Publications by authors named "Marieluise Kirchner"

26 Publications

  • Page 1 of 1

Surface AMP deaminase 2 as a novel regulator modifying extracellular adenine nucleotide metabolism.

FASEB J 2021 07;35(7):e21684

Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.

Adenine nucleotides represent crucial immunomodulators in the extracellular environment. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thus promoting an anti-inflammatory milieu induced by the "adenosine halo". AMPD2 intracellularly mediates AMP deamination to IMP, thereby both enhancing the degradation of inflammatory ATP and reducing the formation of anti-inflammatory adenosine. Here, we show that this enzyme is expressed on the surface of human immune cells and its predominance may modify inflammatory states by altering the extracellular milieu. Surface AMPD2 (eAMPD2) expression on monocytes was verified by immunoblot, surface biotinylation, mass spectrometry, and immunofluorescence microscopy. Flow cytometry revealed enhanced monocytic eAMPD2 expression after TLR stimulation. PBMCs from patients with rheumatoid arthritis displayed significantly higher levels of eAMPD2 expression compared with healthy controls. Furthermore, the product of AMPD2-IMP-exerted anti-inflammatory effects, while the levels of extracellular adenosine were not impaired by an increased eAMPD2 expression. In summary, our study identifies eAMPD2 as a novel regulator of the extracellular ATP-adenosine balance adding to the immunomodulatory CD39-CD73 system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202002658RRDOI Listing
July 2021

Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories.

Nat Commun 2021 06 11;12(1):3576. Epub 2021 Jun 11.

German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany.

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a tandem mass tag (TMT) approach. With a microscaled approach, we quantify 7,000 phosphosites, enabling the analysis of FFPE biopsies with limited tissue amounts. We also investigate the influence of pre-analytical variables including fixation time and heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for the most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. Finally, we present general guidelines to which methods are best suited for different applications, highlighting TMT methods for comprehensive (phospho-)proteome profiling for focused clinical studies and label-free methods for large cohorts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-23855-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196151PMC
June 2021

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

Nat Commun 2020 06 19;11(1):3133. Epub 2020 Jun 19.

Neuromuscular and Cardiovascular Cell Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Robert Rössle Strasse, 1013125, Berlin, Germany.

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16929-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305127PMC
June 2020

Progression-Dependent Altered Metabolism in Osteosarcoma Resulting in Different Nutrient Source Dependencies.

Cancers (Basel) 2020 May 27;12(6). Epub 2020 May 27.

Berlin Institute of Health Metabolomics Platform, Berlin Institute of Health (BIH), 13125 Berlin, Germany.

Osteosarcoma (OS) is a primary malignant bone tumor and OS metastases are mostly found in the lung. The limited understanding of the biology of metastatic processes in OS limits the ability for effective treatment. Alterations to the metabolome and its transformation during metastasis aids the understanding of the mechanism and provides information on treatment and prognosis. The current study intended to identify metabolic alterations during OS progression by using a targeted gas chromatography mass spectrometry approach. Using a female OS cell line model, malignant and metastatic cells increased their energy metabolism compared to benign OS cells. The metastatic cell line showed a faster metabolic flux compared to the malignant cell line, leading to reduced metabolite pools. However, inhibiting both glycolysis and glutaminolysis resulted in a reduced proliferation. In contrast, malignant but non-metastatic OS cells showed a resistance to glycolytic inhibition but a strong dependency on glutamine as an energy source. Our in vivo metabolic approach hinted at a potential sex-dependent metabolic alteration in OS patients with lung metastases (LM), although this will require validation with larger sample sizes. In line with the in vitro results, we found that female LM patients showed a decreased central carbon metabolism compared to metastases from male patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12061371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352851PMC
May 2020

Cold-induced urticarial autoinflammatory syndrome related to factor XII activation.

Nat Commun 2020 01 10;11(1):179. Epub 2020 Jan 10.

Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.

Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1β (IL-1β) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13984-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954242PMC
January 2020

The Translational Landscape of the Human Heart.

Cell 2019 06 30;178(1):242-260.e29. Epub 2019 May 30.

Department of Rheumatology and Clinical Immunology, Sapporo City General Hospital, Sapporo, Hokkaido 060-8604, Japan.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.05.010DOI Listing
June 2019

PRISMA: Protein Interaction Screen on Peptide Matrix Reveals Interaction Footprints and Modifications- Dependent Interactome of Intrinsically Disordered C/EBPβ.

iScience 2019 Mar 1;13:351-370. Epub 2019 Mar 1.

Max Delbrück Center for Molecular Medicine, Robert-Roessle Strasse 10, 13125 Berlin, Germany; Humboldt-University of Berlin, Institute of Biology, 10115 Berlin, Germany. Electronic address:

CCAAT enhancer-binding protein beta (C/EBPβ) is a pioneer transcription factor that specifies cell differentiation. C/EBPβ is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPβ differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPβ alters protein interactions and cell differentiation, suggesting that a C/EBPβ PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPβ using an array technique based on spot-synthesized C/EBPβ-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ∼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPβ acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2019.02.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424098PMC
March 2019

Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress.

Sci Rep 2019 02 28;9(1):3183. Epub 2019 Feb 28.

Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt Universität zu Berlin, and Berlin Institute of Health, Neurocure Cluster of Excellence, Department of Experimental Neurology, Berlin, Germany.

Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10-20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-39438-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395706PMC
February 2019

Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.

Acta Biomater 2019 03 5;86:171-184. Epub 2019 Jan 5.

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Rheumatology and Clinical Immunology, Charitéplatz 1, 10117 Berlin, Germany; German Rheumatism Research Centre (DRFZ) Berlin, A Leibniz Institute, Charitéplatz 1, 10117 Berlin, Germany; Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin Brandenburg Center for Regenerative Therapies, Föhrer Str. 15, 13353 Berlin, Germany. Electronic address:

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2018.12.043DOI Listing
March 2019

Mutations in Disordered Regions Can Cause Disease by Creating Dileucine Motifs.

Cell 2018 09 6;175(1):239-253.e17. Epub 2018 Sep 6.

Proteome Dynamics, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rössle-Str. 10, 13125 Berlin, Germany; Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany. Electronic address:

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.08.019DOI Listing
September 2018

On the relation between filament density, force generation, and protrusion rate in mesenchymal cell motility.

Mol Biol Cell 2018 11 29;29(22):2674-2686. Epub 2018 Aug 29.

Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E18-02-0082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249830PMC
November 2018

Picky: a simple online PRM and SRM method designer for targeted proteomics.

Nat Methods 2018 02;15(3):156-157

Proteome Dynamics, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4607DOI Listing
February 2018

CLCN2 chloride channel mutations in familial hyperaldosteronism type II.

Nat Genet 2018 03 5;50(3):349-354. Epub 2018 Feb 5.

Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.

Primary aldosteronism, a common cause of severe hypertension , features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II) and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-018-0048-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862758PMC
March 2018

Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution.

Nucleic Acids Res 2017 01 6;45(2):938-950. Epub 2016 Sep 6.

Non-coding RNAs and mechanisms of cytoplasmic gene regulation, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin 13125, Germany

Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkw792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314787PMC
January 2017

GABA blocks pathological but not acute TRPV1 pain signals.

Cell 2015 Feb;160(4):759-770

Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. Electronic address:

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2015.01.022DOI Listing
February 2015

Drep-2 is a novel synaptic protein important for learning and memory.

Elife 2014 Nov 13;3. Epub 2014 Nov 13.

Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany.

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.03895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229683PMC
November 2014

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

J Clin Invest 2014 Aug 24;124(8):3419-30. Epub 2014 Jun 24.

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI74523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109538PMC
August 2014

Global characterization of the oocyte-to-embryo transition in Caenorhabditis elegans uncovers a novel mRNA clearance mechanism.

EMBO J 2014 Aug 23;33(16):1751-66. Epub 2014 Jun 23.

Systems Biology of Gene Regulatory Elements, Max Delbrück Center Berlin, Berlin, Germany

The oocyte-to-embryo transition (OET) is thought to be mainly driven by post-transcriptional gene regulation. However, expression of both RNAs and proteins during the OET has not been comprehensively assayed. Furthermore, specific molecular mechanisms that regulate gene expression during OET are largely unknown. Here, we quantify and analyze transcriptome-wide, expression of mRNAs and thousands of proteins in Caenorhabditis elegans oocytes, 1-cell, and 2-cell embryos. This represents a first comprehensive gene expression atlas during the OET in animals. We discovered a first wave of degradation in which thousands of mRNAs are cleared shortly after fertilization. Sequence analysis revealed a statistically highly significant presence of a polyC motif in the 3' untranslated regions of most of these degraded mRNAs. Transgenic reporter assays demonstrated that this polyC motif is required and sufficient for mRNA degradation after fertilization. We show that orthologs of human polyC-binding protein specifically bind this motif. Our data suggest a mechanism in which the polyC motif and binding partners direct degradation of maternal mRNAs. Our data also indicate that endogenous siRNAs but not miRNAs promote mRNA clearance during the OET.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201488769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4195759PMC
August 2014

Conservation of mRNA and protein expression during development of C. elegans.

Cell Rep 2014 Feb 23;6(3):565-77. Epub 2014 Jan 23.

Systems Biology of Gene Regulatory Elements, Max Delbrück Center Berlin, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. Electronic address:

Spatiotemporal control of gene expression is crucial for development and subject to evolutionary changes. Although proteins are the final product of most genes, the developmental proteome of an animal has not yet been comprehensively defined, and the correlation between mRNA and protein abundance during development is largely unknown. Here, we globally measured and compared protein and mRNA expression changes during the life cycle of the nematodes C. elegans and C. briggsae, separated by ~30 million years of evolution. We observed that developmental mRNA and protein changes were highly conserved to a surprisingly similar degree but were poorly correlated within a species, suggesting important and widespread posttranscriptional regulation. Posttranscriptional control was particularly well conserved if mRNA fold changes were buffered on the protein level, indicating a predominant repressive function. Finally, among divergently expressed genes, we identified insulin signaling, a pathway involved in lifespan determination, as a putative target of adaptive evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2014.01.001DOI Listing
February 2014

In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish.

PLoS One 2012 29;7(6):e40000. Epub 2012 Jun 29.

Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040000PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386912PMC
November 2012

In vivo quantitative proteome profiling: planning and evaluation of SILAC experiments.

Methods Mol Biol 2012 ;893:175-99

Cell Signalling and Mass Spectrometry Group, Max Delbrueck Center for Molecular Medicine, Berlin, Germany.

Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. Particularly, quantification based on stable isotope labeling with amino acids (SILAC) has emerged as a robust, reliable and simple method for accurate large-scale protein quantification. The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-885-6_13DOI Listing
September 2012

Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements.

PLoS Biol 2010 Aug 24;8(8). Epub 2010 Aug 24.

Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P(+)GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.1000457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927421PMC
August 2010

Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.

Mol Microbiol 2008 May 4;68(3):749-67. Epub 2008 Mar 4.

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood, Boston, MA 02115, USA.

Listeria monocytogenes is an intracellular bacterial pathogen that causes life-threatening disease. The mechanisms used by L. monocytogenes to invade non-professional phagocytic cells are not fully understood. In addition to the requirement of bacterial determinants, host cell conditions profoundly influence infection. Here, we have shown that inhibition of the RhoA/ROCK pathway by pharmacological inhibitors or RNA interference results in increased L. monocytogenes invasion of murine fibroblasts and hepatocytes. InlF, a member of the internalin multigene family with no known function, was identified as a L. monocytogenes-specific factor mediating increased host cell binding and entry. Conversely, activation of RhoA/ROCK activity resulted in decreased L. monocytogenes adhesion and invasion. Furthermore, virulence of wild-type bacteria during infection of mice was significantly increased upon inhibition of ROCK activity, whereas colonization and virulence of an inlF deletion mutant was not affected, thus supporting a role for InlF as a functional virulence determinant in vivo under specific conditions. In addition, inhibition of ROCK activity in human-derived cells enhanced either bacterial adhesion or adhesion and entry in an InlF-independent manner, further suggesting a host species or cell type-specific role for InlF and that additional bacterial determinants are involved in mediating ROCK-regulated invasion of human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2958.2008.06188.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575752PMC
May 2008

The PilC adhesin of the Neisseria type IV pilus-binding specificities and new insights into the nature of the host cell receptor.

Mol Microbiol 2005 May;56(4):945-57

Department of Molecular Biology, Max Planck Institute for Infection Biology, Schumannstrasse 21/22, 10117 Berlin, Germany.

Type IV pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate the first contact to human mucosal epithelial cells, an interaction which is also critical for the interaction with vascular endothelial cells. The PilC proteins have been characterized as the principal pilus-associated adhesin. Here we show that PilC2 exhibits a defined cell and tissue tropism, as it binds to human epithelial and endothelial cell lines, but not to human T cells or fibroblasts. Piliated gonococci and PilC2 exhibit similar patterns of binding to human epithelial and endothelial cells, supporting the function of PilC as the key pilus adhesin. Although CD46 has previously been suggested to be a pilus receptor, several observations indicate that neisserial type IV pili and the pilus adhesin PilC2 interact with epithelial cells in a CD46 independent manner. Biochemical approaches were used to characterize the nature of host cell factors mediating binding of piliated gonococci and PilC2 protein. Our data indicate that the putative host cell receptor for gonococcal pili and the PilC2 pilus adhesin is a surface protein. Glycostructures were found to not be involved in binding. Moreover, we observed the uptake of purified PilC2 protein together with its receptor via receptor-mediated endocytosis and subsequent receptor re-exposure on the cell surface. Our data support the existence of a specific pilus receptor and provide intriguing information on the nature of the receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2958.2005.04600.xDOI Listing
May 2005

CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells.

Infect Immun 2005 May;73(5):3072-82

Max-Planck Institute for Infection Biology/Department of Molecular Biology, Schumannstrasse 21/22, 10117 Berlin, Germany.

Neisseria gonorrhoeae is a gram-negative bacterial pathogen which infects the human mucosal epithelium. An early critical event in neisserial infection is the type IV pilus-mediated adherence to the host cell. The PilC protein, located on the pilus tip, has earlier been identified as the major pilus adhesin. Previous studies suggested that the cell surface protein CD46 is a pilus receptor for Neisseria. We investigated the role of CD46 in pilus-mediated gonococcal infection of epithelial cells. Differences in binding efficiencies of piliated gonococci as well as purified pilus adhesin PilC2 on human epithelial cell lines did not correlate to the level of surface-expressed CD46. Additionally, no binding of piliated gonococci or PilC2 protein was observed on CD46-transfected CHO and MDCK cells. Furthermore, specific down-regulation of CD46 expression in human epithelial cell lines by RNA interference did not alter the binding efficiency of piliated gonococci or purified PilC2 protein, although other CD46-dependent processes, such as measles virus infection and C3b cleavage, were significantly reduced. These data support the notion that pilus-mediated gonococcal infection of epithelial cells can occur in a CD46-independent manner, thus questioning the function of CD46 as an essential pilus receptor for pathogenic neisseriae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.73.5.3072-3082.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087333PMC
May 2005
-->