Nuclear Parps and Genome Integrity
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CD56+ T-Cells in Relation to Cytomegalovirus in Healthy Subjects and Kidney Transplant Patients
CD56+ T-cells in Relation to Cytomegalovirus in Healthy Subjects and Kidney Transplant Patients Institute of Infection and Global Health Department of Clinical Infection, Microbiology and Immunology Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Mazen Mohammed Almehmadi December 2014 - 1 - Abstract Human T cells expressing CD56 are capable of tumour cell lysis following activation with interleukin-2 but their role in viral immunity has been less well studied. The work described in this thesis aimed to investigate CD56+ T-cells in relation to cytomegalovirus infection in healthy subjects and kidney transplant patients (KTPs). Proportions of CD56+ T cells were found to be highly significantly increased in healthy cytomegalovirus-seropositive (CMV+) compared to cytomegalovirus-seronegative (CMV-) subjects (8.38% ± 0.33 versus 3.29%± 0.33; P < 0.0001). In donor CMV-/recipient CMV- (D-/R-)- KTPs levels of CD56+ T cells were 1.9% ±0.35 versus 5.42% ±1.01 in D+/R- patients and 5.11% ±0.69 in R+ patients (P 0.0247 and < 0.0001 respectively). CD56+ T cells in both healthy CMV+ subjects and KTPs expressed markers of effector memory- RA T-cells (TEMRA) while in healthy CMV- subjects and D-/R- KTPs the phenotype was predominantly that of naïve T-cells. Other surface markers, CD8, CD4, CD58, CD57, CD94 and NKG2C were expressed by a significantly higher proportion of CD56+ T-cells in healthy CMV+ than CMV- subjects. Functional studies showed levels of pro-inflammatory cytokines IFN-γ and TNF-α, as well as granzyme B and CD107a were significantly higher in CD56+ T-cells from CMV+ than CMV- subjects following stimulation with CMV antigens. -
DNA Damage Sensitization of Breast Cancer Cells with PARP10/ARTD10 Inhibitor Mikko Hukkanen
Pro gradu DNA damage sensitization of breast cancer cells with PARP10/ARTD10 inhibitor Mikko Hukkanen University of Oulu Faculty of Biochemistry and Molecular Medicine 2019 This thesis was completed at the Faculty of Biochemistry and Molecular Medicine, University of Oulu. Oulu, Finland Supervisors: Professor Lari Lehtiö, Dr. Jarkko Koivunen and MSc Sudarshan Murthy Acknowledgements The work of this thesis was made in Faculty of Biochemistry and Molecular Medicine (FBMM) of University of Oulu. Firstly, I would like to thank Professor Lari Lehtiö for the opportunity working in his group, and the advice I received for my thesis. My gratitude is expressed also to my other supervisors, Jarkko Koivunen, for all the guidance I received in the cell culture, and Sudarshan Murthy, for the guidance to express and purify protein, and to conclude IC50 analysis. I would also thank all the personnel in LL group for all the advice I received in laboratory. I would especially thank Sven Sowa who made the Mantis run for IC50 analysis possible. Abbreviations 5-FU – 5-fluorouracil aa – Aminoacid ADP – Adenosine diphosphate ADPr – ADP-ribosylation AEC – Anion-exchange chromatography AIF – Apoptosis inducing factor AIM – Auto-induction medium Arg – Arginine ART – ADP-ribosyltransferase ARTD – Diphtheria toxin –like ADP-ribosyltransferase Asp – Aspartate BRCA – Breast cancer gene BRCT – BRCA1 C terminus CPT – Camptothecin CRC – Colorectal cancer CRM1 – Chromosome region maintenance 1 Cys – Cysteine DDR – DNA damage response dH2O – Distilled water DMEM – Dulbecco’s -
Elucidating the Tunability of Binding Behavior for the MERS-Cov Macro Domain with NAD Metabolites
ARTICLE https://doi.org/10.1038/s42003-020-01633-6 OPEN Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites Meng-Hsuan Lin1, Chao-Cheng Cho1,2, Yi-Chih Chiu1, Chia-Yu Chien2,3, Yi-Ping Huang4, Chi-Fon Chang4 & ✉ Chun-Hua Hsu 1,2,3 1234567890():,; The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand- binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions. 1 Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 10617, Taiwan. -
Parps and ADP-Ribosylation: Recent Advances Linking Molecular Functions to Biological Outcomes
Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes Rebecca Gupte,1,2 Ziying Liu,1,2 and W. Lee Kraus1,2 1Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; 2Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA The discovery of poly(ADP-ribose) >50 years ago opened units derived from β-NAD+ to catalyze the ADP-ribosyla- a new field, leading the way for the discovery of the tion reaction. These enzymes include bacterial ADPRTs poly(ADP-ribose) polymerase (PARP) family of enzymes (e.g., cholera toxin and diphtheria toxin) as well as mem- and the ADP-ribosylation reactions that they catalyze. bers of three different protein families in yeast and ani- Although the field was initially focused primarily on the mals: (1) arginine-specific ecto-enzymes (ARTCs), (2) biochemistry and molecular biology of PARP-1 in DNA sirtuins, and (3) PAR polymerases (PARPs) (Hottiger damage detection and repair, the mechanistic and func- et al. 2010). Surprisingly, a recent study showed that the tional understanding of the role of PARPs in different bio- bacterial toxin DarTG can ADP-ribosylate DNA (Jankevi- logical processes has grown considerably of late. This has cius et al. 2016). How this fits into the broader picture of been accompanied by a shift of focus from enzymology to cellular ADP-ribosylation has yet to be determined. -
ADP-Ribosylhydrolase Activity of Chikungunya Virus Macrodomain Is Critical for Virus Replication and Virulence
ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence Robert Lyle McPhersona,1, Rachy Abrahamb,1, Easwaran Sreekumarb,1,2, Shao-En Ongc, Shang-Jung Chenga, Victoria K. Baxterb,d, Hans A. V. Kistemakere, Dmitri V. Filippove, Diane E. Griffinb,3, and Anthony K. L. Leunga,f,3 aDepartment of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; bW. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; cDepartment of Pharmacology, University of Washington, Seattle, WA 98195; dDepartment of Molecular and Comparative Pathobiology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; eDepartment of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands; and fDepartment of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 Contributed by Diane E. Griffin, January 5, 2017 (sent for review October 20, 2016; reviewed by William Lee Kraus and Susan R. Weiss) Chikungunya virus (CHIKV), an Old World alphavirus, is trans- N-terminal domain of nsP2 has helicase, ATPase, GTPase, mitted to humans by infected mosquitoes and causes acute rash methyltransferase, and 5′ triphosphatase activity. nsP4 is the and arthritis, occasionally complicated by neurologic disease and RNA-dependent RNA polymerase (4). chronic arthritis. One determinant of alphavirus virulence is non- The function of nsP3 has been more enigmatic. nsP3 is found structural protein 3 (nsP3) that contains a highly conserved MacroD- in replication complexes and cytoplasmic nonmembranous type macrodomain at the N terminus, but the roles of nsP3 and the granules. -
Regulating New Intracellular Functions of Mono(ADP-Ribosyl)Ation Karla L
PERSPECTIVES to influence different signalling processes, POST-TRANSLATIONAL MODIFICATIONS — OPINION and distinct protein modules are known to bind this modification7,15,16. One such mod- Macrodomain-containing proteins: ule is the macrodomain, which was origi- nally identified in the core histone variant regulating new intracellular functions macroH2A17. Four years ago, macrodomains were shown to be able to bind PAR that has been synthesized in response to DNA dam- of mono(ADP-ribosyl)ation age20,25,26. More recently, the macrodomain of PAR glycohydrolase (PARG) was identified Karla L. H. Feijs, Alexandra H. Forst, Patricia Verheugd and Bernhard Lüscher as a PAR hydrolase21–23, demonstrating that distinct macrodomains can both read and Abstract | ADP-ribosylation of proteins was first described in the early 1960’s, and process PAR in addition to their ability to today the function and regulation of poly(ADP-ribosyl)ation (PARylation) is partially interact with ADP-ribose. We use the term understood. By contrast, little is known about intracellular mono(ADP-ribosyl)ation ‘reader’ throughout this article to indicate (MARylation) by ADP-ribosyl transferase (ART) enzymes, such as ARTD10. Recent proteins that recognize, but do not process, findings indicate that MARylation regulates signalling and transcription by substrates that are modified with ADP- ribose. Furthermore, in our terminology modifying key components in these processes. Emerging evidence also suggests ‘writers’ and ‘erasers’ refer to the enzymes that specific macrodomain-containing proteins, including ARTD8, macroD1, that add and remove ADP-ribosylation, macroD2 and C6orf130, which are distinct from those affecting PARylation, respectively. interact with MARylation on target proteins to ‘read’ and ‘erase’ this modification. -
Downloaded Per Proteome Cohort Via the Web- Site Links of Table 1, Also Providing Information on the Deposited Spectral Datasets
www.nature.com/scientificreports OPEN Assessment of a complete and classifed platelet proteome from genome‑wide transcripts of human platelets and megakaryocytes covering platelet functions Jingnan Huang1,2*, Frauke Swieringa1,2,9, Fiorella A. Solari2,9, Isabella Provenzale1, Luigi Grassi3, Ilaria De Simone1, Constance C. F. M. J. Baaten1,4, Rachel Cavill5, Albert Sickmann2,6,7,9, Mattia Frontini3,8,9 & Johan W. M. Heemskerk1,9* Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome‑wide transcriptomes (57.8 k mRNAs). For 14.8 k protein‑coding transcripts, we assigned the proteins to 21 UniProt‑based classes, based on their preferential intracellular localization and presumed function. This classifed transcriptome‑proteome profle of platelets revealed: (i) Absence of 37.2 k genome‑ wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein‑coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43–0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identifed proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma‑derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identifed proteome of nuclear‑related, membrane and signaling proteins, as well proteins with low‑level transcripts. -
Variable Macro X Domain of SARS-Cov-2 Retains the Ability to Bind ADP-Ribose David N
bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.014639; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Variable Macro X Domain of SARS-CoV-2 Retains the Ability to Bind ADP-ribose David N. Frick,1* Rajdeep S. Virdi,1 Nemanja Vuksanovic,1 Narayan Dahal,2 & Nicholas R Silvaggi1 Departments of Chemistry & Biochemistry,1 and Physics,2 The University of Wisconsin- Milwaukee, Milwaukee, WI 53217 KEYWORDS: COVID-19, Antiviral Drug target, Severe Acute Respiratory Syndrome, Coronavirus. Supporting Information Placeholder ABSTRACT: The virus that causes COVID-19, SARS- to bind ADP-ribose, that will be referred to here as the CoV-2, has a large RNA genome that encodes numerous “macro X” domain, to differentiate it from the two proteins that might be targets for antiviral drugs. Some downstream “SARS unique macrodomains (SUDs),” 3 of these proteins, such as the RNA-dependent RNA pol- which do not bind ADP-ribose. The macro X domain of ymers, helicase and main protease, are well conserved SARS-CoV-1 is also able to catalyze the hydrolysis of 4 between SARS-CoV-2 and the original SARS virus, but ADP-ribose 1’’ phosphate, albeit at a slow rate. several others are not. This study examines one of the All the above differences preclude the use of the most novel proteins encoded by SARS-CoV-2, a SARS-CoV-1 macro X domain structures as scaffolds to macrodomain of nonstructural protein 3 (nsp3). -
ADP-Ribosyl)Hydrolases: Structure, Function, and Biology
Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press SPECIAL SECTION: REVIEW (ADP-ribosyl)hydrolases: structure, function, and biology Johannes Gregor Matthias Rack,1,3 Luca Palazzo,2,3 and Ivan Ahel1 1Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom; 2Institute for the Experimental Endocrinology and Oncology, National Research Council of Italy, 80145 Naples, Italy ADP-ribosylation is an intricate and versatile posttransla- Diversification of NAD+ signaling is particularly apparent tional modification involved in the regulation of a vast in vertebrata, being linked to evolutionary optimization of variety of cellular processes in all kingdoms of life. Its NAD+ biosynthesis and increased (ADP-ribosyl) signaling complexity derives from the varied range of different (Bockwoldt et al. 2019). ADP-ribosylation is used by or- chemical linkages, including to several amino acid side ganisms from all kingdoms of life and some viruses chains as well as nucleic acids termini and bases, it can (Perina et al. 2014; Aravind et al. 2015) and controls a adopt. In this review, we provide an overview of the differ- wide range of cellular processes such as DNA repair, tran- ent families of (ADP-ribosyl)hydrolases. We discuss their scription, cell division, protein degradation, and stress re- molecular functions, physiological roles, and influence sponse to name a few (Bock and Chang 2016; Gupte et al. on human health and disease. Together, the accumulated 2017; Palazzo et al. 2017; Rechkunova et al. 2019). In ad- data support the increasingly compelling view that (ADP- dition to proteins, several in vitro observations strongly ribosyl)hydrolases are a vital element within ADP-ribosyl suggest that nucleic acids, both DNA and RNA, can be signaling pathways and they hold the potential for novel targets of ADP-ribosylation (Nakano et al. -
Mechanistic Insights Into the PARP10-Mediated ADP
Mechanistic insights into the PARP10-mediated ADP-ribosylation reaction and analysis of PARP10’s subcellular localization Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologe Henning Schuchlautz aus Waldbröl Berichter: Universitätsprofessor Dr. Bernhard Lüscher Privatdozent Dr. Christoph Peterhänsel Tag der mündlichen Prüfung: 20. Juni 2008 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. I Abstract ADP-ribosylation controls many cellular processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARPs 6-16, raising questions about how these enzymes function. Therefore the enzymatic properties of PARP10, a representative of the novel PARP enzymes, were analyzed in detail. It was found that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has mono- ADP-ribosyltransferase rather than polymerase activity. Despite this fundamental difference PARP10 also modifies acidic residues. Molecular modeling shows that an acidic residue of the substrate can be placed in a favorable position to stabilize the oxocarbenium transition state of the reaction. This function is normally executed by the catalytic glutamate in ADP-ribosyltransferases and bona fide PARP enzymes. Consequently, a novel catalytic mechanism for PARP10 is proposed, in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate, using substrate-assisted catalysis to transfer ADP-ribose. -
Signature Red Acted
Farnesylation-dependent regulation of transcripts by PARP13 ARCHM MASSACGHUStTSI INSTITUTE by OF TECHNOLOGY Lilen Uchima SEP 17 2015 B.S. Industrial Biotechnology LIBRARIES University of Puerto Rico at MayagOez, 2009 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR IN PHILOSOPHY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY AUGUST 2015 september 20f @ 2015 Massachusetts Institute of Technology. All rights reserved. Signature red acted Signature o Fauthor: IrF I Department of Biology Signature redacted August 31st, 2015 Certified by: Co-Supervisor: Paul Chang _Signature redacted Research Scientist Certified by: Supervisor: Stephen P. Bell Signature redacted Professor of Biology Accepted by Michael Hemann Associate Professor of Biology Co-Chair, Biology Graduate Committee 1 2 Farnesylation-dependent regulation of transcripts by PARP13 By Lilen Uchima Submitted to the Department of Biology on August 3 1 t, 2015 in partial fulfillment of the requirements for the degree of Doctor in Philosophy Abstract PARP1 3 is a RNA-binding and catalytically inactive member of the poly(ADP- ribose) (PARP) family. PARP13 was first identified as a host antiviral factor that selectively binds to viral mRNAs and targets them for degradation by mRNA decay factors. Recently, functions of PARP13 in post-transcriptional regulation of cellular mRNAs were identified. In this context, PARP13 indirectly regulates the miRNA silencing pathway, restricts human retrotransposition by regulating LINE-1 mRNA and downregulates the mRNA levels of TRAIL-R4 -a pro-survival TRAIL ligand receptor. Farnesylation is a post-translational lipid modification that causes proteins to associate to membranes and is required for the activity of some proteins such as Ras. -
Mouse Parp10 Knockout Project (CRISPR/Cas9)
https://www.alphaknockout.com Mouse Parp10 Knockout Project (CRISPR/Cas9) Objective: To create a Parp10 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Parp10 gene (NCBI Reference Sequence: NM_001163576 ; Ensembl: ENSMUSG00000063268 ) is located on Mouse chromosome 15. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000165738). Exon 1~10 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 1 starts from about 0.03% of the coding region. Exon 1~10 covers 97.47% of the coding region. The size of effective KO region: ~9856 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 9 10 Legends Exon of mouse Parp10 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 10 is aligned with itself to determine if there are tandem repeats.