Mouse Gsta4 Knockout Project (CRISPR/Cas9)

Total Page:16

File Type:pdf, Size:1020Kb

Mouse Gsta4 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Gsta4 Knockout Project (CRISPR/Cas9) Objective: To create a Gsta4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Gsta4 gene (NCBI Reference Sequence: NM_010357 ; Ensembl: ENSMUSG00000032348 ) is located on Mouse chromosome 9. 7 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 7 (Transcript: ENSMUST00000034903). Exon 2~6 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: Mice homozygous for disruptions in this gene display increased sensitivity to oxidative stress, increased alanine and aspartate transaminase levels, increased susceptibility to bacterial infection, reduced litter size, and impaired glucose tolerance. Exon 2 starts from the coding region. Exon 2~6 covers 81.98% of the coding region. The size of effective KO region: ~9663 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 Legends Exon of mouse Gsta4 Knockout region Page 2 of 8 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 Exon 2 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 1124 bp section downstream of Exon 6 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.1% 482) | C(20.95% 419) | T(34.55% 691) | G(20.4% 408) Note: The 2000 bp section upstream of Exon 2 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(1124bp) | A(23.93% 269) | C(20.28% 228) | T(33.19% 373) | G(22.6% 254) Note: The 1124 bp section downstream of Exon 6 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr9 + 78196291 78198290 2000 browser details YourSeq 176 193 1153 2000 93.1% chr17 + 71209629 71590785 381157 browser details YourSeq 153 1085 1671 2000 89.6% chr19 + 5614704 5868958 254255 browser details YourSeq 151 158 1155 2000 90.2% chr15 - 102053001 102072783 19783 browser details YourSeq 104 1021 1176 2000 93.4% chr15 + 14985052 14985371 320 browser details YourSeq 99 631 1175 2000 76.1% chr14 - 91925963 91926119 157 browser details YourSeq 98 1029 1155 2000 91.6% chr12 + 31680658 31680789 132 browser details YourSeq 97 1039 1155 2000 95.4% chr3 + 127519424 127519544 121 browser details YourSeq 93 1031 1171 2000 84.3% chr1 + 69824608 69824726 119 browser details YourSeq 92 1046 1172 2000 93.4% chrX - 73530703 73530835 133 browser details YourSeq 91 1071 1645 2000 74.0% chr14 + 55843159 55843392 234 browser details YourSeq 90 1039 1155 2000 91.7% chr10 - 80539445 80539568 124 browser details YourSeq 90 1045 1174 2000 88.8% chr1 + 78291743 78291878 136 browser details YourSeq 89 1038 1155 2000 93.3% chr11 - 74753514 74754030 517 browser details YourSeq 89 1047 1171 2000 94.1% chr10 - 114638919 114639047 129 browser details YourSeq 89 1039 1155 2000 94.2% chr14 + 31122953 31123426 474 browser details YourSeq 87 1038 1155 2000 94.0% chr12 - 76567949 76568069 121 browser details YourSeq 86 1116 1652 2000 75.6% chr11 - 9008214 9008449 236 browser details YourSeq 85 1050 1166 2000 93.8% chr12 - 72093768 72093891 124 browser details YourSeq 83 1039 1155 2000 94.7% chr18 + 35110931 35111053 123 Note: The 2000 bp section upstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 1124 1 1124 1124 100.0% chr9 + 78207934 78209057 1124 browser details YourSeq 119 610 992 1124 89.5% chr17 + 26331691 26332118 428 browser details YourSeq 108 610 931 1124 72.6% chr3 + 67556526 67556829 304 browser details YourSeq 98 664 933 1124 79.4% chr3 - 108608404 108608603 200 browser details YourSeq 77 727 989 1124 79.8% chr4 + 100460109 100460310 202 browser details YourSeq 65 675 940 1124 91.2% chr2 + 51359698 51360057 360 browser details YourSeq 52 874 992 1124 75.9% chr6 - 112558296 112558391 96 browser details YourSeq 38 840 948 1124 95.3% chr4 - 3419225 3419751 527 browser details YourSeq 38 902 951 1124 93.1% chr16 - 44426236 44426289 54 browser details YourSeq 37 696 754 1124 81.4% chr16 + 65029297 65029355 59 browser details YourSeq 34 730 783 1124 89.5% chr18 - 77349236 77349288 53 browser details YourSeq 34 730 793 1124 87.5% chr19 + 36728326 36728388 63 browser details YourSeq 27 612 644 1124 91.0% chr4 - 83953367 83953399 33 browser details YourSeq 27 961 991 1124 96.6% chr16 + 90993695 90993727 33 browser details YourSeq 26 609 640 1124 90.7% chr10 + 99567154 99567185 32 browser details YourSeq 25 268 295 1124 96.5% chr1 + 179485599 179485627 29 browser details YourSeq 24 615 644 1124 90.0% chr5 - 48339128 48339157 30 Note: The 1124 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Gsta4 glutathione S-transferase, alpha 4 [ Mus musculus (house mouse) ] Gene ID: 14860, updated on 1-Oct-2019 Gene summary Official Symbol Gsta4 provided by MGI Official Full Name glutathione S-transferase, alpha 4 provided by MGI Primary source MGI:MGI:1309515 See related Ensembl:ENSMUSG00000032348 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as GST5.7; mGsta4 Expression Biased expression in bladder adult (RPKM 693.7), stomach adult (RPKM 118.2) and 1 other tissue See more Genomic context Location: 9; 9 E1 See Gsta4 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (78191966..78209349) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (78039773..78057156) Chromosome 9 - NC_000075.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Gsta4 ENSMUSG00000032348 Description glutathione S-transferase, alpha 4 [Source:MGI Symbol;Acc:MGI:1309515] Gene Synonyms GST 5.7, mGsta4 Location Chromosome 9: 78,182,774-78,209,349 forward strand. GRCm38:CM001002.2 About this gene This gene has 2 transcripts (splice variants), 214 orthologues, 16 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Gsta4-201 ENSMUST00000034903.6 977 222aa ENSMUSP00000034903.5 Protein coding CCDS23357 P24472 TSL:1 GENCODE basic APPRIS P1 Gsta4-202 ENSMUST00000213215.1 743 105aa ENSMUSP00000149763.1 Protein coding - A0A1L1SS61 CDS 3' incomplete TSL:3 46.58 kb Forward strand 78.18Mb 78.19Mb 78.20Mb 78.21Mb Genes (Comprehensive set... C920006O11Rik-201 >lncRNA Gsta4-201 >protein coding C920006O11Rik-202 >transcribed unprocessed pseudogene Gsta4-202 >protein coding Contigs < AC159313.3 Genes < Rn7sk-201misc RNA < Gm3126-201processed pseudogene (Comprehensive set... Regulatory Build 78.18Mb 78.19Mb 78.20Mb 78.21Mb Reverse strand 46.58 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000034903 17.40 kb Forward strand Gsta4-201 >protein coding ENSMUSP00000034... PDB-ENSP mappings Low complexity (Seg) Superfamily Thioredoxin-like superfamily Glutathione S-transferase, C-terminal domain superfamily SFLD SFLDG01205 Glutathione Transferase family Prints Glutathione S-transferase, alpha class Pfam Glutathione S-transferase, N-terminal Glutathione S-transferase, C-terminal PROSITE profiles Glutathione S-transferase, N-terminal Glutathione S-transferase, C-terminal-like PANTHER PTHR11571 PTHR11571:SF101 Gene3D 1.20.1050.10 3.40.30.10 CDD cd03077 cd03208 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 200 222 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
Recommended publications
  • GSTA4 (NM 001512) Human Tagged ORF Clone – RC202130 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 techsupport@origene.com EU: info-de@origene.com CN: techsupport@origene.cn Product datasheet for RC202130 GSTA4 (NM_001512) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: GSTA4 (NM_001512) Human Tagged ORF Clone Tag: Myc-DDK Symbol: GSTA4 Synonyms: GSTA4-4; GTA4 Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin ORF Nucleotide >RC202130 ORF sequence Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGGCAGCAAGGCCCAAGCTCCACTATCCCAACGGAAGAGGCCGGATGGAGTCCGTGAGATGGGTTTTAG CTGCCGCCGGAGTCGAGTTTGATGAAGAATTTCTGGAAACAAAAGAACAGTTGTACAAGTTGCAGGATGG TAACCACCTGCTGTTCCAACAAGTGCCCATGGTTGAAATTGACGGGATGAAGTTGGTACAGACCCGAAGC ATTCTCCACTACATAGCAGACAAGCACAATCTCTTTGGCAAGAACCTCAAGGAGAGAACCCTGATTGACA TGTACGTGGAGGGGACACTGGATCTGCTGGAACTGCTTATCATGCATCCTTTCTTAAAACCAGATGATCA GCAAAAGGAAGTGGTTAACATGGCCCAGAAGGCTATAATTAGATACTTTCCTGTGTTTGAAAAGATTTTA AGGGGTCACGGACAAAGCTTTCTTGTTGGTAATCAGCTGAGCCTTGCAGATGTGATTTTACTCCAAACCA TTTTAGCTCTAGAAGAGAAAATTCCTAATATCCTGTCTGCATTTCCTTTCCTCCAGGAATACACAGTGAA ACTAAGTAATATCCCTACAATTAAGAGATTCCTTGAACCTGGCAGCAAGAAGAAGCCTCCCCCTGATGAA ATTTATGTGAGAACCGTCTACAACATCTTTAGGCCA ACGCGTACGCGGCCGCTCGAGCAGAAACTCATCTCAGAAGAGGATCTGGCAGCAAATGATATCCTGGATT ACAAGGATGACGACGATAAGGTTTAA This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene
    [Show full text]
  • A Thesis Entitled Identification and Characterization of a Zebrafish
    A Thesis Entitled Identification and Characterization of A Zebrafish Glutathione S-Transferase Pi By Maryam S Abunnaja Submitted to the Graduate Faculty as a Partial Fulfillment of the Requirement for the Master of Science Degree in Pharmacology and Toxicology Dr. Ming-Cheh Liu, Committee Chair Dr. Ezdihar Hassoun, Committee Member Dr. Zahoor Shah, Committee Member Dr. Patricia Komuniecki, Dean College Graduate Studies The University of Toledo May 2013 Copyright 2013, Maryam S Abunnaja This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Identification and Characterization of A Zebrafish Glutathione S-Transferase Pi By Maryam S Abunnaja Submitted to the Graduate Faculty as a Partial Fulfillment of the Requirement for the Master of Science Degree in Pharmacology and Toxicology May 2013 Glutathione S-transferases (GSTs) are a major group of Phase II enzymes involved in the detoxification of certain endogenous compounds such as reactive oxygen species (ROS) generated during metabolism, as well as in the detoxification of xenobiotics including drugs. In recent years, the zebrafish has increasingly been used as a promising animal model for biomedical research. This study is part of an effort to establish the zebrafish as a model for studying GST-mediated glutathione conjugation of xenobiotics. By searching the GenBank database, we have identified sequences encoding fifteen zebrafish GSTs. Using a computer algorithm available at the Genebee website, a dendrogram of the fifteen zebrafish GSTs was generated. A zebrafish GST Pi was subsequently cloned, expressed, and purified. An enzymatic assay for purified GST Pi was established using 1- chloro-2, 4-dinitrobenzene (CDNB) as a substrate.
    [Show full text]
  • Glutathione S-Transferase Enzyme
    Haematologica 2000; 85:573-579 Hematopoiesis & Growth Factors original paper Glutathione S-transferase enzyme expression in hematopoietic cell lines implies a differential protective role for T1 and A1 isoenzymes in erythroid and for M1 in lymphoid lineages LIHUI WANG, MICHAEL J. GROVES, MARY D. HEPBURN, DAVID T. BOWEN Department of Molecular and Cellular Pathology, University of Dundee Medical School, Dundee, UK ABSTRACT Background and Objectives. Glutathione S-trans- exposure to xenobiotics that are substrates for ferases (GSTs) are phase II metabolizing enzymes these enzymes. which catalyze the conjugation of glutathione (GSH) ©2000, Ferrata Storti Foundation to electrophilic substrates and possess selenium- independent glutathione peroxidase activity. The Key words: glutathione S-transferase, genotoxicity, GST enzyme family includes the cytosolic isoforms antioxidant, hematopoietic cell lines GST-α (GSTA), µ (GSTM), π (GSTP), θ (GSTT) and σ (GSTS). GSTT1, P1 and M1 are polymorphic and altered polymorphic frequency of genes encoding igher organisms have evolved complex mech- these proteins has been suggested as a potential anisms by which they can protect themselves risk factor for the development of hematopoietic from environmental challenge. The metabo- malignancies. Overexpression of GSTs has also H lism and detoxification of xenobiotic toxins provides been implicated in chemotherapeutic drug resis- tance. This study was undertaken to elucidate the the first line of intracellular defence and this is medi- potential functional relevance of these genetic poly- ated by enzyme superfamilies, including the cyto- morphisms in hematopoiesis. chrome p450, glutathione S-transferase (GST) and N-acetyltransferases. Glutathione S-transferase Design and Methods. GST genotype of 14 genes encode for five families of cytosolic enzymes: hematopoietic cell lines was determined by poly- GSTs-α (GSTA), µ (GSTM), π (GSTP), θ (GSTT) and merase-chain-reaction (PCR).
    [Show full text]
  • GSTA4 Purified Maxpab Mouse Polyclonal Antibody (B01P)
    GSTA4 purified MaxPab mouse mammalian glutathione S-transferases have been polyclonal antibody (B01P) identified: alpha, kappa, mu, omega, pi, sigma, theta and zeta. This gene encodes a glutathione S-tranferase Catalog Number: H00002941-B01P belonging to the alpha class. The alpha class genes, which are located in a cluster on chromosome 6, are Regulatory Status: For research use only (RUO) highly related and encode enzymes with glutathione peroxidase activity that function in the detoxification of Product Description: Mouse polyclonal antibody raised lipid peroxidation products. Reactive electrophiles against a full-length human GSTA4 protein. produced by oxidative metabolism have been linked to a number of degenerative diseases including Parkinson's Immunogen: GSTA4 (AAH15523, 1 a.a. ~ 222 a.a) disease, Alzheimer's disease, cataract formation, and full-length human protein. atherosclerosis. [provided by RefSeq] Sequence: References: MAARPKLHYPNGRGRMESVRWVLAAAGVEFDEEFLE 1. Glutathione S-transferase alpha 4 induction by TKEQLYKLQDGNHLLFQQVPMVEIDGMKLVQTRSILHY activator protein 1 in colorectal cancer. Yang Y, Huycke IADKHNLFGKNLKERTLIDMYVEGTLDLLELLIMHPFLK MM, Herman TS, Wang X. Oncogene. 2016 Apr 11. PDDQQKEVVNMAQKAIIRYFPVFEKILRGHGQSFLVGN [Epub ahead of print] QLSLADVILLQTILALEEKIPNILSAFPFLQEYTVKLSNIP TIKRFLEPGSKKKPPPDEIYVRTVYNIFRP Host: Mouse Reactivity: Human Applications: WB-Ce, WB-Tr (See our web site product page for detailed applications information) Protocols: See our web site at http://www.abnova.com/support/protocols.asp or product page for detailed protocols Storage Buffer: In 1x PBS, pH 7.4 Storage Instruction: Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Entrez GeneID: 2941 Gene Symbol: GSTA4 Gene Alias: DKFZp686D21185, GSTA4-4, GTA4 Gene Summary: Cytosolic and membrane-bound forms of glutathione S-transferase are encoded by two distinct supergene families.
    [Show full text]
  • Glutathionylated Lipid Aldehydes Are Products of Adipocyte Oxidative Stress and Activators of Macrophage Inflammation
    Page 1 of 42 Diabetes GLUTATHIONYLATED LIPID ALDEHYDES ARE PRODUCTS OF ADIPOCYTE OXIDATIVE STRESS AND ACTIVATORS OF MACROPHAGE INFLAMMATION Brigitte I Frohnert, MD PhD*†, Eric K Long, PhD*‡, Wendy S Hahn‡, David A Bernlohr, PhD‡. †Department of Pediatrics, University of Minnesota, Minneapolis, MN, ‡Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN Running title: Glutathionyl lipid aldehydes and inflammation * These authors contributed equally to this work. Address correspondence to: David A. Bernlohr Biochemistry, Molecular Biology and Biophysics Rm 6-155 JacH 321 Church St SE Minneapolis, MN 55455 Phone: 612-624-2712 Fax: 612-625-2163 Email: bernl001@umn.edu Abstract word count: 197 Word Count: 3996 Tables: 0 Figures: 8 1 Diabetes Publish Ahead of Print, published online September 23, 2013 Diabetes Page 2 of 42 ABSTRACT Obesity-induced insulin resistance has been linked to adipose tissue lipid aldehyde production and protein carbonylation. Trans-4-hydroxy-2-nonenal (4-HNE) is the most abundant lipid aldehyde in murine adipose tissue and is metabolized by glutathione S-transferase A4 (GSTA4) producing glutathionyl-HNE (GS-HNE) and its metabolite glutathionyl-1,4-dihydroxynonene (GS-DHN). The objective of this study was to evaluate adipocyte production of GS-HNE and GS-DHN and their effect on macrophage inflammation. Compared to lean controls, GS-HNE and GS-DHN were more abundant in visceral adipose tissue of ob/ob mice and diet-induced obese, insulin resistant mice. High glucose and oxidative stress induced production of GS-HNE and GS-DHNE by 3T3-L1 adipocytes in a GSTA4-dependent manner and both glutathionylated metabolites induced secretion of TNFα from RAW264.7 and primary peritoneal macrophages.
    [Show full text]
  • Frequent Loss-Of-Heterozygosity in CRISPR-Cas9–Edited Early Human Embryos
    Frequent loss-of-heterozygosity in CRISPR-Cas9–edited COLLOQUIUM PAPER early human embryos Gregorio Alanis-Lobatoa, Jasmin Zohrenb, Afshan McCarthya, Norah M. E. Fogartya,c, Nada Kubikovad,e, Emily Hardmana, Maria Grecof, Dagan Wellsd,g, James M. A. Turnerb, and Kathy K. Niakana,h,1 aHuman Embryo and Stem Cell Laboratory, The Francis Crick Institute, NW1 1AT London, United Kingdom; bSex Chromosome Biology Laboratory, The Francis Crick Institute, NW1 1AT London, United Kingdom; cCentre for Stem Cells and Regenerative Medicine, Guy’s Campus, King’s College London, SE1 9RT London, United Kingdom; dNuffield Department of Women’s and Reproductive Health, John Radcliffe Hospital, University of Oxford, OX3 9DU Oxford, United Kingdom; eJesus College, University of Oxford, OX1 3DW Oxford, United Kingdom; fAncient Genomics Laboratory, The Francis Crick Institute, NW1 1AT London, United Kingdom; gJuno Genetics, OX4 4GE Oxford, United Kingdom; and hThe Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, CB2 3EG Cambridge, United Kingdom Edited by Barbara J. Meyer, University of California, Berkeley, CA, and approved October 31, 2020 (received for review June 5, 2020) CRISPR-Cas9 genome editing is a promising technique for clinical homozygous WT embryos in both cases was not associated with applications, such as the correction of disease-associated alleles in use of the provided repair template for gene correction. Instead, somatic cells. The use of this approach has also been discussed in the authors suggest that in edited embryos the WT maternal the context of heritable editing of the human germ line. However, allele served as a template for the high-fidelity homology di- studies assessing gene correction in early human embryos report rected repair (HDR) pathway to repair the double-strand lesion low efficiency of mutation repair, high rates of mosaicism, and the caused by the Cas9 protein in the paternal allele (8).
    [Show full text]
  • Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Article Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases Christian Gu,1 D. Alexander Shannon,5 Tom Colby,2 Zheming Wang,1 Mohammed Shabab,6 Selva Kumari,1 Joji Grace Villamor,1 Christopher J. McLaughlin,5 Eranthie Weerapana,3,5 Markus Kaiser,4 Benjamin F. Cravatt,3 and Renier A.L. van der Hoorn1,* 1The Plant Chemetics Laboratory 2Proteomics Service Center Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany 3Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA 4Zentrum fu¨ r Medizinische Biotechnologie, Fakulta¨ tfu¨ r Biologie, Universita¨ t Duisburg-Essen, Universita¨ tsstr. 2, 45117 Essen, Germany 5Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02466, USA 6Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Kno¨ ll-strasse 8, 07745 Jena, Germany *Correspondence: hoorn@mpipz.mpg.de http://dx.doi.org/10.1016/j.chembiol.2013.01.016 SUMMARY for posttranslational modifications such as acylation, glycosyla- tion, and prenylation (Heal and Tate, 2010). Chemical probes have great potential for identifying Recently, chemical proteomics has been expanded further by functional residues in proteins in crude proteomes. including reactivity probes, which carry only a reactive group Here we studied labeling sites of chemical probes (e.g., Weerapana et al., 2008). When used properly, reactivity based on sulfonyl fluorides (SFs) on plant and animal probes globally identify hyperreactive amino acids in proteomes.
    [Show full text]
  • Snps in Genes Coding for ROS Metabolism and Signalling in Association with Docetaxel Clearance
    The Pharmacogenomics Journal (2010) 10, 513–523 & 2010 Macmillan Publishers Limited. All rights reserved 1470-269X/10 www.nature.com/tpj ORIGINAL ARTICLE SNPs in genes coding for ROS metabolism and signalling in association with docetaxel clearance H Edvardsen1,2, PF Brunsvig3, The dose of docetaxel is currently calculated based on body surface area 1,4 5 and does not reflect the pharmacokinetic, metabolic potential or genetic H Solvang , A Tsalenko , background of the patients. The influence of genetic variation on the 6 7 A Andersen , A-C Syvanen , clearance of docetaxel was analysed in a two-stage analysis. In step one, 583 Z Yakhini5, A-L Børresen-Dale1,2, single-nucleotide polymorphisms (SNPs) in 203 genes were genotyped on H Olsen6, S Aamdal3 and samples from 24 patients with locally advanced non-small cell lung cancer. 1,2 We found that many of the genes harbour several SNPs associated with VN Kristensen clearance of docetaxel. Most notably these were four SNPs in EGF, three SNPs 1Department of Genetics, Institute of Cancer in PRDX4 and XPC, and two SNPs in GSTA4, TGFBR2, TNFAIP2, BCL2, DPYD Research, Oslo University Hospital Radiumhospitalet, and EGFR. The multiple SNPs per gene suggested the existence of common Oslo, Norway; 2Institute of Clinical Medicine, haplotypes associated with clearance. These were confirmed with detailed 3 University of Oslo, Oslo, Norway; Cancer Clinic, haplotype analysis. On the basis of analysis of variance (ANOVA), quantitative Oslo University Hospital Radiumhospitalet, Oslo, Norway; 4Institute of
    [Show full text]
  • Deletion of GSTA4-4 Results in Increased Mitochondrial Post-Translational Modification of Proteins by Reactive Aldehydes Following Chronic Ethanol Consumption in Mice
    Redox Biology 7 (2016) 68–77 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research paper Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice Colin T. Shearn a,n, Kristofer S. Fritz a, Alisabeth H. Shearn b, Laura M. Saba a, Kelly E. Mercer c, Bridgette Engi c, James J. Galligan e, Piotr Zimniak c, David J. Orlicky d, Martin J. Ronis f, Dennis R. Petersen a,nn a Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, United States b Alpine Achievement Systems Inc., Englewood, CO, United States c Department of Pediatrics, Arkansas Children's Nutrition Center, Little Rock, AR, United States d Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO, United States e Department of Biochemistry, Vanderbilt, Nashville, TN, United States f Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA, United States article info abstract Article history: Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive Received 20 October 2015 electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary me- Received in revised form chanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic con- 23 November 2015 jugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, Accepted 25 November 2015 À À we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4 / mice is due to Available online 27 November 2015 enhanced mitochondrial protein modification by reactive aldehydes.
    [Show full text]
  • Itraq-Based Quantitative Proteomic Analysis Reveals Important
    www.nature.com/scientificreports OPEN iTRAQ-based quantitative proteomic analysis reveals important metabolic pathways for Received: 22 September 2017 Accepted: 31 January 2018 arsenic-induced liver fbrosis in rats Published: xx xx xxxx Shunhua Wu 1, Jing Li1 & Xiang Jin1,2 Long-term consumption of sodium arsenite contaminated water can cause endemic arsenic disease. The proteome profle changes of liver fbrosis after exposure to arsenite containing water remain unclear. In this study, Sprague-Dawley (SD) male rats were treated with sodium arsenite (iAs3+), using a daily dose of 1.36 mg/kg body weight (medium dose group, M), 2.73 mg/kg body weight (high dose group, H) or deionized water (control group, C). Isobaric tags for relative and absolute quantitation (iTRAQ) were used to identify the diferent abundant proteins (DAPs) after arsenic-induced liver fbrosis. A total of 2987 high-quality proteins were detected (95% confdent peptides ≥ 2), 608 of which were diferentially expressed (fold change > 2 and p < 0.05) in M group and 475 in H group. Moreover, 431 DAPs were found in both M and H groups and used in subsequent bioinformatic analyses. Gene ontology (GO) analysis revealed 4,709 GO terms could be mapped, among which purine binding, actin flament binding and protein kinase binding were the most enriched terms for molecular function category. In addition, protein-protein interaction analysis showed six clusters of interaction networks. Our data provided new insights into the proteome changes after arsenic-induced liver fbrosis in model rats. In nature, trivalent arsenite [As (III)] is the most common oxidation states for soluble arsenic1.
    [Show full text]
  • 4-Hydroxy-2-Nonenal Antimicrobial Toxicity Is Neutralized by an Intracellular Pathogen
    RESEARCH ARTICLE 4-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen Hannah Tabakh1, Adelle P McFarland1,2, Maureen K Thomason1, Alex J Pollock1, Rochelle C Glover1, Shivam A Zaver1,2, Joshua J Woodward1* 1Department of Microbiology, University of Washington, Seattle, United States; 2Molecular and Cellular Biology Program, University of Washington, Seattle, United States Abstract Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Although studied extensively in the context of sterile inflammation, research into 4-HNE’s role during infection remains limited. Here, we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen Listeria monocytogenes induces many genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes lmo0103 and lmo0613, deemed rha1 and rha2 (reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on L. monocytogenes bacterial burdens during murine or tissue culture infection. However, heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE in vitro and promoted bacterial survival following phagocytosis by murine macrophages in an ROS-dependent manner. Thus, Rha1 and Rha2 are not necessary for 4- HNE resistance in L. monocytogenes but are sufficient to confer resistance to an otherwise *For correspondence: jjwoodwa@uw.edu sensitive organism in vitro and in host cells.
    [Show full text]
  • Recombinant Human GSTA4 Protein Catalog Number: ATGP3141
    Recombinant human GSTA4 protein Catalog Number: ATGP3141 PRODUCT INPORMATION Expression system E.coli Domain 1-222aa UniProt No. O15217 NCBI Accession No. NP_001503 Alternative Names Glutathione S-transferase alpha 4, GSTA4-4, GTA4 PRODUCT SPECIFICATION Molecular Weight 28.3 kDa (246aa) confirmed by MALDI-TOF Concentration 1mg/ml (determined by Bradford assay) Formulation Liquid in. 20mM Tris-HCl buffer (pH 8.0) 2mM DTT, 20% glycerol,100mM NaCl Purity > 95% by SDS-PAGE Biological Activity Specific activity is > 4,000pmol/min/ug, and is defined as the amount of enzyme that conjugate 1.0 u mole of 1- chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione per minute at pH 6.5 at 25C. Tag His-Tag Application Enzyme Activity,SDS-PAGE Storage Condition Can be stored at +2C to +8C for 1 week. For long term storage, aliquot and store at -20C to -80C. Avoid repeated freezing and thawing cycles. BACKGROUND Description GSTA4, also known as glutathione S-transferase A4, belongs to the GST superfamily. This enzyme is involved in cellular defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds. GSTA4 shows very high activity with reactive carbonyl compounds such as alk-2-enals. GSTA4 is highly effective in 1 Recombinant human GSTA4 protein Catalog Number: ATGP3141 catalyzing the conjugate addition of reduced glutathione to 4-hydroxynonenal, an important product of peroxidative degradation of arachidonic acid and a commonly used biomarker for oxidative damage in tissue. This enzyme is expressed at a high level in brain, placenta, and skeletal muscle and much lower in lung and liver. Recombinant human GSTA4 protein, fused to His-tag at N-terminus, was expressed in E.
    [Show full text]