Center for Molecular Medicine Cologne

Köln, Germany

Center for Molecular Medicine Cologne

Köln, Germany
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Hornig-Do H.-T.,University of Cologne | Tatsuta T.,University of Cologne | Buckermann A.,University of Cologne | Bust M.,University of Cologne | And 6 more authors.
EMBO Journal | Year: 2012

Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly. © 2012 European Molecular Biology Organization | All Rights Reserved.

Lehr S.,Institute of Clinical Biochemistry and Pathobiochemistry | Hartwig S.,Institute of Clinical Biochemistry and Pathobiochemistry | Lamers D.,Paul Langerhans Group | Famulla S.,Paul Langerhans Group | And 8 more authors.
Molecular and Cellular Proteomics | Year: 2012

Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, termed adipokines, via which adipose tissue communicates with other organs. Expansion of adipose tissue in obesity alters adipokine secretion, which may contribute to the development of metabolic diseases. Although recent profiling studies have identified numerous adipokines, the amount of overlap from these studies indicates that the adipokinome is still incompletely characterized. Therefore, we conducted a complementary protein profiling on concentrated conditioned medium derived from primary human adipocytes. SDS-PAGE/liquid chromatography-electrospray ionization tandem MS and two-dimensional SDS-PAGE/matrix-assisted laser desorption ionization/time of flight MS identified 347 proteins, 263 of which were predicted to be secreted. Fourty-four proteins were identified as novel adipokines. Furthermore, we validated the regulation and release of selected adipokines in primary human adipocytes and in serum and adipose tissue biopsies from morbidly obese patients and normal-weight controls. Validation experiments conducted for complement factor H, αB-crystallin, cartilage intermediate-layer protein, and heme oxygenase-1 show that the release and expression of these factors in adipocytes is regulated by differentiation and stimuli, which affect insulin sensitivity, as well as by obesity. Heme oxygenase-1 especially reveals to be a novel adipokine of interest. In vivo, circulating levels and adipose tissue expression of heme oxygenase-1 are significantly increased in obese subjects compared with lean controls. Collectively, our profiling study of the human adipokinome expands the list of adipokines and further highlights the pivotal role of adipokines in the regulation of multiple biological processes within adipose tissue and their potential dysregulation in obesity. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Noetel A.,University of Cologne | Elfimova N.,University of Cologne | Altmuller J.,University of Cologne | Becker C.,University of Cologne | And 9 more authors.
Journal of Hepatology | Year: 2013

Background & Aims: After myofibroblastic transdifferentiation, hepatic stellate cells (HSC), mainly involved in liver fibrosis by extracellular matrix production, exhibit an altered growth factor profile including increased expression of neuronal mediators. Here, we analyzed putative targets of neuronal microRNAs miR-9, miR-125b, and miR-128 by deep sequencing of the transcript population, interacting with the miRNA/Argonaute 2 (Ago2) complex in myofibroblastic HSC. Methods: MicroRNA expression was quantified by real-time PCR in primary HSC, isolated from the rat or human liver. Myofibroblastic HSC were transfected either with mimics or inhibitors of miR-9, miR-125b, and miR-128. RNA from immunoprecipitated Ago2-miRNA/transcript complexes was purified and used for next generation sequencing. Additionally, gene expression was investigated in quiescent and activated primary HSC, treated with the miR-128 mimic or inhibitor, by microarray analysis. Results: During myofibroblastic transdifferentiation of HSC, miR-9, miR-125b, and miR-128 expression was markedly increased. Transcriptome analysis of Ago2 bound mRNA by deep sequencing identified a broad spectrum of transcripts that interact with neuronal miRNAs in myofibroblastic HSC. In particular, in HSC overexpressing miR-128, many members of the chemokine family were bound to the Ago2 repression complex. Furthermore, a comprehensive profiling of gene expression demonstrates the high impact of neuronal miRNAs on the chemokine network. Conclusions: Ago2 immunoprecipitation followed by deep sequencing is a useful tool to identify novel miRNA targets. Upregulation of neuronal miR-9, miR-125b, and miR-128 during myofibroblastic transition and the identified interaction with a wide range of chemokines and chemokine receptors suggest a prominent role of neuronal miRNAs in the inflammatory response of HSC during fibrosis. © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Brinkmann K.,Center for Molecular Medicine Cologne | Brinkmann K.,University of Cologne | Kashkar H.,Center for Molecular Medicine Cologne | Kashkar H.,University of Cologne
Cell Death and Disease | Year: 2014

Acquired resistance toward apoptosis represents one of the hallmarks of human cancer and a major cause of the inefficacy of most anticancer treatment regimens. Based on its ability to inhibit apoptosis, the B-cell lymphoma/leukemia 2 (Bcl-2) protein family has garnered the most attention as a promising therapeutic target in cancer. Accordingly, efforts have lately been focused on the development of drugs targeting Bcl-2 proteins with considerable therapeutic success, particularly in hematologic malignancies. Here, we review the previous studies and highlight the pivotal role of the Bcl-2 protein family in the homeostasis of hematologic tissue compartment. This knowledge provides more insight into why some cancers are more sensitive to Bcl-2 targeting than others and will foster the clinical evaluation of Bcl-2-targeting strategies in cancer by avoiding severe on-target side effects in the development of healthy tissues. © 2014 Macmillan Publishers Limited All rights reserved.

Dogan S.A.,University of Cologne | Pujol C.,University of Cologne | Maiti P.,University of Cologne | Kukat A.,University of Cologne | And 9 more authors.
Cell Metabolism | Year: 2014

Adaptive stress responses activated upon mitochondrial dysfunction are assumed to arise in order to counteract respiratory chain deficiency. Here, we demonstrate that loss of DARS2 (mitochondrial aspartyl-tRNA synthetase) leads to the activation of various stress responses in a tissue-specific manner independently of respiratory chain deficiency. DARS2 depletion in heart and skeletal muscle leads to the severe deregulation of mitochondrial protein synthesis followed by a strong respiratory chain deficit in both tissues, yet the activation of adaptive responses is observed predominantly in cardiomyocytes. We show that the impairment of mitochondrial proteostasis in the heart activates the expression of mitokine FGF21, which acts as a signal for cell-autonomous and systemic metabolic changes. Conversely, skeletal muscle has an intrinsic mechanism relying on the slow turnover of mitochondrial transcripts and higher proteostatic buffering capacity. Our results show that mitochondrial dysfunction is sensed independently of respiratory chain deficiency, questioning the current view on the role of stress responses in mitochondrial diseases. © 2014 Elsevier Inc.

Anton F.,University of Cologne | Fres J.M.,University of Cologne | Fres J.M.,Center for Molecular Medicine Cologne | Schauss A.,University of Cologne | And 8 more authors.
Journal of Cell Science | Year: 2011

Dynamin-related GTPase proteins (DRPs) are main players in membrane remodelling. Conserved DRPs called mitofusins (Mfn1/Mfn2/Fzo1) mediate the fusion of mitochondrial outer membranes (OM). OM fusion depends on self-assembly and GTPase activity of mitofusins as well as on two other proteins, Ugo1 and Mdm30. Here, we define distinct steps of the OM fusion cycle using in vitro and in vivo approaches. We demonstrate that yeast Fzo1 assembles into homo-dimers, depending on Ugo1 and on GTP binding to Fzo1. Fzo1 homo-dimers further associate upon formation of mitochondrial contacts, allowing membrane tethering. Subsequent GTP hydrolysis is required for Fzo1 ubiquitylation by the F-box protein Mdm30. Finally, Mdm30-dependent degradation of Fzo1 completes Fzo1 function in OM fusion. Our results thus unravel functions of Ugo1 and Mdm30 at distinct steps during OM fusion and suggest that protein clearance confers a non-cycling mechanism to mitofusins, which is distinct from other cellular membrane fusion events. © 2011. Published by The Company of Biologists Ltd.

Steculorum S.M.,University of Cologne | Steculorum S.M.,Center for Molecular Medicine Cologne | Steculorum S.M.,Max Planck Institute for Neurological Research | Solas M.,University of Cologne | And 5 more authors.
Alzheimer's and Dementia | Year: 2014

During past decades, ever-increasing life expectancy, despite the development of a sedentary lifestyle and altered eating habits, has led to a dramatic parallel increase in the prevalence of age-related diseases such as type 2 diabetes mellitus (T2DM) and neurodegenerative disorders. Converging evidence from animal and human studies has indicated that insulin resistance in the central nervous system (CNS) is observed in both T2DM and neurodegenerative disorders such as Alzheimer's disease (AD), leading to the hypothesis that impaired neuronal insulin action might be a unifying pathomechanism in the development of both diseases. This assumption, however, is in striking contrast to the evolutionary conserved, protective role of impaired insulin/insulin-like growth factor 1 signaling (IIS) in aging and in protein aggregation-associated diseases, such as AD. Thus, this review summarizes our current understanding of the physiological role of insulin action in various regions of the CNS to regulate neuronal function, learning, and memory, and to control peripheral metabolism. We also discuss mechanisms and clinical outcomes of neuronal insulin resistance and address the seeming paradox of how impaired neuronal IIS can protect from the development of neurodegenerative disorders. © 2014 The Alzheimer's Association. All rights reserved.

Talbot J.J.,University of California at Santa Barbara | Song X.,University of Toronto | Wang X.,Mayo Medical School | Rinschen M.M.,Center for Molecular Medicine Cologne | And 7 more authors.
Journal of the American Society of Nephrology | Year: 2014

Polycystin-1 (PC1) mutations result in proliferative renal cyst growth and progression to renal failure in autosomal dominant polycystic kidney disease (ADPKD). The transcription factor STAT3 (signal transducer and activator of transcription 3) was shown to be activated in cyst-lining cells in ADPKD and PKD mouse models and may drive renal cyst growth, but the mechanisms leading to persistent STAT3 activation are unknown. A proteolytic fragment of PC1 corresponding to the cytoplasmic tail, PC1-p30, is overexpressed in ADPKD. Here, we showthat PC1-p30 interactswith the nonreceptor tyrosine kinase Src, resulting in Srcdependent activation of STAT3 by tyrosine phosphorylation. The PC1-p30-mediated activation of Src/ STAT3 was independent of JAK family kinases and insensitive to the STAT3 inhibitor suppressor of cytokine signaling 3. Signaling by the EGF receptor (EGFR) or cAMP amplified the activation of Src/STAT3 by PC1-p30. Expression of PC1-p30 changed the cellular response to cAMP signaling. In the absence of PC1-p30, cAMP dampened EGFR- or IL-6-dependent activation of STAT3; in the presence of PC1-p30, cAMP amplified Src-dependent activation of STAT3. In the polycystic kidney (PCK) ratmodel, activation of STAT3 in renal cystic cells depended on vasopressin receptor 2 (V2R) signaling, which increased cAMP levels. Genetic inhibition of vasopressin expression or treatment with a pharmacologic V2R inhibitor strongly suppressed STAT3 activation and reduced renal cyst growth. These results suggest that PC1, via its cleaved cytoplasmic tail, integrates signaling inputs from EGFR and cAMP< resulting in Src-dependent activation of STAT3 and a proliferative response. Copyright © 2014 by the American Society of Nephrology.

Joshi A.R.,University of Cologne | Joshi A.R.,Center for Molecular Medicine Cologne | Bobylev I.,University of Cologne | Bobylev I.,Center for Molecular Medicine Cologne | And 4 more authors.
Experimental Neurology | Year: 2015

The small GTPase RhoA and its down-stream effector Rho-kinase (ROCK) are important effector molecules of the neuronal cytoskeleton. Modulation of the RhoA/ROCK pathway has been shown to promote axonal regeneration, however in vitro and animal studies are inconsistent regarding the extent of axonal outgrowth induced by pharmacological inhibition of ROCK. We hypothesized that injury to sensory and motor nerves result in diverse activation levels of RhoA, which may impact the response of those nerve fiber modalities to ROCK inhibition. We therefore examined the effects of Y-27632, a chemical ROCK inhibitor, on the axonal outgrowth of peripheral sensory and motor neurons grown in the presence of growth-inhibiting chondroitin sulfate proteoglycans (CSPGs). In addition we examined the effects of three different doses of Y-27632 on nerve regeneration of motor and sensory nerves in animal models of peripheral nerve crush. In vitro, sensory neurons were less responsive to Y-27632 compared to motor neurons in a non-growth permissive environment. These differences were associated with altered expression and activation of RhoA in sensory and motor axons. In vivo, systemic treatment with high doses of Y-27632 significantly enhanced the regeneration of motor axons over short distances, while the regeneration of sensory fibers remained largely unchanged. Our results support the concept that in a growth non-permissive environment, the regenerative capacity of sensory and motor axons is differentially affected by the RhoA/ROCK pathway, with motor neurons being more responsive compared to sensory. Future treatments, that are aimed to modulate RhoA activity, should consider this functional diversity. © 2014 Elsevier Inc.

Fres J.M.,Institute for Genetics | Muller S.,Center for Molecular Medicine Cologne | Praefcke G.J.K.,Institute for Genetics
Journal of Lipid Research | Year: 2010

Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein.jlr Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.

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