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News Article | October 7, 2016
Site: phys.org

The MDC research team headed by Dr. Oliver Rocks is investigating the mechanisms that control the remodeling of the cell skeleton. During a screen of proteins involved in this process the scientists made an interesting discovery - they noticed that one of the proteins binds the catalytic subunit of the PKA. "We were surprised to find the catalytic subunit of PKA, because normally control of this pathway is through the regulatory subunit," says the researcher. In the classical model of PKA regulation, the regulatory subunits dock onto the catalytic ones and stop them sending signals. They only release the catalytic subunits when the cell receives a signal that increases the levels of the cellular chemical cAMP. cAMP clips onto the regulatory subunits and force them to set the catalytic subunits free. In the screen the catalytic subunit of PKA (PKAC) was binding to a protein called ARHGAP36. For her PhD research in Dr. Rocks's lab, Rebecca Eccles investigated how ARHGAP36 interacts with PKAC. She worked with other scientists at the MDC, the Berlin Institute of Health (BIH), as well as partners at the University of Liverpool. Eccles found that ARHGAP36 can turn off PKAC in two ways: by binding to it and blocking its action, and by sending it on the path to one of the cell's degradation centers. PKA's job in the cell is to pass on signals, which the catalytic subunit does by its kinase action - kinases attach a phosphate molecule to their target proteins (substrates). ARHGAP36 stops PKAC from binding its substrates in much the same way as a key stuck in a lock prevents you opening a door. Because PKA occurs in almost all tissues, the researchers wanted to identify where and when it is inhibited by ARHGAP36. "ARGAP36 is a strong inhibitor, so you wouldn't want it turning off PKA everywhere," Rocks explains. He and his team found that ARHGAP36 is not present in all cells all the time - in fact its expression is quite limited, for example to embryonic muscle cells. Abnormally high levels of ARHGAP3 are also found in at least one of the four subtypes of medulloblastoma, the most common childhood brain cancer, as well as in neuroblastoma, another frequent cancer of the nervous system in children. The exact biological role of ARHGAP36 is not yet understood, but it may well play a role in muscle development and in tumor progression in some cancers. For example, changes in PKA signaling could influence tumor growth in many types of cancer. Understanding how signaling pathways are controlled may also be useful in drug development since it opens up opportunities for regulating proteins indirectly and thus blocking enzymes that are otherwise hard to manipulate. Explore further: Study reveals the regulatory mechanism of key enzyme More information: Rebecca L. Eccles et al, Bimodal antagonism of PKA signalling by ARHGAP36, Nature Communications (2016). DOI: 10.1038/NCOMMS12963


PubMed | German Cancer Consortium DKTK, Humboldt University of Berlin, German Rheumatism Research Center, Berlin Institute of Health and 5 more.
Type: | Journal: Nature communications | Year: 2017

Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal-distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC.


Chu V.T.,Max Delbrück Center for Molecular Medicine | Weber T.,Max Delbrück Center for Molecular Medicine | Wefers B.,Helmholtz Center for Environmental Research | Wefers B.,Deutsches Zentrum fur Neurodegenerative Erkrankungen E.V. | And 8 more authors.
Nature Biotechnology | Year: 2015

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells. © 2015 Nature America, Inc. All rights reserved.


Ebert A.D.,Stanford University | Diecke S.,Max Delbrück Center for Molecular Medicine | Diecke S.,Berlin Institute of Health | Chen I.Y.,Stanford University | Wu J.C.,Stanford University
EMBO Molecular Medicine | Year: 2015

Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options. © 2015 The Authors.


Mackowiak S.D.,Max Delbrück Center for Molecular Medicine | Zauber H.,Max Delbrück Center for Molecular Medicine | Bielow C.,Max Delbrück Center for Molecular Medicine | Bielow C.,Berlin Institute of Health | And 8 more authors.
Genome Biology | Year: 2015

Background: There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging. Results: Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates. Conclusions: Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo. © 2015 Mackowiak et al.


Bielow C.,Max Delbrück Center for Molecular Medicine | Bielow C.,Berlin Institute of Health | Mastrobuoni G.,Max Delbrück Center for Molecular Medicine | Kempa S.,Max Delbrück Center for Molecular Medicine | Kempa S.,Berlin Institute of Health
Journal of Proteome Research | Year: 2016

Mass spectrometry-based proteomics coupled to liquid chromatography has matured into an automatized, high-throughput technology, producing data on the scale of multiple gigabytes per instrument per day. Consequently, an automated quality control (QC) and quality analysis (QA) capable of detecting measurement bias, verifying consistency, and avoiding propagation of error is paramount for instrument operators and scientists in charge of downstream analysis. We have developed an R-based QC pipeline called Proteomics Quality Control (PTXQC) for bottom-up LC-MS data generated by the MaxQuant1 software pipeline. PTXQC creates a QC report containing a comprehensive and powerful set of QC metrics, augmented with automated scoring functions. The automated scores are collated to create an overview heatmap at the beginning of the report, giving valuable guidance also to nonspecialists. Our software supports a wide range of experimental designs, including stable isotope labeling by amino acids in cell culture (SILAC), tandem mass tags (TMT), and label-free data. Furthermore, we introduce new metrics to score MaxQuant's Match-between-runs (MBR) functionality by which peptide identifications can be transferred across Raw files based on accurate retention time and m/z. Last but not least, PTXQC is easy to install and use and represents the first QC software capable of processing MaxQuant result tables. PTXQC is freely available at https://github.com/cbielow/PTXQC. © 2015 American Chemical Society.


Kuhnen P.,Charité - Medical University of Berlin | Clement K.,University Pierre and Marie Curie | Wiegand S.,Charité - Medical University of Berlin | Blankenstein O.,Charité - Medical University of Berlin | And 7 more authors.
New England Journal of Medicine | Year: 2016

Patients with rare defects in the gene encoding proopiomelanocortin (POMC) have extreme early-onset obesity, hyperphagia, hypopigmentation, and hypocortisolism, resulting from the lack of the proopiomelanocortin-derived peptides melanocyte-stimulating hormone and corticotropin. In such patients, adrenal insufficiency must be treated with hydrocortisone early in life. No effective pharmacologic treatments have been available for the hyperphagia and obesity that characterize the condition. In this investigator-initiated, open-label study, two patients with proopio-melanocortin deficiency were treated with setmelanotide, a new melanocortin-4 receptor agonist. The patients had a sustainable reduction in hunger and substantial weight loss (51.0 kg after 42 weeks in Patient 1 and 20.5 kg after 12 weeks in Patient 2). Copyright © 2016 Massachusetts Medical Society.


Holman C.,Charité - Medical University of Berlin | Piper S.K.,Charité - Medical University of Berlin | Grittner U.,Charité - Medical University of Berlin | Diamantaras A.A.,Charité - Medical University of Berlin | And 5 more authors.
PLoS Biology | Year: 2016

Given small sample sizes, loss of animals in preclinical experiments can dramatically alter results. However, effects of attrition on distortion of results are unknown. We used a simulation study to analyze the effects of random and biased attrition. As expected, random loss of samples decreased statistical power, but biased removal, including that of outliers, dramatically increased probability of false positive results. Next, we performed a meta-analysis of animal reporting and attrition in stroke and cancer. Most papers did not adequately report attrition, and extrapolating from the results of the simulation data, we suggest that their effect sizes were likely overestimated. © 2016 Holman et al.


Dragun D.,Campus Virchow Klinikum and Center for Cardiovascular Research | Dragun D.,Berlin Institute of Health | Catar R.,Campus Virchow Klinikum and Center for Cardiovascular Research | Catar R.,Berlin Institute of Health | Philippe A.,Campus Virchow Klinikum and Center for Cardiovascular Research
Kidney International | Year: 2016

Detrimental actions of donor-specific antibodies (DSAs) directed against both major histocompatibility antigens (human leukocyte antigen [HLA]) and specific non-HLA antigens expressed on the allograft endothelium are a flourishing research area in kidney transplantation. Newly developed solid-phase assays enabling detection of functional non-HLA antibodies targeting G protein–coupled receptors such as angiotensin type I receptor and endothelin type A receptor were instrumental in providing long-awaited confirmation of their broad clinical relevance. Numerous recent clinical studies implicate angiotensin type I receptor and endothelin type A receptor antibodies as prognostic biomarkers for earlier occurrence and severity of acute and chronic immunologic complications in solid organ transplantation, stem cell transplantation, and systemic autoimmune vascular disease. Angiotensin type 1 receptor and endothelin type A receptor antibodies exert their pathophysiologic effects alone and in synergy with HLA-DSA. Recently identified antiperlecan antibodies are also implicated in accelerated allograft vascular pathology. In parallel, protein array technology platforms enabled recognition of new endothelial surface antigens implicated in endothelial cell activation. Upon target antigen recognition, non-HLA antibodies act as powerful inducers of phenotypic perturbations in endothelial cells via activation of distinct intracellular cell-signaling cascades. Comprehensive diagnostic assessment strategies focusing on both HLA-DSA and non-HLA antibody responses could substantially improve immunologic risk stratification before transplantation, help to better define subphenotypes of antibody-mediated rejection, and lead to timely initiation of targeted therapies. Better understanding of similarities and dissimilarities in HLA-DSA and distinct non-HLA antibody–related mechanisms of endothelial damage should facilitate discovery of common downstream signaling targets and pave the way for the development of endothelium-centered therapeutic strategies to accompany intensified immunosuppression and/or mechanical removal of antibodies. © 2016 International Society of Nephrology


PubMed | Berlin Institute of Health and Campus Virchow Klinikum and Center for Cardiovascular Research
Type: Journal Article | Journal: Kidney international | Year: 2016

Detrimental actions of donor-specific antibodies (DSAs) directed against both major histocompatibility antigens (human leukocyte antigen [HLA]) and specific non-HLA antigens expressed on the allograft endothelium are a flourishing research area in kidney transplantation. Newly developed solid-phase assays enabling detection of functional non-HLA antibodies targeting G protein-coupled receptors such as angiotensin type I receptor and endothelin type A receptor were instrumental in providing long-awaited confirmation of their broad clinical relevance. Numerous recent clinical studies implicate angiotensin type I receptor and endothelin type A receptor antibodies as prognostic biomarkers for earlier occurrence and severity of acute andchronic immunologic complications in solid organ transplantation, stem cell transplantation, and systemic autoimmune vascular disease. Angiotensin type 1 receptor and endothelin type A receptor antibodies exert their pathophysiologic effects alone and in synergy with HLA-DSA. Recently identified antiperlecan antibodies are also implicated in accelerated allograft vascular pathology. In parallel, protein array technology platforms enabled recognition of new endothelial surface antigens implicated in endothelial cell activation. Upon target antigen recognition, non-HLA antibodies act as powerful inducers ofphenotypic perturbations in endothelial cells via activation of distinct intracellular cell-signaling cascades. Comprehensive diagnostic assessment strategies focusing on both HLA-DSA and non-HLA antibody responses could substantially improve immunologic risk stratification before transplantation, help to better define subphenotypes of antibody-mediated rejection, and lead to timely initiation oftargeted therapies. Better understanding of similarities anddissimilarities in HLA-DSA and distinct non-HLA antibody-related mechanisms of endothelial damage should facilitate discovery of common downstream signaling targets and pave the way for the development of endothelium-centered therapeutic strategies to accompany intensified immunosuppression and/or mechanical removal of antibodies.

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