10 results on '"Johanna Heideker"'
Search Results
2. SUMO-targeted ubiquitin ligase, Rad60, and Nse2 SUMO ligase suppress spontaneous Top1-mediated DNA damage and genome instability.
- Author
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Johanna Heideker, John Prudden, J Jefferson P Perry, John A Tainer, and Michael N Boddy
- Subjects
Genetics ,QH426-470 - Abstract
Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)-mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl-DNA phosphodiesterase 1 (Tdp1). This is notable, as cells lacking Tdp1 are largely aphenotypic in the vegetative cell cycle due to the existence of alternative pathways for the removal of covalent Top1-DNA adducts (Top1cc). We further identify Rad60, a SUMO mimetic and STUbL-interacting protein, and the SUMO E3 ligase Nse2 as critical Top1cc repair factors in cells lacking Tdp1. Detection of Top1ccs using chromatin immunoprecipitation and quantitative PCR shows that they are elevated in cells lacking Tdp1 and STUbL, Rad60, or Nse2 SUMO ligase activity. These unrepaired Top1ccs are shown to cause DNA damage, hyper-recombination, and checkpoint-mediated cell cycle arrest. We further determine that Tdp1 and the nucleotide excision repair endonuclease Rad16-Swi10 initiate the major Top1cc repair pathways of fission yeast. Tdp1-based repair is the predominant activity outside S phase, likely acting on transcription-coupled Top1cc. Epistasis analyses suggest that STUbL, Rad60, and Nse2 facilitate the Rad16-Swi10 pathway, parallel to Tdp1. Collectively, these results reveal a unified role for STUbL, Rad60, and Nse2 in protecting genome stability against spontaneous Top1-mediated DNA damage.
- Published
- 2011
- Full Text
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3. USP7 small-molecule inhibitors interfere with ubiquitin binding
- Author
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William F. Forrest, Sumit Prakash, Vickie Tsui, Adam R. Renslo, Richard Pastor, Christiaan Klijn, Frank Peale, Mark McCleland, Lorna Kategaya, Carsten Schwerdtfeger, Zachary Stiffler, Matthias Trost, Frederick Cohen, Priyadarshini Jaishankar, Kevin R Clark, Paola Di Lello, Bradley B. Brasher, Florian Gnad, Michael C. M. Kwok, Johanna Heideker, Jeremy Murray, Jason Drummond, Xiaojing Wang, Maria Stella Ritorto, Till Maurer, Maureen Beresini, Matthew T. Chang, James A. Ernst, Taylur P. Ma, Robert A. Blake, Elizabeth Blackwood, Dario R. Alessi, Michelle R. Arkin, Lionel Rouge, Kebing Yu, Brian R. Hearn, Travis W. Bainbridge, Eva Lin, Tracy Kleinheinz, Yinyan Tang, Chudi Ndubaku, Scott E. Martin, John-Paul Upton, and Ingrid E. Wertz
- Subjects
0301 basic medicine ,Multidisciplinary ,biology ,Ubiquitin binding ,Plasma protein binding ,Ubiquitin-conjugating enzyme ,Small molecule ,Ubiquitin ligase ,Deubiquitinating enzyme ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Biochemistry ,Proteasome ,Ubiquitin ,030220 oncology & carcinogenesis ,biology.protein - Abstract
The development of selective ubiquitin-specific protease-7 (USP7) inhibitors GNE-6640 and GNE-6776, which induce tumour cell death and reveal differential kinetics of Lys-48 and Lys-63-linked ubiquitin chain depolymerization by USP7. Deubiquitinating enzymes remove the small modifier protein ubiquitin from target substrates regulating their stability. One such enzyme, USP7, is a potential target for anti-cancer therapy, as its inhibition would result in the degradation of the ubiquitinated oncoprotein MDM2, leading to reactivation of the tumour suppressor protein p53. However, selective inhibitors of USP7 have remained elusive. Here, Ingrid Wertz and team develop two USP7 inhibitors, providing structural insights into the mode of action of these compounds and demonstrating their toxicity towards tumour cells. Elsewhere in this issue, David Komander and colleagues independently report the identification of two small molecules that inhibit USP7 with high affinity and specificity both in vitro and within cells, also demonstrating their ability to inhibit tumour growth. The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation1. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease2; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival3. However, developing selective deubiquitinase inhibitors has been challenging4 and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 A distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain5, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
- Published
- 2017
4. DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus
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Artem Y. Lyubimov, James M. Berger, Natasha K. Vidangos, Yi-Xin Huo, Jimmy Ton, David E. Wemmer, Johanna Heideker, Meindert H. Lamers, Jeffrey G. Pelton, and Jay D. Gralla
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Magnetic Resonance Spectroscopy ,Transcription, Genetic ,Crystallography, X-Ray ,sequence-specific recognition ,Structural Biology ,Factor For Inversion Stimulation Protein ,Crystallography ,biology ,Escherichia coli Proteins ,Cell biology ,Transcription ,Transcriptional Activation ,Protein Structure ,Biochemistry & Molecular Biology ,HMG-box ,PII Nitrogen Regulatory Proteins ,Microbiology ,Article ,Medicinal and Biomolecular Chemistry ,Bacterial Proteins ,Genetic ,Genetics ,Deoxyribonuclease I ,Binding site ,DNA-binding domain ,DNA complex ,Molecular Biology ,Transcription factor ,Aquifex aeolicus ,Binding Sites ,Bacteria ,Fis ,Proteins ,Hydrogen Bonding ,DNA ,biology.organism_classification ,Molecular biology ,Protein Structure, Tertiary ,DNA binding site ,X-Ray ,NtrC ,Nucleic Acid Conformation ,Generic health relevance ,Biochemistry and Cell Biology ,RNA Polymerase Sigma 54 ,Tertiary ,Transcription Factors - Abstract
Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (−145 and −85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the −145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.
- Published
- 2014
5. Abstract SY23-03: Development and mechanistic characterization of USP7 deubiquitinase inhibitors
- Author
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Maureen Beresini, Matthew T. Chang, Brian R. Hearn, Bradley B. Brasher, Mark McCleland, Ingrid E. Wertz, Lionel Rouge, Tracy Kleinheinz, Kebing Yu, Yinyan Tang, Richard Pastor, Jason Drummond, Chudi Ndubaku, James A. Ernst, William F. Forrest, Scott E. Martin, Christiaan Klijn, Frederick Cohen, John-Paul Upton, Taylur P. Ma, Dario R. Alessi, Carsten Schwerdtfeger, Paola Di Lello, Robert A. Blake, Eva Lin, Travis W. Bainbridge, Sumit Prakash, Adam R. Renslo, Vickie Tsui, Zachary Stiffler, Frank Peale, Maria Stella Ritorto, Till Maurer, Florian Gnad, Jeremy Murray, Matthias Trost, Elizabeth Blackwood, Michael C. Kwok, Priya Jaishanker, Xiaojing Wang, Lorna Kategaya, Kevin R Clark, Johanna Heideker, and Michelle R. Arkin
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chemistry.chemical_classification ,Cancer Research ,biology ,Ubiquitin binding ,Kinase ,Preferential binding ,Deubiquitinating enzyme ,Cell biology ,Deubiquitinase activity ,Enzyme ,Oncology ,Ubiquitin ,chemistry ,biology.protein ,Cysteine - Abstract
The ubiquitin system regulates the majority of cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains, and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates, including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumor suppressor and other proteins critical for tumor cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance (NMR)-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumor cell death and enhance cytotoxicity with chemotherapeutics and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 noncovalently target USP7 12Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate H-bond interactions with the ubiquitin Lys-48 side-chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties having free Lys-48 side-chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding via NMR, a study that substantiated our hypothesis. This preferential binding significantly protracted the depolymerization kinetics of Lys-48-linked ubiquitin chains relative to Lys-63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity. [LK, PDL, and LR contributed equally to this work.] Citation Format: Ingrid Wertz, Lorna Kategaya, Paola Di Lello, Lionel Rouge, Richard Pastor, Kevin R. Clark, Jason Drummond, Tracy Kleinheinz, Eva Lin, John-Paul Upton, Sumit Prakash, Johanna Heideker, Mark McCleland, Maria Stella Ritorto, Dario R. Alessi, Matthias Trost, Travis W. Bainbridge, Michael C. Kwok, Taylur P. Ma, Zachary Stiffler, Bradley Brasher, Yinyan Tang, Priya Jaishanker, Brian Hearn, Adam R. Renslo, Michelle R. Arkin, Frederick Cohen, Kebing Yu, Frank Peale, Florian Gnad, Matthew T. Chang, Christiaan Klijn, Elizabeth Blackwood, Scott E. Martin, William F. Forrest, James A. Ernst, Chudi Ndubaku, Xiaojing Wang, Maureen H. Beresini, Vickie Tsui, Carsten Schwerdtfeger, Robert A. Blake, Jeremy Murray, Till Maurer. Development and mechanistic characterization of USP7 deubiquitinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY23-03.
- Published
- 2018
6. Structure and Regulatory Mechanism of Aquifex aeolicus NtrC4: Variability and Evolution in Bacterial Transcriptional Regulation
- Author
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Joseph D. Batchelor, Sacha De Carlo, David E. Wemmer, Johanna Heideker, Michaeleen Doucleff, Chul-Jin Lee, Meindert H. Lamers, Jeffrey G. Pelton, and Koshi Matsubara
- Subjects
DNA, Bacterial ,Magnetic Resonance Spectroscopy ,Transcription, Genetic ,Molecular Sequence Data ,Protein Data Bank (RCSB PDB) ,Sequence alignment ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Protein Structure, Secondary ,Evolution, Molecular ,Adenosine Triphosphate ,Protein structure ,Bacterial Proteins ,Structural Biology ,Amino Acid Sequence ,Protein Structure, Quaternary ,Molecular Biology ,Peptide sequence ,Aquifex aeolicus ,Bacteria ,Sequence Homology, Amino Acid ,Hydrolysis ,Computational Biology ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Protein Structure, Tertiary ,Solutions ,Response regulator ,Biochemistry ,Trans-Activators ,Biophysics ,Dimerization ,Sequence Alignment ,Heteronuclear single quantum coherence spectroscopy ,Protein Binding - Abstract
Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.
- Published
- 2008
7. Crystal Structure of Archaeal Ribonuclease P Protein aRpp29 from Archaeoglobus fulgidus
- Author
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Johanna Heideker, David W. Hoffman, and David J. Sidote
- Subjects
Circular dichroism ,RNase P ,Archaeal Proteins ,Molecular Sequence Data ,Biology ,Crystallography, X-Ray ,Antiparallel (biochemistry) ,Biochemistry ,Protein Structure, Secondary ,Ribonuclease P ,Ribonucleases ,Bacterial transcription ,Hydrolase ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Conserved Sequence ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Circular Dichroism ,Archaeoglobus fulgidus ,Hyperthermophile ,Amino acid ,Solutions ,Crystallography ,Ribonucleoproteins ,chemistry ,Crystallization - Abstract
The crystal structure of ribonuclease P protein aRpp29 from the sulfate-reducing hyperthermophile Archaeoglobus fulgidus was determined at 1.7 A resolution using X-ray diffraction methods. The central feature of this archaeal protein is a sheet of six antiparallel beta-strands twisted around a conserved hydrophobic core. Residues near the N- and C-termini form helical structures that are oriented in an antiparallel manner. A comparison of conserved amino acids indicates that archaeal aRpp29 is homologous to human ribonuclease P protein Rpp29. The aRpp29 protein is structurally similar to bacterial transcription factors Hfq and NusG, as well as the Sm and Sm-like RNA-associated proteins from eukarya. The crystal structure of A. fulgidus aRpp29 differs from the previously reported solution structure, where NMR data did not detect the helices and indicated that approximately 40% of the residues are relatively flexible or disordered. Circular dichroism data indicate that the protein has less helical content than the amount observed in the crystal, suggesting that in solution the helical regions are unfolded or in equilibrium between folded and unfolded forms; this hypothesis is consistent with amide proton exchange rate data. Surface residues that are conserved from archaea to humans and are likely to interact with the ribonuclease P RNA or other protein subunits are identified in the structure. The model of the aRpp29 protein defined by this work provides an essential step toward eventually understanding the overall architecture of ribonuclease P.
- Published
- 2004
8. Dual Recruitment of Cdc48 (p97)-Ufd1-Npl4 Ubiquitin-selective Segregase by Small Ubiquitin-like Modifier Protein (SUMO) and Ubiquitin in SUMO-targeted Ubiquitin Ligase-mediated Genome Stability Functions*
- Author
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Aaron Aslanian, Ajay A. Vashisht, Johanna Heideker, Minghua Nie, James A. Wohlschlegel, John R. Yates, John Prudden, and Michael N. Boddy
- Subjects
SUMO-1 Protein ,Ubiquitin binding ,DNA Repair ,Ubiquitin-Protein Ligases ,genetic processes ,Amino Acid Motifs ,SUMO protein ,SUMO enzymes ,Cell Cycle Proteins ,macromolecular substances ,Biochemistry ,environment and public health ,Genomic Instability ,Ubiquitin ,Valosin Containing Protein ,Schizosaccharomyces ,DNA, Fungal ,Molecular Biology ,Adenosine Triphosphatases ,biology ,RNF4 ,Ubiquitination ,Cell Biology ,Ubiquitin ligase ,Cell biology ,enzymes and coenzymes (carbohydrates) ,health occupations ,biology.protein ,Schizosaccharomyces pombe Proteins ,Carrier Proteins ,Signal Transduction ,Protein Binding - Abstract
Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that “read” their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the “ubiquitin-selective” segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets. Indeed, we define key cooperative DNA repair functions for Cdc48-Ufd1-Npl4 and STUbL, thereby revealing a new signaling mechanism involving dual recruitment by SUMO and ubiquitin for Cdc48-Ufd1-Npl4 functions in maintaining genome stability.
- Published
- 2012
9. SUMO-targeted ubiquitin ligase, Rad60, and Nse2 SUMO ligase suppress spontaneous Top1-mediated DNA damage and genome instability
- Author
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John A. Tainer, Johanna Heideker, J. Jefferson P. Perry, John Prudden, Michael N. Boddy, and Heyer, Wolf-Dietrich
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Genome instability ,Cancer Research ,lcsh:QH426-470 ,SUMO ligase activity ,DNA Repair ,Chromosomal Proteins, Non-Histone ,DNA damage ,DNA repair ,Ubiquitin-Protein Ligases ,1.1 Normal biological development and functioning ,SUMO-1 Protein ,SUMO protein ,Type I ,Genomic Instability ,Fungal Proteins ,03 medical and health sciences ,DNA Adducts ,0302 clinical medicine ,Underpinning research ,Schizosaccharomyces ,Genetics ,2.1 Biological and endogenous factors ,Aetiology ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,0303 health sciences ,Molecular Biology/DNA Repair ,biology ,Phosphoric Diester Hydrolases ,DNA replication ,Non-Histone ,Molecular biology ,3. Good health ,Ubiquitin ligase ,Genetics and Genomics/Chromosome Biology ,Chromosomal Proteins ,lcsh:Genetics ,DNA Topoisomerases, Type I ,030220 oncology & carcinogenesis ,biology.protein ,Schizosaccharomyces pombe Proteins ,DNA Topoisomerases ,Research Article ,Nucleotide excision repair ,DNA Damage ,Signal Transduction ,Developmental Biology - Abstract
Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)–mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl–DNA phosphodiesterase 1 (Tdp1). This is notable, as cells lacking Tdp1 are largely aphenotypic in the vegetative cell cycle due to the existence of alternative pathways for the removal of covalent Top1–DNA adducts (Top1cc). We further identify Rad60, a SUMO mimetic and STUbL-interacting protein, and the SUMO E3 ligase Nse2 as critical Top1cc repair factors in cells lacking Tdp1. Detection of Top1ccs using chromatin immunoprecipitation and quantitative PCR shows that they are elevated in cells lacking Tdp1 and STUbL, Rad60, or Nse2 SUMO ligase activity. These unrepaired Top1ccs are shown to cause DNA damage, hyper-recombination, and checkpoint-mediated cell cycle arrest. We further determine that Tdp1 and the nucleotide excision repair endonuclease Rad16-Swi10 initiate the major Top1cc repair pathways of fission yeast. Tdp1-based repair is the predominant activity outside S phase, likely acting on transcription-coupled Top1cc. Epistasis analyses suggest that STUbL, Rad60, and Nse2 facilitate the Rad16-Swi10 pathway, parallel to Tdp1. Collectively, these results reveal a unified role for STUbL, Rad60, and Nse2 in protecting genome stability against spontaneous Top1-mediated DNA damage., Author Summary The failure of cellular DNA repair mechanisms can lead to cancer, neurodegeneration, or premature aging. Although much is known about specific DNA repair mechanisms, an understanding of how these processes are critically orchestrated by post-translational modifiers such as SUMO and ubiquitin is in its infancy. We identified an intriguing family of E3 ubiquitin ligases called STUbLs that act at the interface between the SUMO and ubiquitin pathways, and through undefined proteins and pathways maintain genome stability. Here we show that dysfunction of STUbL, an associated SUMO-like protein called Rad60, or the Nse2 SUMO E3 ligase converts the normally benign topoisomerase I (Top1) activity into a genome destabilizing genotoxin. Normally, Top1 transiently introduces a break in one strand of the DNA duplex allowing DNA to unwind. However, these transient breaks are converted into recombinogenic DNA lesions when STUbL, Rad60, Nse2, and parallel pathways that we identify are compromised. This study reveals important regulatory circuits reliant on STUbL, Rad60, and Nse2 that insulate the genome from the potentially harmful effects of Top1, which may otherwise promote cancer or neurodegeneration. Furthermore, Top1 is a major chemotherapeutic target, and so our findings may aid in the development of more efficacious Top1-based therapies.
- Published
- 2011
10. Variability analysis of human plasma and cerebral spinal fluid reveals statistical significance of changes in mass spectrometry-based metabolomics data
- Author
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Ewa Kalisiak, Johanna Heideker, Gary J. Patti, Gary Siuzdak, Hin-Koon Woo, William R. Wikoff, and Bridgit Crews
- Subjects
Databases, Factual ,Coefficient of variation ,Metabolite ,Mass spectrometry ,Article ,Mass Spectrometry ,Analytical Chemistry ,chemistry.chemical_compound ,Metabolomics ,Cerebrospinal fluid ,Humans ,Biomarker discovery ,Cerebrospinal Fluid ,Reproducibility ,Analysis of Variance ,Principal Component Analysis ,Chromatography ,Reproducibility of Results ,Fold change ,chemistry ,Nonlinear Dynamics ,Biomarkers ,Blood Chemical Analysis ,Software ,Chromatography, Liquid - Abstract
Analytical and biological variability are issues of central importance to human metabolomics studies. Here both types of variation are examined in human plasma and cerebrospinal fluid (CSF) using a global liquid chromatography-mass spectrometry (LC/MS) metabolomics strategy. The platform shows small analytical variation with a median coefficient of variation (CV) of 15–16% for both plasma and CSF sample matrices when the integrated area of each peak in the mass spectra is considered. Analysis of biological variation shows that human CSF has a median CV of 35% and plasma has a median CV of 46%. To understand the difference in CV between the biofluids, we compared plasma and CSF independently obtained from different healthy humans. Additionally, we analyzed another group of patients from whom we compared matched CSF and plasma (plasma and CSF obtained from the same human subject). A similar number of features was observed in both biofluids, although the majority of features appeared with greater intensity in plasma. More than a dozen metabolites shared between the human CSF and plasma metabolomes were identified based on accurate mass measurements, retention times, and MS/MS spectra. The fold change in these metabolites was consistent with the median biological CV determined for all peaks. The measured median biological CV together with analysis of intra-group variation of healthy individuals suggests that fold changes above 2 in metabolomics studies investigating plasma or CSF are statistically relevant with respect to the inherent variability of a healthy control group. These data demonstrate the reproducibility of the global metabolomics platform using LC/MS and reveal the robustness of the approach for biomarker discovery.
- Published
- 2009
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