Publications by authors named "Michael L Nielsen"

83 Publications

Identification of Protein Direct Interactome with Genetic Code Expansion and Search Engine OpenUaa.

Adv Biol (Weinh) 2021 Mar 8;5(3):e2000308. Epub 2021 Feb 8.

MOE Laboratory of Biosystem Homeostasis and Protection and Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China.

Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage. Integrating GECX with OpenUaa, the direct interactome of thioredoxin is identified in Escherichia coli cells, yielding 289 crosslinked peptides and corresponding to 205 direct binding protein of thioredoxin. These identified direct binders provide evidence for thioredoxin's regulation of redox state and mitochondria energy metabolism. When identifying endogenous conjugation of small ubiquitin-like modifier (SUMO), OpenUaa also markedly improves coverage of SUMOylated peptides by ≈92%, revealing new SUMO targets. GECX-OpenUaa will enable efficient identification of direct interactomes of various proteins in live cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adbi.202000308DOI Listing
March 2021

DIVERSIFY: A Fungal Multispecies Gene Expression Platform.

ACS Synth Biol 2021 03 2;10(3):579-588. Epub 2021 Mar 2.

Eukaryotic Molecular Cell Biology, Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kongens Lyngby, Denmark.

Recent sequencing of numerous fungal species revealed large repertoires of putative biotechnologically relevant genes and secondary metabolite gene clusters. However, often the commercial potential of these species is impeded by difficulties to predict host physiological and metabolic compatibility with a given product, and lack of adequate genetic tools. Consequently, most heterologous production is performed in standard hosts where genetic tools and experience are in place. However, these species may not be suitable for all products. To increase chances of successful heterologous production, we have created a flexible platform, DIVERSIFY, for multispecies heterologous gene expression. This reduces the workload to construction of a single gene expression cassette, used to transform all DIVERSIFY strains in order to identify the optimal cell factory host. As proof of principle of the DIVERSIFY concept, we present the first version of our platform, DIVERSIFY 1.0, which we have successfully used for the production of three proteins and a metabolite in four different Aspergilli species, and for the identification of the best producer for each of the products. Moreover, we show that DIVERSIFY 1.0 is compatible with marker-free gene targeting induced by the CRISPR nucleases Cas9 and MAD7.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssynbio.0c00587DOI Listing
March 2021

MaxQuant.Live Enables Enhanced Selectivity and Identification of Peptides Modified by Endogenous SUMO and Ubiquitin.

J Proteome Res 2021 Apr 4;20(4):2042-2055. Epub 2021 Feb 4.

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

Small ubiquitin-like modifiers (SUMO) and ubiquitin are frequent post-translational modifications of proteins that play pivotal roles in all cellular processes. We previously reported mass spectrometry-based proteomics methods that enable profiling of lysines modified by endogenous SUMO or ubiquitin in an unbiased manner, without the need for genetic engineering. Here we investigated the applicability of precursor mass filtering enabled by MaxQuant.Live to our SUMO and ubiquitin proteomics workflows, which efficiently avoided sequencing of precursors too small to be modified but otherwise indistinguishable by mass-to-charge ratio. Using precursor mass filtering, we achieved a much higher selectivity of modified peptides, ultimately resulting in up to 30% more SUMO and ubiquitin sites identified from replicate samples. Real-time exclusion of unmodified peptides by MQL resulted in 90% SUMO-modified precursor selectivity from a 25% pure sample, demonstrating great applicability for digging deeper into ubiquitin-like modificomes. We adapted the precursor mass filtering strategy to the new Exploris 480 mass spectrometer, achieving comparable gains in SUMO precursor selectivity and identification rates. Collectively, precursor mass filtering via MQL significantly increased identification rates of SUMO- and ubiquitin-modified peptides from the exact same samples, without the requirement for prior knowledge or spectral libraries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.0c00892DOI Listing
April 2021

Chemical genetics and proteome-wide site mapping reveal cysteine MARylation by PARP-7 on immune-relevant protein targets.

Elife 2021 Jan 21;10. Epub 2021 Jan 21.

Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, United States.

Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.60480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880690PMC
January 2021

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Mol Cell 2021 02 14;81(3):442-458.e9. Epub 2020 Dec 14.

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864614PMC
February 2021

Mapping Physiological ADP-Ribosylation Using Activated Ion Electron Transfer Dissociation.

Cell Rep 2020 09;32(12):108176

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

ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and electron-transfer higher-energy collisional dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508052PMC
September 2020

Extensive SUMO Modification of Repressive Chromatin Factors Distinguishes Pluripotent from Somatic Cells.

Cell Rep 2020 09;32(11):108146

Nuclear Organization and Oncogenesis Unit, Institut Pasteur, Équipe Labellisée Ligue Nationale Contre le Cancer, 75015 Paris, France; INSERM, U993, 75015 Paris, France. Electronic address:

Post-translational modification by SUMO is a key regulator of cell identity. In mouse embryonic fibroblasts (MEFs), SUMO impedes reprogramming to pluripotency, while in embryonic stem cells (ESCs), it represses the emergence of totipotent-like cells, suggesting that SUMO targets distinct substrates to preserve somatic and pluripotent states. Using MS-based proteomics, we show that the composition of endogenous SUMOylomes differs dramatically between MEFs and ESCs. In MEFs, SUMO2/3 targets proteins associated with canonical SUMO functions, such as splicing, and transcriptional regulators driving somatic enhancer selection. In contrast, in ESCs, SUMO2/3 primarily modifies highly interconnected repressive chromatin complexes, thereby preventing chromatin opening and transitioning to totipotent-like states. We also characterize several SUMO-modified pluripotency factors and show that SUMOylation of Dppa2 and Dppa4 impedes the conversion to 2-cell-embryo-like states. Altogether, we propose that rewiring the repertoire of SUMO target networks is a major driver of cell fate decision during embryonic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495044PMC
September 2020

Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites.

J Biol Chem 2020 06 29;295(25):8350-8362. Epub 2020 Apr 29.

Ubiquitin Signaling Group, Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen, Denmark

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307195PMC
June 2020

Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation.

Nat Commun 2020 01 8;11(1):123. Epub 2020 Jan 8.

Department of Gynecology, University of Zurich, Wagistrasse 14, CH-8952, Schlieren, Switzerland.

Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13981-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949271PMC
January 2020

Regulation of GLI1 by cis DNA elements and epigenetic marks.

DNA Repair (Amst) 2019 07 2;79:10-21. Epub 2019 May 2.

Developmental Biology Program, Stanley Manne Children's Research Institute, Department of Pediatrics, Ann & Robert H. Lurie Children's Hospital, Northwestern University Feinberg School of Medicine, USA. Electronic address:

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5' exons and large introns 5' of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2019.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570425PMC
July 2019

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells.

Exp Cell Res 2019 08 9;381(1):129-138. Epub 2019 May 9.

Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, Stockholm, Sweden. Electronic address:

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role for Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at gene regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interacts with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but could rather regulate other processes in the nucleoplasm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2019.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994247PMC
August 2019

PAD2-Mediated Citrullination Contributes to Efficient Oligodendrocyte Differentiation and Myelination.

Cell Rep 2019 04;27(4):1090-1102.e10

Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden; Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, 171 77 Stockholm, Sweden. Electronic address:

Citrullination, the deimination of peptidylarginine residues into peptidylcitrulline, has been implicated in the etiology of several diseases. In multiple sclerosis, citrullination is thought to be a major driver of pathology through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show that citrullination by peptidylarginine deiminase 2 (PAD2) contributes to normal oligodendrocyte differentiation, myelination, and motor function. We identify several targets for PAD2, including myelin and chromatin-related proteins, implicating PAD2 in epigenomic regulation. Accordingly, we observe that PAD2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PAD2 display motor dysfunction and a decreased number of myelinated axons in the corpus callosum. We conclude that citrullination contributes to proper oligodendrocyte lineage progression and myelination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.03.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486480PMC
April 2019

Citrullination of HP1γ chromodomain affects association with chromatin.

Epigenetics Chromatin 2019 Apr 2;12(1):21. Epub 2019 Apr 2.

The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

Background: Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance.

Results: We find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation. Peptidylarginine deiminase 4, a known regulator of pluripotency, citrullinates HP1γ in vitro. This requires R38 and R39 within the HP1γ chromodomain, and the catalytic activity is enhanced by trimethylated H3K9 (H3K9me3) peptides. Mutation of R38 and R39, designed to mimic citrullination, affects HP1γ binding to H3K9me3-containing peptides. Using live-cell single-particle tracking, we demonstrate that R38 and R39 are important for HP1γ binding to chromatin in vivo. Furthermore, their mutation reduces the residence time of HP1γ on chromatin in differentiating mESCs.

Conclusion: Citrullination is a novel post-translational modification of the structural heterochromatin protein HP1γ in mESCs that is dynamically regulated during mESC differentiation. The citrullinated residues lie within the HP1γ chromodomain and are important for H3K9me3 binding in vitro and chromatin association in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13072-019-0265-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444592PMC
April 2019

SUMOylation promotes protective responses to DNA-protein crosslinks.

EMBO J 2019 04 26;38(8). Epub 2019 Mar 26.

Ubiquitin Signaling Group, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, the mechanisms by which cells respond to and overcome DPCs remain incompletely understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-regulated manner. Here, we show that chemically induced and defined enzymatic DPCs trigger potent chromatin SUMOylation responses targeting the crosslinked proteins and associated factors. Consequently, inhibiting SUMOylation compromises DPC clearance and cellular fitness. We demonstrate that ACRC/GCNA family SprT proteases interact with SUMO and establish important physiological roles of GCNA-1 and SUMOylation in promoting germ cell and embryonic survival upon DPC formation. Our findings provide first global insights into signaling responses to DPCs and reveal an evolutionarily conserved function of SUMOylation in facilitating responses to these lesions in metazoans that may complement replication-coupled DPC resolution processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019101496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463212PMC
April 2019

An Advanced Strategy for Comprehensive Profiling of ADP-ribosylation Sites Using Mass Spectrometry-based Proteomics.

Mol Cell Proteomics 2019 05 23;18(5):1010-1026. Epub 2019 Feb 23.

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

ADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. Although ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.TIR119.001315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495254PMC
May 2019

Systems-wide Analysis of Serine ADP-Ribosylation Reveals Widespread Occurrence and Site-Specific Overlap with Phosphorylation.

Cell Rep 2018 08;24(9):2493-2505.e4

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

ADP-ribosylation (ADPr) is a reversible posttranslational modification involved in a range of cellular processes. Here, we report system-wide identification of serine ADPr in human cells upon oxidative stress. High-resolution mass spectrometry and unrestricted data processing confirm that serine residues are the major target of ADPr in HeLa cells. Proteome-wide analysis identifies 3,090 serine ADPr sites, with 97% of acceptor sites modulating more than 2-fold upon oxidative stress, while treatment with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib abrogates this induction. Serine ADPr predominantly targets nuclear proteins, while structural-predictive analyses reveal that serine ADPr preferentially targets disordered protein regions. The identified ADP-ribosylated serines significantly overlap with known phosphorylated serines, and large-scale phosphoproteomics analysis provides evidence for site-specific crosstalk between serine ADPr and phosphorylation. Collectively, we demonstrate that serine ADPr is a widespread modification and a major nuclear signaling response to oxidative stress, with a regulatory scope comparable to other extensive posttranslational modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.07.083DOI Listing
August 2018

Site-specific characterization of endogenous SUMOylation across species and organs.

Nat Commun 2018 06 25;9(1):2456. Epub 2018 Jun 25.

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

Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO2/3 has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2. Using a peptide-level immunoprecipitation enrichment strategy, we identify 14,869 endogenous SUMO2/3 sites in human cells during heat stress and proteasomal inhibition, and quantitatively map 1963 SUMO sites across eight mouse tissues. Characterization of the SUMO equilibrium highlights striking differences in SUMO metabolism between cultured cancer cells and normal tissues. Targeting preferences of SUMO2/3 vary across different organ types, coinciding with markedly differential SUMOylation states of all enzymes involved in the SUMO conjugation cascade. Collectively, our systemic investigation details the SUMOylation architecture across species and organs and provides a resource of endogenous SUMOylation sites on factors important in organ-specific functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-04957-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018634PMC
June 2018

Integrative Characterization of the R6/2 Mouse Model of Huntington's Disease Reveals Dysfunctional Astrocyte Metabolism.

Cell Rep 2018 05;23(7):2211-2224

Proteomics Program, The Novo Nordisk Foundation Centre for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Huntington's disease is a fatal neurodegenerative disease, where dysfunction and loss of striatal and cortical neurons are central to the pathogenesis of the disease. Here, we integrated quantitative studies to investigate the underlying mechanisms behind HD pathology in a systems-wide manner. To this end, we used state-of-the-art mass spectrometry to establish a spatial brain proteome from late-stage R6/2 mice and compared this with wild-type littermates. We observed altered expression of proteins in pathways related to energy metabolism, synapse function, and neurotransmitter homeostasis. To support these findings, metabolic C labeling studies confirmed a compromised astrocytic metabolism and regulation of glutamate-GABA-glutamine cycling, resulting in impaired release of glutamine and GABA synthesis. In recent years, increasing attention has been focused on the role of astrocytes in HD, and our data support that therapeutic strategies to improve astrocytic glutamine homeostasis may help ameliorate symptoms in HD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.04.052DOI Listing
May 2018

Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography-Tandem Mass Spectrometry.

Methods Mol Biol 2017 ;1608:149-162

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

ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-6993-7_11DOI Listing
April 2018

Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification.

J Proteome Res 2017 08 7;16(8):2762-2772. Epub 2017 Jul 7.

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

RNA-binding proteins (RBPs) allow cells to carry out pre-RNA processing and post-transcriptional regulation of gene expression, and aberrations in RBP functions have been linked to many diseases, including neurological disorders and cancer. Human cells encode thousands of RNA-binding proteins with unique RNA-binding properties. These properties are regulated through modularity of a large variety of RNA-binding domains, rendering RNA-protein interactions difficult to study. Recently, the introduction of proteomics methods has provided novel insights into RNA-binding proteins at a systems level. However, determining the exact protein sequence regions that interact with RNA remains challenging and laborious, especially considering that many RBPs lack canonical RNA-binding domains. Here we describe a streamlined proteomic workflow called peptide cross-linking and affinity purification (pCLAP) that allows rapid characterization of RNA-binding regions in proteins. pCLAP is based upon the combined use of UV cross-linking and enzymatic digestion of RNA-bound proteins followed by single-shot mass spectrometric analysis. To benchmark our method, we identified the binding regions for polyadenylated RNA-binding proteins in HEK293 cells, allowing us to map the mRNA interaction regions of more than 1000 RBPs with very high reproducibility from replicate single-shot analyses. Our results show specific enrichment of many known RNA-binding regions on many known RNA-binding proteins, confirming the specificity of our approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.7b00042DOI Listing
August 2017

An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes.

Cell Syst 2017 06 7;4(6):587-599.e4. Epub 2017 Jun 7.

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

This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including ∼7,000 N-acetylation sites and ∼10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cels.2017.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493283PMC
June 2017

Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature.

J Proteome Res 2017 06 7;16(6):2307-2317. Epub 2017 Apr 7.

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

Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than -2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the coelution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.6b01055DOI Listing
June 2017

Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation.

Nat Struct Mol Biol 2017 03 23;24(3):325-336. Epub 2017 Jan 23.

Department of Proteomics, Novo Nordisk Foundation Center for Protein Research (NNF-CPR), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Small ubiquitin-like modifiers (SUMOs) are post-translational modifications (PTMs) that regulate nuclear cellular processes. Here we used an augmented K0-SUMO proteomics strategy to identify 40,765 SUMO acceptor sites and quantify their fractional contribution for 6,747 human proteins. Structural-predictive analyses revealed that lysines residing in disordered regions are preferentially targeted by SUMO, in notable contrast to other widespread lysine modifications. In our data set, we identified 807 SUMOylated peptides that were co-modified by phosphorylation, along with dozens of SUMOylated peptides that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between SUMOylation and other major PTMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.3366DOI Listing
March 2017

Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue.

Nat Commun 2016 Sep 30;7:12917. Epub 2016 Sep 30.

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

Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms12917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056437PMC
September 2016

Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

Sci Signal 2016 08 30;9(443):rs9. Epub 2016 Aug 30.

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

The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.aaf7329DOI Listing
August 2016

Large-Scale Identification of the Arginine Methylome by Mass Spectrometry.

Curr Protoc Protein Sci 2015 Nov 2;82:24.7.1-24.7.17. Epub 2015 Nov 2.

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

The attachment of one or more methylation groups to the side chain of arginine residues is a regulatory mechanism for cellular proteins. Recent advances in mass spectrometry-based characterization allow comprehensive identification of arginine methylation sites by peptide-level enrichment strategies. Described in this unit is a 4-day protocol for enrichment of arginine-methylated peptides and subsequent identification of thousands of distinct sites by mass spectrometry. Specifically, the protocol explains step-by-step sample preparation, enrichment using commercially available antibodies, prefractionation using strong cation exchange, and identification using liquid chromatography coupled to tandem mass spectrometry. A strategy for relative quantification is described using stable isotope labeling by amino acids in cell culture (SILAC). Approaches for analysis of arginine methylation site occupancy are also discussed. Collectively, the unit describes the essential parameters required for a successful and comprehensive experiment detailing the arginine methylome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/0471140864.ps2407s82DOI Listing
November 2015

Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p.

Nat Commun 2015 Jul 9;6:7726. Epub 2015 Jul 9.

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

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms8726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510963PMC
July 2015

A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast.

J Biol Chem 2015 Aug 7;290(34):21141-21153. Epub 2015 Jul 7.

Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark. Electronic address:

A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.662312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543670PMC
August 2015

The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage.

Nat Cell Biol 2014 Aug 27;16(8):792-803. Epub 2014 Jul 27.

1] Department of Gynecology, University Hospital and University of Zurich, Wagistrasse 14, CH-8952 Schlieren, Switzerland [2] Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb3007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962910PMC
August 2014