Publications by authors named "Simon Bekker-Jensen"

63 Publications

Ribosomal stress-surveillance: three pathways is a magic number.

Nucleic Acids Res 2020 11;48(19):10648-10661

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

Cells rely on stress response pathways to uphold cellular homeostasis and limit the negative effects of harmful environmental stimuli. The stress- and mitogen-activated protein (MAP) kinases, p38 and JNK, are at the nexus of numerous stress responses, among these the ribotoxic stress response (RSR). Ribosomal impairment is detrimental to cell function as it disrupts protein synthesis, increase inflammatory signaling and, if unresolved, lead to cell death. In this review, we offer a general overview of the three main translation surveillance pathways; the RSR, Ribosome-associated Quality Control (RQC) and the Integrated Stress Response (ISR). We highlight recent advances made in defining activation mechanisms for these pathways and discuss their commonalities and differences. Finally, we reflect on the physiological role of the RSR and consider the therapeutic potential of targeting the sensing kinase ZAKα for treatment of ribotoxin exposure.
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http://dx.doi.org/10.1093/nar/gkaa757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641731PMC
November 2020

ZAKα Recognizes Stalled Ribosomes through Partially Redundant Sensor Domains.

Mol Cell 2020 05 13;78(4):700-713.e7. Epub 2020 Apr 13.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.
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http://dx.doi.org/10.1016/j.molcel.2020.03.021DOI Listing
May 2020

The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3.

J Immunol 2019 10 16;203(8):2291-2300. Epub 2019 Sep 16.

Institute of Cell Biochemistry, Center of Biochemistry, Hannover Medical School, D-30625 Hannover, Germany;

Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38/MAPKAPK2/3-mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow-derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa. The comparisons reveal that MK2/3 are the only LPS-induced kinases for S52 and S178 of TTP and the role of MK2 and MK3 in the regulation of TNF biosynthesis is not restricted to phosphorylation of TTP at S52/S178 but includes independent processes, which could involve other TTP phosphorylations (such as S316) or other substrates of MK2/3 or p38 Furthermore, we found differences in the dependence of various cytokines on the cooperation between MK2/3 deletion and TTP mutation ex vivo. In the cecal ligation and puncture model of systemic inflammation, a dramatic decrease of cytokine production in MK2/3 DKO, TTPaa, and DKO/TTPaa mice compared with wild-type animals is observed, thus confirming the role of the MK2/3/TTP signaling axis in cytokine production also in vivo. These findings improve our understanding of this signaling axis and could be of future relevance in the treatment of inflammation.
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http://dx.doi.org/10.4049/jimmunol.1801221DOI Listing
October 2019

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites.

Nat Commun 2019 09 13;10(1):4176. Epub 2019 Sep 13.

Cell Stress and Survival Unit, Center for Autophagy, Recycling and Disease (CARD), Danish Cancer Society Research Center, Copenhagen, 2100, Denmark.

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.
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http://dx.doi.org/10.1038/s41467-019-12094-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744468PMC
September 2019

Alternative Translation Initiation Generates a Functionally Distinct Isoform of the Stress-Activated Protein Kinase MK2.

Cell Rep 2019 06;27(10):2859-2870.e6

Institute of Cell Biochemistry, Hannover Medical School (MHH), Carl-Neuberg-Str. 1, 30625 Hannover, Germany; Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. Electronic address:

Alternative translation is an important mechanism of post-transcriptional gene regulation leading to the expression of different protein isoforms originating from the same mRNA. Here, we describe an abundant long isoform of the stress/p38-activated protein kinase MK2. This isoform is constitutively translated from an alternative CUG translation initiation start site located in the 5' UTR of its mRNA. The RNA helicase eIF4A1 is needed to ensure translation of the long and the known short isoforms of MK2, of which the molecular properties were determined. Only the short isoform phosphorylated Hsp27 in vivo, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling revealed short-isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains at least one additional phosphorylatable serine in its unique N terminus. In sum, our data reveal a longer isoform of MK2 with distinct physiological properties.
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http://dx.doi.org/10.1016/j.celrep.2019.05.024DOI Listing
June 2019

GIGYF1/2-Driven Cooperation between ZNF598 and TTP in Posttranscriptional Regulation of Inflammatory Signaling.

Cell Rep 2019 03;26(13):3511-3521.e4

Department of Cellular and Molecular Medicine, Center for Healthy Aging, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

Inflammatory signaling is restricted through degradation and the translational repression of cytokine mRNAs. A key factor in this regulation is tristetraprolin (TTP), an RNA-binding protein (RBP) that recruits RNA-destabilizing factors and the translation inhibitory complex 4EHP-GIGYF1/2 to AU-rich element (ARE)-containing mRNAs. Here, we show that the RBP ZNF598 contributes to the same regulatory module in a TTP-like manner. Similar to TTP, ZNF598 harbors three proline-rich motifs that bind the GYF domain of GIGYF1. RNA sequencing experiments showed that ZNF598 is required for the regulation of known TTP targets, including IL-8 and CSF2 mRNA. Furthermore, we demonstrate that ZNF598 binds to IL-8 mRNA, but not TNF mRNA. Collectively, our findings highlight that ZNF598 functions as an RBP that buffers the level of a range of mRNAs. We propose that ZNF598 is a TTP-like factor that can contribute to the regulation of the inflammatory potential of cytokine-producing cells.
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http://dx.doi.org/10.1016/j.celrep.2019.03.006DOI Listing
March 2019

Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation.

Mol Cell Proteomics 2019 05 4;18(5):1027-1035. Epub 2019 Mar 4.

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

Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.
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http://dx.doi.org/10.1074/mcp.TIR118.001270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495262PMC
May 2019

Osmotic Stress Blocks Mobility and Dynamic Regulation of Centriolar Satellites.

Cells 2018 Jun 22;7(7). Epub 2018 Jun 22.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark.

Centriolar satellites (CS) are small proteinaceous granules that cluster around the centrosome and serve as cargo vehicles for centrosomal proteins. It is generally accepted that CS support a number of canonical and specialized centrosome functions. Consequently, these highly dynamic structures are the target of regulation by several cellular signalling pathways. Two decades of research have led to the identification of a large number of molecular components and new biological roles of CS. Here, we summarize the latest advances in the continuous efforts to uncover the compositional, functional, dynamic and regulatory aspects of CS. We also report on our discovery that osmotic stress conditions render CS immobile and insensitive to remodelling. Upon a range of p38-activating stimuli, MK2 phosphorylates the CS component CEP131, resulting in 14-3-3 binding and a block to CS formation. This normally manifests as a rapid cellular depletion of satellites. In the case of osmotic stress, a potent inducer of p38 activity, CS translocation and dissolution is blocked, with the net result that satellites persist in an immobile state directly adjacent to the centrosome. Our results highlight a unique scenario where p38 activation and CS depletion is uncoupled, with potential implications for physiological and pathological osmotic stress responses.
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http://dx.doi.org/10.3390/cells7070065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070812PMC
June 2018

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Nat Commun 2018 03 9;9(1):1017. Epub 2018 Mar 9.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins as primary substrates and 14-3-3 as direct readers of p38-MK2-dependent phosphorylation induced by UV light. Mechanistically, we show that MK2 phosphorylates the RNA-binding subunit of the NELF complex NELFE on Serine 115. NELFE phosphorylation promotes the recruitment of 14-3-3 and rapid dissociation of the NELF complex from chromatin, which is accompanied by RNA polymerase II elongation.
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http://dx.doi.org/10.1038/s41467-018-03417-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845016PMC
March 2018

SCAI promotes DNA double-strand break repair in distinct chromosomal contexts.

Nat Cell Biol 2016 Dec 7;18(12):1357-1366. Epub 2016 Nov 7.

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

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.
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http://dx.doi.org/10.1038/ncb3436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278951PMC
December 2016

Activation of the ATR kinase by the RPA-binding protein ETAA1.

Nat Cell Biol 2016 Nov 10;18(11):1196-1207. Epub 2016 Oct 10.

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

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress.
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http://dx.doi.org/10.1038/ncb3422DOI Listing
November 2016

H4K20me0 marks post-replicative chromatin and recruits the TONSL–MMS22L DNA repair complex.

Nature 2016 06 22;534(7609):714-718. Epub 2016 Jun 22.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSL–MMS22L binds new histones H3–H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.
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http://dx.doi.org/10.1038/nature18312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939875PMC
June 2016

SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling.

PLoS One 2016 25;11(5):e0156081. Epub 2016 May 25.

Department of Medicine, Division of Nephrology, Boston Children's Hospital, Boston, Massachusetts, United States of America.

Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC1), and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14) at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156081PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880186PMC
July 2017

Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers.

Nat Rev Mol Cell Biol 2016 05;17(6):379-94

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate lesion repair and protection of genome integrity in mammalian cells. These advances offer new therapeutic opportunities for diseases linked to genetic instability.
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http://dx.doi.org/10.1038/nrm.2016.58DOI Listing
May 2016

Structural Analysis of a Complex between Small Ubiquitin-like Modifier 1 (SUMO1) and the ZZ Domain of CREB-binding Protein (CBP/p300) Reveals a New Interaction Surface on SUMO.

J Biol Chem 2016 Jun 25;291(24):12658-12672. Epub 2016 Apr 25.

From the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark,; Amgen Inc., Thousand Oaks, California 91320. Electronic address:

We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.
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http://dx.doi.org/10.1074/jbc.M115.711325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933466PMC
June 2016

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress.

J Cell Biol 2016 Jan 28;212(1):63-75. Epub 2015 Dec 28.

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.
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http://dx.doi.org/10.1083/jcb.201506071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700480PMC
January 2016

p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1.

Nat Commun 2015 Nov 30;6:10075. Epub 2015 Nov 30.

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

Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress.
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http://dx.doi.org/10.1038/ncomms10075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674683PMC
November 2015

RNF138 joins the HR team.

Nat Cell Biol 2015 Nov;17(11):1375-7

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.

Two studies show that the E3 ubiquitin ligase RNF138 is recruited to DNA double-strand break sites, where it ubiquitylates key repair factors to promote DNA-end resection and homologous recombination. These findings add insights into the multilayered regulatory mechanisms underlying DNA double-strand break repair pathway choice in mammalian cells.
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http://dx.doi.org/10.1038/ncb3262DOI Listing
November 2015

Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage.

Nature 2015 Nov 21;527(7578):389-93. Epub 2015 Oct 21.

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

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8-UBC13 in DSB signalling and expand the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.
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http://dx.doi.org/10.1038/nature15401DOI Listing
November 2015

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

Science 2015 May 30;348(6234):1253671. Epub 2015 Apr 30.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. Novo Nordisk Foundation Center for Protein Research, Proteomics Program, University of Copenhagen, DK-2200 Copenhagen, Denmark.

DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.
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http://dx.doi.org/10.1126/science.1253671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331883PMC
May 2015

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

EMBO J 2015 May 9;34(10):1385-98. Epub 2015 Apr 9.

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

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.
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http://dx.doi.org/10.15252/embj.201490376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491998PMC
May 2015

Lamin A/C-dependent interaction with 53BP1 promotes cellular responses to DNA damage.

Aging Cell 2015 Apr 23;14(2):162-9. Epub 2015 Jan 23.

School of Biological and Biomedical Sciences, Durham University, Mountjoy Science Park, Durham, DH1 3LE, UK; Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DK-2200, Denmark.

Lamins A/C have been implicated in DNA damage response pathways. We show that the DNA repair protein 53BP1 is a lamin A/C binding protein. In undamaged human dermal fibroblasts (HDF), 53BP1 is a nucleoskeleton protein. 53BP1 binds to lamins A/C via its Tudor domain, and this is abrogated by DNA damage. Lamins A/C regulate 53BP1 levels and consequently lamin A/C-null HDF display a 53BP1 null-like phenotype. Our data favour a model in which lamins A/C maintain a nucleoplasmic pool of 53BP1 in order to facilitate its rapid recruitment to sites of DNA damage and could explain why an absence of lamin A/C accelerates aging.
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http://dx.doi.org/10.1111/acel.12258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364828PMC
April 2015

Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

Mol Cell 2015 Jan 31;57(1):150-64. Epub 2014 Dec 31.

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

We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage.
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http://dx.doi.org/10.1016/j.molcel.2014.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416315PMC
January 2015

Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells.

DNA Repair (Amst) 2015 Jan 25;25:84-96. Epub 2014 Nov 25.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark; Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. Electronic address:

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational repair. We have also shown that deficiency of POLD3 leads to an increase in RPA-bound ssDNA when cells are under replication stress, suggesting that POLD3 plays a role in the cellular response to DNA replication stress. Considering that DNA replication stress is a source of genome instability, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology.
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http://dx.doi.org/10.1016/j.dnarep.2014.10.011DOI Listing
January 2015

Centriolar satellites: key mediators of centrosome functions.

Cell Mol Life Sci 2015 Jan 31;72(1):11-23. Epub 2014 Aug 31.

Faculty of Health Sciences, Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200, Copenhagen N, Denmark.

Centriolar satellites are small, microscopically visible granules that cluster around centrosomes. These structures, which contain numerous proteins directly involved in centrosome maintenance, ciliogenesis, and neurogenesis, have traditionally been viewed as vehicles for protein trafficking towards the centrosome. However, the recent identification of several new centriolar satellite components suggests that this model offers only an incomplete picture of their cellular functions. While the mechanisms controlling centriolar satellite status and function are not yet understood in detail, emerging evidence points to these structures as important hubs for dynamic, multi-faceted regulation in response to a variety of cues. In this review, we summarize the current knowledge of the roles of centriolar satellites in regulating centrosome functions, ciliogenesis, and neurogenesis. We also highlight newly discovered regulatory mechanisms targeting centriolar satellites and their functional status, and we discuss how defects in centriolar satellite components are intimately linked to a wide spectrum of human diseases.
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http://dx.doi.org/10.1007/s00018-014-1711-3DOI Listing
January 2015

UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells.

EMBO Rep 2014 Sep 4;15(9):956-64. Epub 2014 Aug 4.

Ubiquitin Signaling Group, Department of Disease Biology, The Novo Nordisk Foundation Center for Protein Research University of Copenhagen, Copenhagen, Denmark

UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre-mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre-mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.
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http://dx.doi.org/10.15252/embr.201438679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198039PMC
September 2014

Renal-retinal ciliopathy gene Sdccag8 regulates DNA damage response signaling.

J Am Soc Nephrol 2014 Nov 10;25(11):2573-83. Epub 2014 Apr 10.

Division of Nephrology, Boston Children's Hospital, Boston, Massachusetts; Howard Hughes Medical Institute, Chevy Chase, Maryland

Nephronophthisis-related ciliopathies (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, and intellectual disability. We recently identified mutations in a gene encoding the centrosomal protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8(gt/gt) mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration. These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to progressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys. Instead, renal pathology was associated with elevated levels of DNA damage response signaling activity. Cell culture studies confirmed the aberrant activation of DNA damage response in Sdccag8(gt/gt)-derived cells, characterized by elevated levels of γH2AX and phosphorylated ATM and cell cycle profile abnormalities. Our analysis of Sdccag8(gt/gt) mice indicates that the pleiotropic phenotypes in these mice may arise through multiple tissue-specific disease mechanisms.
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http://dx.doi.org/10.1681/ASN.2013050565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214515PMC
November 2014

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

Cell 2013 Nov;155(5):1088-103

Chromosome Stability and Dynamics Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.
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http://dx.doi.org/10.1016/j.cell.2013.10.043DOI Listing
November 2013

A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis.

EMBO J 2013 Nov 11;32(23):3029-40. Epub 2013 Oct 11.

Ubiquitin Signaling Group, Department of Disease Biology, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

Centriolar satellites are small, granular structures that cluster around centrosomes, but whose biological function and regulation are poorly understood. We show that centriolar satellites undergo striking reorganization in response to cellular stresses such as UV radiation, heat shock, and transcription blocks, invoking acute and selective displacement of the factors AZI1/CEP131, PCM1, and CEP290 from this compartment triggered by activation of the stress-responsive kinase p38/MAPK14. We demonstrate that the E3 ubiquitin ligase MIB1 is a new component of centriolar satellites, which interacts with and ubiquitylates AZI1 and PCM1 and suppresses primary cilium formation. In response to cell stress, MIB1 is abruptly inactivated in a p38-independent manner, leading to loss of AZI1, PCM1, and CEP290 ubiquitylation and concomitant stimulation of ciliogenesis, even in proliferating cells. Collectively, our findings uncover a new two-pronged signalling response, which by coupling p38-dependent phosphorylation with MIB1-catalysed ubiquitylation of ciliogenesis-promoting factors plays an important role in controlling centriolar satellite status and key centrosomal functions in a cell stress-regulated manner.
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http://dx.doi.org/10.1038/emboj.2013.223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844950PMC
November 2013

Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling.

EMBO Mol Med 2013 Aug 1;5(8):1278-95. Epub 2013 Jul 1.

Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling.
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http://dx.doi.org/10.1002/emmm.201303090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944466PMC
August 2013