Publications by authors named "Jesper V Olsen"

181 Publications

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures.

Nat Commun 2021 05 4;12(1):2507. Epub 2021 May 4.

Proteomics Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.
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http://dx.doi.org/10.1038/s41467-021-22787-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097059PMC
May 2021

Proteomic investigation of Cbl and Cbl-b in neuroblastoma cell differentiation highlights roles for SHP-2 and CDK16.

iScience 2021 Apr 17;24(4):102321. Epub 2021 Mar 17.

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

Neuroblastoma is a highly heterogeneous embryonal solid tumor of the sympathetic nervous system. As some tumors can be treated to undergo differentiation, investigating this process can guide differentiation-based therapies of neuroblastoma. Here, we studied the role of E3 ubiquitin ligases Cbl and Cbl-b in regulation of long-term signaling responses associated with extracellular signal-regulated kinase phosphorylation and neurite outgrowth, a morphological marker of neuroblastoma cell differentiation. Using quantitative mass spectrometry (MS)-based proteomics, we analyzed how the neuroblastoma cell line proteome, phosphoproteome, and ubiquitylome were affected by Cbl and Cbl-b depletion. To quantitatively assess neurite outgrowth, we developed a high-throughput microscopy assay that was applied in combination with inhibitor studies to pinpoint signaling underlying neurite outgrowth and to functionally validate proteins identified in the MS data sets. Using this combined approach, we identified a role for SHP-2 and CDK16 in Cbl/Cbl-b-dependent regulation of extracellular signal-regulated kinase phosphorylation and neurite outgrowth, highlighting their involvement in neuroblastoma cell differentiation.
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http://dx.doi.org/10.1016/j.isci.2021.102321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050387PMC
April 2021

Quantitative proteome comparison of human hearts with those of model organisms.

PLoS Biol 2021 Apr 19;19(4):e3001144. Epub 2021 Apr 19.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Delineating human cardiac pathologies and their basic molecular mechanisms relies on research conducted in model organisms. Yet translating findings from preclinical models to humans present a significant challenge, in part due to differences in cardiac protein expression between humans and model organisms. Proteins immediately determine cellular function, yet their large-scale investigation in hearts has lagged behind those of genes and transcripts. Here, we set out to bridge this knowledge gap: By analyzing protein profiles in humans and commonly used model organisms across cardiac chambers, we determine their commonalities and regional differences. We analyzed cardiac tissue from each chamber of human, pig, horse, rat, mouse, and zebrafish in biological replicates. Using mass spectrometry-based proteomics workflows, we measured and evaluated the abundance of approximately 7,000 proteins in each species. The resulting knowledgebase of cardiac protein signatures is accessible through an online database: atlas.cardiacproteomics.com. Our combined analysis allows for quantitative evaluation of protein abundances across cardiac chambers, as well as comparisons of cardiac protein profiles across model organisms. Up to a quarter of proteins with differential abundances between atria and ventricles showed opposite chamber-specific enrichment between species; these included numerous proteins implicated in cardiac disease. The generated proteomics resource facilitates translational prospects of cardiac studies from model organisms to humans by comparisons of disease-linked protein networks across species.
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http://dx.doi.org/10.1371/journal.pbio.3001144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084454PMC
April 2021

Faecal proteomics as a novel method to study mammalian behaviour and physiology.

Mol Ecol Resour 2021 Aug 8;21(6):1808-1819. Epub 2021 Apr 8.

Evolutionary Genomics Section, The Globe Institute, University of Copenhagen, Copenhagen, Denmark.

Mammalian faeces can be collected noninvasively during field research and provide valuable information on the ecology and evolution of the source individuals. Undigested food remains, genome/metagenome, steroid hormones, and stable isotopes obtained from faecal samples provide evidence on diet, host/symbiont genetics, and physiological status of the individuals. However, proteins in mammalian faeces have hardly been studied, which hinders the molecular investigations into the behaviour and physiology of the source individuals. Here, we apply mass spectrometry-based proteomics to faecal samples (n = 10), collected from infant, juvenile, and adult captive Japanese macaques (Macaca fuscata), to describe the proteomes of the source individual, of the food it consumed, and its intestinal microbes. The results show that faecal proteomics is a useful method to: (i) investigate dietary changes along with breastfeeding and weaning, (ii) reveal the taxonomic and histological origin of the food items consumed, and (iii) estimate physiological status inside intestinal tracts. These types of insights are difficult or impossible to obtain through other molecular approaches. Most mammalian species are facing extinction risk and there is an urgent need to obtain knowledge on their ecology and evolution for better conservation strategy. The faecal proteomics framework we present here is easily applicable to wild settings and other mammalian species, and provides direct evidence of their behaviour and physiology.
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http://dx.doi.org/10.1111/1755-0998.13380DOI Listing
August 2021

Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function.

Nucleic Acids Res 2021 04;49(6):3185-3203

Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316 Oslo, Norway.

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.
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http://dx.doi.org/10.1093/nar/gkab088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034639PMC
April 2021

The methyltransferase METTL9 mediates pervasive 1-methylhistidine modification in mammalian proteomes.

Nat Commun 2021 02 9;12(1):891. Epub 2021 Feb 9.

Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, 0316, Oslo, Norway.

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
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http://dx.doi.org/10.1038/s41467-020-20670-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873184PMC
February 2021

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Mol Cell 2021 02;81(3):426-441.e8

Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.
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http://dx.doi.org/10.1016/j.molcel.2021.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211091PMC
February 2021

Causal integration of multi-omics data with prior knowledge to generate mechanistic hypotheses.

Mol Syst Biol 2021 01;17(1):e9730

Faculty of Medicine, and Heidelberg University Hospital, Institute for Computational Biomedicine, Heidelberg University, Heidelberg, Germany.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.
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http://dx.doi.org/10.15252/msb.20209730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838823PMC
January 2021

FRMD6 has tumor suppressor functions in prostate cancer.

Oncogene 2021 01 28;40(4):763-776. Epub 2020 Nov 28.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.

Available tools for prostate cancer (PC) prognosis are suboptimal but may be improved by better knowledge about genes driving tumor aggressiveness. Here, we identified FRMD6 (FERM domain-containing protein 6) as an aberrantly hypermethylated and significantly downregulated gene in PC. Low FRMD6 expression was associated with postoperative biochemical recurrence in two large PC patient cohorts. In overexpression and CRISPR/Cas9 knockout experiments in PC cell lines, FRMD6 inhibited viability, proliferation, cell cycle progression, colony formation, 3D spheroid growth, and tumor xenograft growth in mice. Transcriptomic, proteomic, and phospho-proteomic profiling revealed enrichment of Hippo/YAP and c-MYC signaling upon FRMD6 knockout. Connectivity Map analysis and drug repurposing experiments identified pyroxamide as a new potential therapy for FRMD6 deficient PC cells. Finally, we established orthotropic Frmd6 and Pten, or Pten only (control) knockout in the ROSA26 mouse prostate. After 12 weeks, Frmd6/Pten double knockouts presented high-grade prostatic intraepithelial neoplasia (HG-PIN) and hyperproliferation, while Pten single-knockouts developed only regular PIN lesions and displayed lower proliferation. In conclusion, FRMD6 was identified as a novel tumor suppressor gene and prognostic biomarker candidate in PC.
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http://dx.doi.org/10.1038/s41388-020-01548-wDOI Listing
January 2021

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping.

Mol Cell Proteomics 2020 12 5;19(12):2139-2157. Epub 2020 Oct 5.

Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.
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http://dx.doi.org/10.1074/mcp.TIR120.002129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710147PMC
December 2020

Multi-omic detection of in archaeological human dental calculus.

Philos Trans R Soc Lond B Biol Sci 2020 11 5;375(1812):20190584. Epub 2020 Oct 5.

Section for Evolutionary Genomics, GLOBE Institute, Faculty of Health and Medical Sciences, University of Stavanger, Stavanger, Norway.

Mineralized dental plaque (calculus) has proven to be an excellent source of ancient biomolecules. Here we present a genome (6.6-fold), the causative agent of leprosy, recovered via shotgun sequencing of sixteenth-century human dental calculus from an individual from Trondheim, Norway. When phylogenetically placed, this genome falls in branch 3I among the diversity of other contemporary ancient strains from Northern Europe. Moreover, ancient mycobacterial peptides were retrieved via mass spectrometry-based proteomics, further validating the presence of the pathogen. can readily be detected in the oral cavity and associated mucosal membranes, which likely contributed to it being incorporated into this individual's dental calculus. This individual showed some possible, but not definitive, evidence of skeletal lesions associated with early-stage leprosy. This study is the first known example of successful multi-omics retrieval of from archaeological dental calculus. Furthermore, we offer new insights into dental calculus as an alternative sample source to bones or teeth for detecting and molecularly characterizing in individuals from the archaeological record. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.
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http://dx.doi.org/10.1098/rstb.2019.0584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702802PMC
November 2020

Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation.

Biomedicines 2020 Jul 25;8(8). Epub 2020 Jul 25.

Department of Clinical Biochemistry, Aalborg University Hospital, DK-9000 Aalborg, Denmark.

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells' biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.
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http://dx.doi.org/10.3390/biomedicines8080246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459681PMC
July 2020

Effects of active farnesoid X receptor on GLUTag enteroendocrine L cells.

Mol Cell Endocrinol 2020 11 20;517:110923. Epub 2020 Jul 20.

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200, Copenhagen, Denmark; Dept. of Health Technology, Technical University of Denmark, DK-2800, Lyngby, Denmark. Electronic address:

Activated transcription factor (TF) farnesoid X receptor (FXR) represses glucagon-like peptide-1 (GLP-1) secretion in enteroendocrine L cells. This, in turn, reduces insulin secretion, which is triggered when β cells bind GLP-1. Preventing FXR activation could boost GLP-1 production and insulin secretion. Yet, FXR's broader role in L cell biology still lacks understanding. Here, we show that FXR is a multifaceted TF in L cells using proteomics and gene expression data generated on GLUTag L cells. Most striking, 252 proteins regulated upon glucose stimulation have their abundances neutralized upon FXR activation. Mitochondrial repression or glucose import block are likely mechanisms of this. Further, FXR physically targets bile acid metabolism proteins, growth factors and other TFs, regulates ChREBP, while extensive text-mining found 30 FXR-regulated proteins to be well-known in L cell biology. Taken together, this outlines FXR as a powerful TF, where GLP-1 secretion block is just one of many downstream effects.
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http://dx.doi.org/10.1016/j.mce.2020.110923DOI Listing
November 2020

Multi-protease analysis of Pleistocene bone proteomes.

J Proteomics 2020 09 9;228:103889. Epub 2020 Jul 9.

Evolutionary Genomics Section, Globe Institute, University of Copenhagen, Copenhagen, Denmark; Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. Electronic address:

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics. SIGNIFICANCE: Maximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.
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http://dx.doi.org/10.1016/j.jprot.2020.103889DOI Listing
September 2020

The dental proteome of Homo antecessor.

Nature 2020 04 1;580(7802):235-238. Epub 2020 Apr 1.

Evolutionary Genomics Section, Globe Institute, University of Copenhagen, Copenhagen, Denmark.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain) and Homo erectus from Dmanisi (Georgia), two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.
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http://dx.doi.org/10.1038/s41586-020-2153-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582224PMC
April 2020

Proteome and Phosphoproteome Changes Associated with Prognosis in Acute Myeloid Leukemia.

Cancers (Basel) 2020 Mar 17;12(3). Epub 2020 Mar 17.

Department of Clinical Science, University of Bergen, 5021 Bergen, Norway.

Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for the majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has become of major clinical interest in AML. We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a five-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells. This suggests that therapy against the upregulated kinases could also target the factors inducing their upregulation rather than their activity. This study, therefore, presents markers that could help predict AML relapse and direct therapeutic strategies.
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http://dx.doi.org/10.3390/cancers12030709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140113PMC
March 2020

Quantitative phosphoproteomics to unravel the cellular response to chemical stressors with different modes of action.

Arch Toxicol 2020 05 18;94(5):1655-1671. Epub 2020 Mar 18.

Department of Human Genetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.

Damage to cellular macromolecules and organelles by chemical exposure evokes activation of various stress response pathways. To what extent different chemical stressors activate common and stressor-specific pathways is largely unknown. Here, we used quantitative phosphoproteomics to compare the signaling events induced by four stressors with different modes of action: the DNA damaging agent: cisplatin (CDDP), the topoisomerase II inhibitor: etoposide (ETO), the pro-oxidant: diethyl maleate (DEM) and the immunosuppressant: cyclosporine A (CsA) administered at an equitoxic dose to mouse embryonic stem cells. We observed major differences between the stressors in the number and identity of responsive phosphosites and the amplitude of phosphorylation. Kinase motif and pathway analyses indicated that the DNA damage response (DDR) activation by CDDP occurs predominantly through the replication-stress-related Atr kinase, whereas ETO triggers the DDR through Atr as well as the DNA double-strand-break-associated Atm kinase. CsA shares with ETO activation of CK2 kinase. Congruent with their known modes of action, CsA-mediated signaling is related to down-regulation of pathways that control hematopoietic differentiation and immunity, whereas oxidative stress is the most prominent initiator of DEM-modulated stress signaling. This study shows that even at equitoxic doses, different stressors induce distinctive and complex phosphorylation signaling cascades.
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http://dx.doi.org/10.1007/s00204-020-02712-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261734PMC
May 2020

Deciphering the human phosphoproteome.

Nat Biotechnol 2020 03;38(3):285-286

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

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http://dx.doi.org/10.1038/s41587-020-0441-3DOI Listing
March 2020

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients.

Mol Cell Proteomics 2020 04 12;19(4):716-729. Epub 2020 Feb 12.

The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DENMARK. Electronic address:

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.
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http://dx.doi.org/10.1074/mcp.TIR119.001906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124470PMC
April 2020

Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries.

Nat Commun 2020 02 7;11(1):787. Epub 2020 Feb 7.

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

Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation.
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http://dx.doi.org/10.1038/s41467-020-14609-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005859PMC
February 2020

Molecular Basis of the Mechanisms Controlling MASTL.

Mol Cell Proteomics 2020 02 18;19(2):326-343. Epub 2019 Dec 18.

The Novo Nordisk Foundation Center for Protein Research, Protein Structure & Function Programme, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.
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http://dx.doi.org/10.1074/mcp.RA119.001879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000116PMC
February 2020

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA.

Nucleic Acids Res 2020 01;48(2):830-846

Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo 0316, Norway.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.
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http://dx.doi.org/10.1093/nar/gkz1147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954407PMC
January 2020

Enamel proteome shows that Gigantopithecus was an early diverging pongine.

Nature 2019 12 13;576(7786):262-265. Epub 2019 Nov 13.

Evolutionary Genomics Section, Globe Institute, University of Copenhagen, Copenhagen, Denmark.

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information.
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http://dx.doi.org/10.1038/s41586-019-1728-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908745PMC
December 2019

Dynamic lineage priming is driven via direct enhancer regulation by ERK.

Nature 2019 11 6;575(7782):355-360. Epub 2019 Nov 6.

The Novo Nordisk Foundation Center for Stem Cell Biology, Copenhagen, Denmark.

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.
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http://dx.doi.org/10.1038/s41586-019-1732-zDOI Listing
November 2019

Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Cell 2019 10;179(2):543-560.e26

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

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.
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http://dx.doi.org/10.1016/j.cell.2019.09.008DOI Listing
October 2019

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

Nature 2019 10 11;574(7776):103-107. Epub 2019 Sep 11.

Evolutionary Genomics Section, Globe Institute, University of Copenhagen, Copenhagen, Denmark.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa. However, the irreversible post-mortem degradation of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr). By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I, and suggested the presence of protein residues in fossils of the Cretaceous period-although with limited phylogenetic use. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia). Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.
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http://dx.doi.org/10.1038/s41586-019-1555-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894936PMC
October 2019

Palaeoproteomic identification of breast milk protein residues from the archaeological skeletal remains of a neonatal dog.

Sci Rep 2019 09 6;9(1):12841. Epub 2019 Sep 6.

Section for Evolutionary Genomics, The GLOBE Institute, University of Copenhagen, Øster Voldgade 5-7, 1350, Copenhagen, Denmark.

Accurate postmortem estimation of breastfeeding status for archaeological or forensic neonatal remains is difficult. Confident identification of milk-specific proteins associated with these remains would provide direct evidence of breast milk consumption. We used liquid chromatography coupled to tandem mass spectrometry (MS) to confidently identify beta-lactoglobulin-1 (LGB1) and whey acidic protein (WAP), major whey proteins associated with a neonatal dog (Canis lupus familiaris) skeleton (430-960 cal AD), from an archaeological site in Hokkaido, Japan. The age at death of the individual was estimated to be approximately two weeks after birth. Protein residues extracted from rib and vertebra fragments were analyzed and identified by matching tandem MS spectra against the dog reference proteome. A total of 200 dog protein groups were detected and at least one peptide from canine LGB1 and two peptides from canine WAP were confidently identified. These milk proteins most probably originated from the mother's breast milk, ingested by the neonate just before it died. We suggest the milk diffused outside the digestive apparatus during decomposition, and, by being absorbed into the bones, it partially preserved. The result of this study suggests that proteomic analysis can be used for postmortem reconstruction of the breastfeeding status at the time of death of neonatal mammalian, by analyzing their skeletal archaeological remains. This method is also applicable to forensic and wildlife studies.
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http://dx.doi.org/10.1038/s41598-019-49183-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731306PMC
September 2019

Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability.

Nucleic Acids Res 2019 Sep;47(15):8332

Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Copenhagen 2200, Denmark.

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http://dx.doi.org/10.1093/nar/gkz591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736055PMC
September 2019

Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability.

Nucleic Acids Res 2019 09;47(15):8004-8018

Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Copenhagen 2200, Denmark.

Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.
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http://dx.doi.org/10.1093/nar/gkz510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735892PMC
September 2019