Publications by authors named "Heiko Horn"

24 Publications

  • Page 1 of 1

Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy.

Cancer Discov 2021 Feb 6;11(2):384-407. Epub 2020 Nov 6.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of and overexpression of and , which was contrasted with an identical model carrying wild-type . For homologous recombination-proficient tumors, we constructed genotypes combining loss of and overexpression of , and driven by or or overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC. SIGNIFICANCE: We engineered a panel of murine fallopian tube epithelial cells bearing mutations typical of HGSC and capable of forming tumors in syngeneic immunocompetent hosts. These models recapitulate tumor microenvironments and drug responses characteristic of human disease. In a -overexpressing model, immune-checkpoint resistance was driven by follistatin..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-0818DOI Listing
February 2021

Prediction of cancer driver genes through network-based moment propagation of mutation scores.

Bioinformatics 2020 07;36(Suppl_1):i508-i515

Department of Biosystems Science and Engineering, Machine Learning and Computational Biology Lab, ETH Zürich, Basel 4058, Switzerland.

Motivation: Gaining a comprehensive understanding of the genetics underlying cancer development and progression is a central goal of biomedical research. Its accomplishment promises key mechanistic, diagnostic and therapeutic insights. One major step in this direction is the identification of genes that drive the emergence of tumors upon mutation. Recent advances in the field of computational biology have shown the potential of combining genetic summary statistics that represent the mutational burden in genes with biological networks, such as protein-protein interaction networks, to identify cancer driver genes. Those approaches superimpose the summary statistics on the nodes in the network, followed by an unsupervised propagation of the node scores through the network. However, this unsupervised setting does not leverage any knowledge on well-established cancer genes, a potentially valuable resource to improve the identification of novel cancer drivers.

Results: We develop a novel node embedding that enables classification of cancer driver genes in a supervised setting. The embedding combines a representation of the mutation score distribution in a node's local neighborhood with network propagation. We leverage the knowledge of well-established cancer driver genes to define a positive class, resulting in a partially labeled dataset, and develop a cross-validation scheme to enable supervised prediction. The proposed node embedding followed by a supervised classification improves the predictive performance compared with baseline methods and yields a set of promising genes that constitute candidates for further biological validation.

Availability And Implementation: Code available at https://github.com/BorgwardtLab/MoProEmbeddings.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btaa452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355253PMC
July 2020

Components of genetic associations across 2,138 phenotypes in the UK Biobank highlight adipocyte biology.

Nat Commun 2019 09 6;10(1):4064. Epub 2019 Sep 6.

Department of Biomedical Data Science, School of Medicine, Stanford University, Stanford, CA, USA.

Population-based biobanks with genomic and dense phenotype data provide opportunities for generating effective therapeutic hypotheses and understanding the genomic role in disease predisposition. To characterize latent components of genetic associations, we apply truncated singular value decomposition (DeGAs) to matrices of summary statistics derived from genome-wide association analyses across 2,138 phenotypes measured in 337,199 White British individuals in the UK Biobank study. We systematically identify key components of genetic associations and the contributions of variants, genes, and phenotypes to each component. As an illustration of the utility of the approach to inform downstream experiments, we report putative loss of function variants, rs114285050 (GPR151) and rs150090666 (PDE3B), that substantially contribute to obesity-related traits and experimentally demonstrate the role of these genes in adipocyte biology. Our approach to dissect components of genetic associations across the human phenome will accelerate biomedical hypothesis generation by providing insights on previously unexplored latent structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11953-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731283PMC
September 2019

Single-cell sequencing of neonatal uterus reveals an Misr2+ endometrial progenitor indispensable for fertility.

Elife 2019 06 24;8. Epub 2019 Jun 24.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, United States.

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.46349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650247PMC
June 2019

GeNets: a unified web platform for network-based genomic analyses.

Nat Methods 2018 07 18;15(7):543-546. Epub 2018 Jun 18.

Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.

Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-018-0039-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450090PMC
July 2018

NetSig: network-based discovery from cancer genomes.

Nat Methods 2018 01 4;15(1):61-66. Epub 2017 Dec 4.

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Methods that integrate molecular network information and tumor genome data could complement gene-based statistical tests to identify likely new cancer genes; but such approaches are challenging to validate at scale, and their predictive value remains unclear. We developed a robust statistic (NetSig) that integrates protein interaction networks with data from 4,742 tumor exomes. NetSig can accurately classify known driver genes in 60% of tested tumor types and predicts 62 new driver candidates. Using a quantitative experimental framework to determine in vivo tumorigenic potential in mice, we found that NetSig candidates induce tumors at rates that are comparable to those of known oncogenes and are ten-fold higher than those of random genes. By reanalyzing nine tumor-inducing NetSig candidates in 242 patients with oncogene-negative lung adenocarcinomas, we find that two (AKT2 and TFDP2) are significantly amplified. Our study presents a scalable integrated computational and experimental workflow to expand discovery from cancer genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985961PMC
January 2018

Heterogeneous contribution of microdeletions in the development of common generalised and focal epilepsies.

J Med Genet 2017 09 29;54(9):598-606. Epub 2017 Jul 29.

Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany.

Background: Microdeletions are known to confer risk to epilepsy, particularly at genomic rearrangement 'hotspot' loci. However, microdeletion burden not overlapping these regions or within different epilepsy subtypes has not been ascertained.

Objective: To decipher the role of microdeletions outside hotspots loci and risk assessment by epilepsy subtype.

Methods: We assessed the burden, frequency and genomic content of rare, large microdeletions found in a previously published cohort of 1366 patients with genetic generalised epilepsy (GGE) in addition to two sets of additional unpublished genome-wide microdeletions found in 281 patients with rolandic epilepsy (RE) and 807 patients with adult focal epilepsy (AFE), totalling 2454 cases. Microdeletions were assessed in a combined and subtype-specific approaches against 6746 controls.

Results: When hotspots are considered, we detected an enrichment of microdeletions in the combined epilepsy analysis (adjusted p=1.06×10,OR 1.89, 95% CI 1.51 to 2.35). Epilepsy subtype-specific analyses showed that hotspot microdeletions in the GGE subgroup contribute most of the overall signal (adjusted p=9.79×10, OR 7.45, 95% CI 4.20-13.5). Outside hotspots , microdeletions were enriched in the GGE cohort for neurodevelopmental genes (adjusted p=9.13×10,OR 2.85, 95% CI 1.62-4.94). No additional signal was observed for RE and AFE. Still, gene-content analysis identified known (, and ) and novel () candidate genes across epilepsy subtypes that were not deleted in controls.

Conclusions: Our results show a heterogeneous effect of recurrent and non-recurrent microdeletions as part of the genetic architecture of GGE and a minor contribution in the aetiology of RE and AFE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jmedgenet-2016-104495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574393PMC
September 2017

A scored human protein-protein interaction network to catalyze genomic interpretation.

Nat Methods 2017 01 28;14(1):61-64. Epub 2016 Nov 28.

Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.

Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap, or InWeb_IM) with severalfold more interactions (>500,000) and better functional biological relevance than comparable resources. We illustrate that InWeb_InBioMap enables functional interpretation of >4,700 cancer genomes and genes involved in autism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839635PMC
January 2017

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers.

Cell Syst 2016 09;3(3):302-316.e4

Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cels.2016.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455996PMC
September 2016

CD44 Splice Variant v8-10 as a Marker of Serous Ovarian Cancer Prognosis.

PLoS One 2016 2;11(6):e0156595. Epub 2016 Jun 2.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Department of Surgery, Boston, Massachusetts, United States of America.

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156595PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890777PMC
July 2017

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles.

Cancer Discov 2016 07 4;6(7):714-26. Epub 2016 May 4.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Unlabelled: Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies.

Significance: Experimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714-26. ©2016 AACR.See related commentary by Cho and Collisson, p. 694This article is highlighted in the In This Issue feature, p. 681.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-16-0160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930723PMC
July 2016

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

Sci Signal 2015 Apr 28;8(374):ra40. Epub 2015 Apr 28.

Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.2005769DOI Listing
April 2015

KinomeXplorer: an integrated platform for kinome biology studies.

Nat Methods 2014 Jun;11(6):603-4

Cellular Signal Integration Group (C-SIG), Center for Biological Sequence Analysis (CBS), Department of Systems Biology, Technical University of Denmark (DTU), Lyngby, Denmark.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.2968DOI Listing
June 2014

Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense.

Proc Natl Acad Sci U S A 2014 May 12;111(21):7741-6. Epub 2014 May 12.

Broad Institute, Cambridge, MA 02142;Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114;Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114;Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA 02114;

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1β production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1β production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1407001111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040621PMC
May 2014

Predicting kinase activity in angiotensin receptor phosphoproteomes based on sequence-motifs and interactions.

PLoS One 2014 10;9(4):e94672. Epub 2014 Apr 10.

Department of Clinical Biochemistry, Glostrup Research Institute, Glostrup Hospital, Glostrup, Denmark; Cellular and Metabolic Research Section, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.

Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094672PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983226PMC
June 2015

DoReMi: context-based prioritization of linear motif matches.

PeerJ 2014 20;2:e315. Epub 2014 Mar 20.

NNF Center for Protein Research, University of Copenhagen , Denmark.

Many protein domains bind to short peptide sequences, called linear motifs. Data on their sequence specificities is sparse, which is why biologists usually resort to basic pattern searches to identify new putative binding sites for experimental follow-up. Most motifs have poor specificity and prioritization of the matches is thus crucial when scanning a full proteome with a pattern. Here we present a generic method to prioritize motif occurrence predictions by using cellular contextual information. We take 2 parameters as input: the motif occurrences and one or more of the interacting domains. The potential hits are ranked based on how strongly the context network associates them with a protein containing one of the specified domains, which leads to an increased predictive performance. The method is available through a web interface at doremi.jensenlab.org, which allows for an easy application of the method. We show that this approach leads to improved predictions of binding partners for PDZ domains and the SUMO binding domain. This is consistent with the earlier observation that coupling sequence motifs with network information improves kinase-specific substrate predictions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7717/peerj.315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970808PMC
April 2014

Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest.

Mol Cell Proteomics 2014 Aug 21;13(8):2072-88. Epub 2014 Feb 21.

From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark;

The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.O113.032748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125738PMC
August 2014

TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells.

J Proteome Res 2013 Sep 21;12(9):4136-51. Epub 2013 Aug 21.

Institute of Veterinary Disease Biology, Faculty of Health and Medical Sciences and Sino-Danish Breast Cancer Research Centre, University of Copenhagen, Dyrlægevej 88, 1., 1870 Frederiksberg C, Denmark.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr400457uDOI Listing
September 2013

In vivo phosphoproteomics analysis reveals the cardiac targets of β-adrenergic receptor signaling.

Sci Signal 2013 Jun 4;6(278):rs11. Epub 2013 Jun 4.

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark.

β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5'-monophosphate-activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine monophosphate-dependent protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase type II). We found specific regulation of phosphorylation sites on six ion channels and transporters that mediate increased ion fluxes at higher heart rates, and we showed that phosphorylation of one of these, Ser(92) of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility, suggesting new potential targets for the treatment of heart disease and hypertension.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.2003506DOI Listing
June 2013

The 3rd DBCLS BioHackathon: improving life science data integration with Semantic Web technologies.

J Biomed Semantics 2013 Feb 11;4(1). Epub 2013 Feb 11.

Database Center for Life Science, Research Organization of Information and Systems, 2-11-16, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Background: BioHackathon 2010 was the third in a series of meetings hosted by the Database Center for Life Sciences (DBCLS) in Tokyo, Japan. The overall goal of the BioHackathon series is to improve the quality and accessibility of life science research data on the Web by bringing together representatives from public databases, analytical tool providers, and cyber-infrastructure researchers to jointly tackle important challenges in the area of in silico biological research.

Results: The theme of BioHackathon 2010 was the 'Semantic Web', and all attendees gathered with the shared goal of producing Semantic Web data from their respective resources, and/or consuming or interacting those data using their tools and interfaces. We discussed on topics including guidelines for designing semantic data and interoperability of resources. We consequently developed tools and clients for analysis and visualization.

Conclusion: We provide a meeting report from BioHackathon 2010, in which we describe the discussions, decisions, and breakthroughs made as we moved towards compliance with Semantic Web technologies - from source provider, through middleware, to the end-consumer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/2041-1480-4-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598643PMC
February 2013

DistiLD Database: diseases and traits in linkage disequilibrium blocks.

Nucleic Acids Res 2012 Jan 3;40(Database issue):D1036-40. Epub 2011 Nov 3.

Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Genome-wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) associated with the risk of hundreds of diseases. However, there is currently no database that enables non-specialists to answer the following simple questions: which SNPs associated with diseases are in linkage disequilibrium (LD) with a gene of interest? Which chromosomal regions have been associated with a given disease, and which are the potentially causal genes in each region? To answer these questions, we use data from the HapMap Project to partition each chromosome into so-called LD blocks, so that SNPs in LD with each other are preferentially in the same block, whereas SNPs not in LD are in different blocks. By projecting SNPs and genes onto LD blocks, the DistiLD database aims to increase usage of existing GWAS results by making it easy to query and visualize disease-associated SNPs and genes in their chromosomal context. The database is available at http://distild.jensenlab.org/.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkr899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245128PMC
January 2012

Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation.

Sci Signal 2011 Jul 26;4(183):ra48. Epub 2011 Jul 26.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.

Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines. With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines were significantly more conserved than were nonacetylated lysines. Bioinformatics analysis using Gene Ontology terms suggested that the proteins with conserved acetylation control cellular processes such as protein translation, protein folding, DNA packaging, and mitochondrial metabolism. We found that acetylation of ubiquitin-conjugating E2 enzymes was evolutionarily conserved, and mutation of a conserved acetylation site impaired the function of the human E2 enzyme UBE2D3. This systems-level analysis of comparative posttranslational modification showed that acetylation is an anciently conserved modification and suggests that phosphorylation sites may have evolved faster than acetylation sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.2001902DOI Listing
July 2011

Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level.

Mol Cell Proteomics 2011 Mar 7;10(3):M110.003590. Epub 2010 Dec 7.

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen.

The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.M110.003590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047152PMC
March 2011