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Braig D.,Albert Ludwigs University of Freiburg | Kaiser B.,Albert Ludwigs University of Freiburg | Thiele J.R.,Albert Ludwigs University of Freiburg | Bannasch H.,Albert Ludwigs University of Freiburg | And 4 more authors.
International Immunology | Year: 2014

Tissue damage in burn injury leads to a rapid increase of leukocytes and acute phase reactants. Plasma levels of C-reactive protein (CRP) rise within hours after the insult. No deficiency of this protein has been reported in humans, suggesting it plays a pivotal role in innate immunity. CRP in circulation is composed of five identical subunits [pentameric CRP (pCRP)]. Recently, deposits of structurally modified CRP (mCRP) have been found in inflammatory diseases. Little is known about this structural change and how it affects CRP functions. We analyzed CRP deposits in burn wounds and serum by immunohistochemistry, western blot and dot blot analysis. CRP was deposited in necrotic and inflamed tissue, but not in adjacent healthy tissue. Tissue deposited CRP was detected by mCRP-specific antibodies and structurally different from serum pCRP. mCRP but not pCRP induced reactive oxygen species production by monocytes and facilitated uptake of necrotic Jurkat cells by macrophages. In addition, it accelerated migration of keratinocytes in a scratch wound assay. The structural changes that occur in pCRP upon localization to damaged and inflamed tissue in burn wounds result in a functionally altered protein with distinct functions. mCRP exhibits opsonic, proinflammatory and promigratory properties which modulate wound healing. Source


Ott C.,University of Wurzburg | Dorsch E.,University of Wurzburg | Fraunholz M.,University of Wurzburg | Straub S.,University of Wurzburg | And 2 more authors.
PLoS ONE | Year: 2015

Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components. © 2015 Ott et al. Source


Prabakaran S.,Proteomics Center | Prabakaran S.,Harvard University | Prabakaran S.,Tufts University | Hemberg M.,Boston Childrens Hospital | And 18 more authors.
Nature Communications | Year: 2014

Only a small fraction of the mammalian genome codes for messenger RNAs destined to be translated into proteins, and it is generally assumed that a large portion of transcribed sequences - including introns and several classes of noncoding RNAs (ncRNAs) - do not give rise to peptide products. A systematic examination of translation and physiological regulation of ncRNAs has not been conducted. Here we use computational methods to identify the products of non-canonical translation in mouse neurons by analysing unannotated transcripts in combination with proteomic data. This study supports the existence of non-canonical translation products from both intragenic and extragenic genomic regions, including peptides derived from antisense transcripts and introns. Moreover, the studied novel translation products exhibit temporal regulation similar to that of proteins known to be involved in neuronal activity processes. These observations highlight a potentially large and complex set of biologically regulated translational events from transcripts formerly thought to lack coding potential. © 2014 Macmillan Publishers Limited. Source


Schleicher R.I.,Section for Cardioimmunology | Schleicher R.I.,University Hospital of Tuebingen | Reichenbach F.,Institute for Biochemistry and Molecular Biology | Reichenbach F.,Albert Ludwigs University of Freiburg | And 22 more authors.
Blood | Year: 2015

After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells,andmouseembryonic fibroblasts.Membraneprotein fromPLTslackingmembranebound FasL (FasL Δm/Δm) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-D-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. (Blood. 2015;126(12):1483-1493). © 2015 by The American Society of Hematology. Source


Muller C.S.,Albert Ludwigs University of Freiburg | Bildl W.,Albert Ludwigs University of Freiburg | Haupt A.,Albert Ludwigs University of Freiburg | Ellenrieder L.,Institute for Biochemistry and Molecular Biology | And 9 more authors.
Molecular and Cellular Proteomics | Year: 2016

Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryoslicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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