciences | Konvalinka J.,Charles University | Krausslich H.-G.,University of Heidelberg | Krausslich H.-G.,Molecular Medicine Partnership Unit | And 2 more authors.
Virology | Year: 2015

Proteolytic processing of viral polyproteins is essential for retrovirus infectivity. Retroviral proteases (PR) become activated during or after assembly of the immature, non-infectious virion. They cleave viral polyproteins at specific sites, inducing major structural rearrangements termed maturation. Maturation converts retroviral enzymes into their functional form, transforms the immature shell into a metastable state primed for early replication events, and enhances viral entry competence. Not only cleavage at all PR recognition sites, but also an ordered sequence of cleavages is crucial. Proteolysis is tightly regulated, but the triggering mechanisms and kinetics and pathway of morphological transitions remain enigmatic.Here, we outline PR structures and substrate specificities focusing on HIV PR as a therapeutic target. We discuss design and clinical success of HIV PR inhibitors, as well as resistance development towards these drugs. Finally, we summarize data elucidating the role of proteolysis in maturation and highlight unsolved questions regarding retroviral maturation. © 2015 Elsevier Inc.


Altamura S.,University of Heidelberg | Altamura S.,Molecular Medicine Partnership Unit | Kessler R.,University of Heidelberg | Kessler R.,Molecular Medicine Partnership Unit | And 7 more authors.
Cell Metabolism | Year: 2014

Summary The regulatory axis between the iron hormone hepcidin and its receptor, the iron exporter ferroportin (FPN), is central to iron homeostasis. Mutations preventing hepcidin-mediated degradation of FPN cause systemic iron overload. We have introduced a point mutation (C326S) into the murine Fpn locus, resembling human hereditary hemochromatosis type 4, including elevated plasma iron and ferritin levels, high transferrin saturation, hepatic iron overload, and iron depletion of duodenal enterocytes and reticuloendothelial macrophages. Unlike other mouse models of iron overload, homozygous C326S mice die between 7 and 14 months of age. Pancreatic acinar cells display marked iron accumulation, oxidative damage and degeneration, associated with failure of the exocrine pancreas and severe body weight loss. Rescue experiments reveal iron overload and exocrine pancreatic failure as leading causes of death. This work uncovers the critical importance of the hepcidin-ferroportin regulatory axis for life and unveils the sensitivity of the exocrine pancreas to iron overload. © 2014 Elsevier Inc.


Selvaraj D.,University of Heidelberg | Gangadharan V.,University of Heidelberg | Michalski C.W.,University of Heidelberg | Kurejova M.,University of Heidelberg | And 13 more authors.
Cancer Cell | Year: 2015

Cancer pain is a debilitating disorder and a primary determinant of the poor quality of life. Here, we report a non-vascular role for ligands of the Vascular Endothelial Growth Factor (VEGF) family in cancer pain. Tumor-derived VEGF-A, PLGF-2, and VEGF-B augment pain sensitivity through selective activation of VEGF receptor 1 (VEGFR1) expressed in sensory neurons in human cancer and mouse models. Sensory-neuron-specific genetic deletion/silencing or local or systemic blockade of VEGFR1 prevented tumor-induced nerve remodeling and attenuated cancer pain in diverse mouse models invivo. These findings identify a therapeutic potential for VEGFR1-modifying drugs in cancer pain and suggest a palliative effect for VEGF/VEGFR1-targeting anti-angiogenic tumor therapies. © 2015 The Authors.


Casanovas G.,University of Heidelberg | Casanovas G.,Molecular Medicine Partnership Unit | Casanovas G.,European Molecular Biology Laboratory | Banerji A.,Institute of Molecular Biology IMB | And 7 more authors.
PLoS Computational Biology | Year: 2014

Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signaling cascades. Deregulation of either pathway causes iron-related diseases such as hemochromatosis or anemia of inflammation. We quantitatively analyzed how BMP6 and IL6 control hepcidin expression. Transcription factor (TF) phosphorylation and reporter gene expression were measured under co-stimulation conditions, and the promoter was perturbed by mutagenesis. Using mathematical modeling, we systematically analyzed potential mechanisms of cooperative and competitive promoter regulation by the transcription factors, and experimentally validated the model predictions. Our results reveal that hepcidin cross-regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signaling crosstalk is insignificant. We find that the presence of two BMP-responsive elements enhances the steepness of the promoter response towards the iron-sensing BMP signaling axis, which promotes iron homeostasis in vivo. IL6 co-stimulation reduces the promoter sensitivity towards the BMP signal, because the SMAD and STAT transcription factors compete for recruiting RNA polymerase to the transcription start site. This may explain why inflammatory signals disturb iron homeostasis in anemia of inflammation. Taken together, our results reveal why the iron homeostasis circuit is sensitive to perturbations implicated in disease. © 2014 Casanovas et al.


Song K.,University of Heidelberg | Song K.,Max Delbrück Center for Molecular Medicine | Wang H.,University of Heidelberg | Kamm G.B.,University of Heidelberg | And 7 more authors.
Science | Year: 2016

Body temperature homeostasis is critical for survival and requires precise regulation by the nervous system.The hypothalamus serves as the principal thermostat that detects and regulates internal temperature.We demonstrate that the ion channel TRPM2 [of the transient receptor potential (TRP) channel family] is a temperature sensor in a subpopulation of hypothalamic neurons.TRPM2 limits the fever response and may detect increased temperatures to prevent overheating. Furthermore, chemogenetic activation and inhibition of hypothalamic TRPM2-expressing neurons in vivo decreased and increased body temperature, respectively. Such manipulation may allow analysis of the beneficial effects of altered body temperature on diverse disease states. Identification of a functional role for TRP channels in monitoring internal body temperature should promote further analysis of molecular mechanisms governing thermoregulation and foster the genetic dissection of hypothalamic circuits involved with temperature homeostasis.


Guida C.,University of Heidelberg | Guida C.,European Molecular Biology Laboratory | Altamura S.,University of Heidelberg | Altamura S.,Molecular Medicine Partnership Unit | And 8 more authors.
Blood | Year: 2015

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recyclingmay represent a first-line response to restrict iron access for numerous pathogens. © 2015 by The American Society of Hematology.


Lodrini M.,German Cancer Research Center | Oehme I.,German Cancer Research Center | Schroeder C.,German Cancer Research Center | Milde T.,German Cancer Research Center | And 16 more authors.
Nucleic Acids Research | Year: 2013

MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function. © 2013 The Author(s) 2013. Published by Oxford University Press.


Schur F.K.M.,Structural and Computational Biology Unit | Schur F.K.M.,Molecular Medicine Partnership Unit | Dick R.A.,Cornell University | Hagen W.J.H.,Structural and Computational Biology Unit | And 3 more authors.
Journal of Virology | Year: 2015

The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason- Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ~8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation.


Mleczko-Sanecka K.,University of Heidelberg | Mleczko-Sanecka K.,Molecular Medicine Partnership Unit | Mleczko-Sanecka K.,European Molecular Biology Laboratory EMBL | Casanovas G.,University of Heidelberg | And 11 more authors.
Blood | Year: 2010

Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin deficiency causes common iron overload syndromes whereas its overexpression is responsible for microcytic anemias. Hepcidin transcription is activated by the bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways, whereas comparatively little is known about how hepcidin expression is inhibited. By using high-throughput siRNA screening we identified SMAD7 as a potent hepcidin suppressor. SMAD7 is an inhibitory SMAD protein that mediates a negative feedback loop to both transforming growth factor-β and BMP signaling and that recently was shown to be coregulated with hepcidin via SMAD4 in response to altered iron availability in vivo. We show that SMAD7 is coregulated with hepcidin byBMPsin primary murine hepatocytes and that SMAD7 overexpression completely abolishes hepcidin activation by BMPsand transforming growth factor-β.We identify a distinct SMAD regulatory motif (GTCAAGAC) within the hepcidin promoter involved in SMAD7-dependent hepcidin suppression, demonstrating that SMAD7 does not simply antagonize the previously reported hemojuvelin/BMP-responsive elements. This work identifies a potent inhibitory factor for hepcidin expression and uncovers a negative feedback pathway for hepcidin regulation, providing insight into a mechanism how hepcidin expression may be limited to avoid iron deficiency. © 2010 by The American Society of Hematology.

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