Packer Redox Cell Biology and Genetics, Part B

1. Auflage 2002
ISBN: 978-0-08-049700-6
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, Band Volume 353, 634 Seiten

Reihe: Methods in Enzymology

The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today-truly an essential publication for researchers in all fields of life sciences. - Protein Structure and Function - Nucleic Acids and Genes

Autoren/Hrsg.

Packer, Lester

Inhaltsverzeichnis

1;Cover;1
2;TIC$Table of Contents;6
3;Contributors to Volume 353;13
4;Preface;18
5;Volumes in Series;20
6;Section I: Protein Structure and Function;40
6.1;Chapter 1. Mammalian Two-Hybrid Assay Showing Redox Control of HIF-Like Factor;42
6.2;Chapter 2. Predicting Redox State of Cysteines in Proteins;49
6.3;Chapter 3. Enzyme-Linked Immunospot Assay for Detection of Thioredoxin and Thioredoxin Reductase Secretion from Cells;61
6.4;Chapter 4. Determination of Redox Properties of Protein Disulfide Bonds by Radiolytic Methods;74
6.5;Chapter 5. Crystal Structures of Oxidized and Reduced Forms of NADH Peroxidase;83
6.6;Chapter 6. Redox Control of Zinc Finger Proteins;93
6.7;Chapter 7. Quantification of Intracellular Calcineurin Activity and H2O2-Induced Oxidative Stress;109
6.8;Chapter 8. High-Performance Affinity Beads for Identifying Anti-NF-K B Drug Receptors;120
6.9;Chapter 9. Regulation of Protein Kinase C Isozyme Activity by S-Glutathiolation;128
6.10;Chapter 10. Detection and Affinity Purification of Oxidant- Sensitive Proteins Using Biotinylated Glutathione Ethyl Ester;140
6.11;Chapter 11. Redox Role for Tetrahydrobiopterin in Nitric Oxide Synthase Catalysis: Low-Temperature Optical Absorption Spectral Detection;153
6.12;Chapter 12. Lysozyme–Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria;160
6.13;Chapter 13. Alterations in Membrane Cholesterol That Affect Structure and Function of Caveolae;170
6.14;Chapter 14. Contribution of Neelaredoxin to Oxygen Tolerance by Treponema pallidum;179
6.15;Chapter 15. Redox Control of Integrin Adhesion Receptors;195
6.16;Chapter 16. Heme Oxygenase 1 in Regulation of Inflammation and Oxidative Damage;202
6.17;Chapter 17. Redox Properties of Vanillyl-Alcohol Oxidase;216
6.18;Chapter 18. Anaerobic Oxidations of Myoglobin and Hemoglobin by Spectroelectrochemistry;226
6.19;Chapter 19. Cysteine–Nitric Oxide Interaction and Olfactory Function;248
6.20;Chapter 20. Functional Evaluation of Nonphagocytic NAD(P)H Oxidases;259
6.21;Chapter 21. Purification and Assessment of Proteins Associated with Nitric Oxide Synthase;272
6.22;Chapter 22. Detection of Redox Sensor of Ryanodine Receptor Complexes;279
6.23;Chapter 23. Redox Control of 20S Proteasome;292
6.24;Chapter 24. Measuring Reactive Oxygen Species Inhibition of Endothelin-Converting Enzyme;302
6.25;Chapter 25. Redox Sensor Function of Metallothioneins;307
6.26;Chapter 26. SIR2 Family of NAD+-Dependent Protein Deacetylases;321
6.27;Chapter 27. Defining Redox State of X-Ray Crystal Structures by Single-Crystal Ultraviolet–Visible Microspectrophotometry;340
7;Section II: Nucleic Acids and Genes;358
7.1;Chapter 28. Model System for Developing Gene Therapy Approaches for Myocardial Ischemia–Reperfusion Injury;360
7.2;Chapter 29. Cloning and Characterization of Soluble Decoy Receptors;376
7.3;Chapter 30. Using Genetically Engineered Mice to Study Myocardial Ischemia–Reperfusion Injury;385
7.4;Chapter 31. Transgenic Model for the Study of Oxidative Damage in Huntington’s Disease;404
7.5;Chapter 32. Heme Oxygenase 1 Transgenic Mice as a Model to Study Neuroprotection;413
7.6;Chapter 33. Copper/Zinc Superoxide Dismutase Transgenic Brain in Neonatal Hypoxia–Ischemia;428
7.7;Chapter 34. Manganese Superoxide Dismutase Transgenic Mice: Characteristics and Implications;437
7.8;Chapter 35. Tissue-Specific Knockout Model for Study of Mitochondrial DNA Mutation Disorders;448
7.9;Chapter 36. Antisense Oligodeoxyribonucleotides. A Better Way to Inhibit Monocyte Superoxide Anion Production? ERIK A. BEY AND MARTHA K. CATHCART;460
7.10;Chapter 37. Transgenic Shuttle Vector Assays for Determining Genetic Differences in Oxidative B Cell Mutagenesis in Vivo;473
7.11;Chapter 38. Redox Control of Cell Cycle-Coupled Topoisomerase IIa Gene Expression;487
7.12;Chapter 39. Chemokine Expression in Transgenic Mice Over-producing Human Glutathione Peroxidases;499
7.13;Chapter 40. Analysis of Promoter Methylation and Its Role in Silencing Metallothionein I Gene Expression in Tumor Cells;515
7.14;Chapter 41. Functional Genomics: High-Density Oligonucleotide Arrays;526
7.15;Chapter 42. Reporter Transgenes for Study of Oxidant Stress in Caenorhabditis elegans;536
7.16;Chapter 43. Detection of DNA Base Mismatches Using DNA Intercalators;545
7.17;Chapter 44. Deoxyguanosine Adducts of tert-4-Hydroxy- 2-nonenal as Markers of Endogenous DNA Lesions;562
7.18;Chapter 45. Transcription-Coupled Repair of 8-Oxoguanine in Human Cells;575
7.19;Chapter 46. Nucleobase and 5'-Terminal Probes for DNA Redox Chemistry;587
7.20;Chapter 47. Expressed Sequence Tag Database Screening for Identification of Human Genes;605
8;Author Index;614
9;Subject Index;656

Leseproben

[1] Mammalian Two-Hybrid Assay Showing Redox Control of HIF-Like Factor
David Lando; Daniel J. Peet; Ingemar Pongratz; Murray L. Whitelaw Introduction
A growing number of transcription factors including activating protein-1 (AP-1), Myb, activating transcription factor/cAMP response element-binding protein (ATF/CREB), nuclear factor-?B (NF-?B), and p53 have been shown to have their DNA-binding activities modulated by changes in redox status (reviewed by Morel and Barouki1). For the Fos–Jun heterodimer that constitutes the AP-1 complex, the nuclear redox protein Ref-1 imparts DNA-binding activity by reducing specific cysteine sulfhydryl groups in the basic DNA-binding regions of Fos and Jun. Although the precise mechanism by which Ref-1 reduces Fos and Jun is not known, chemical cross-linking studies with bacterially expressed proteins have suggested that a direct cysteine-mediated interaction may occur between Ref-1 and Jun.2 However, in vivo cellular interactions between Ref-1 and Fos or Jun have not been demonstrated, nor have interactions been shown in cell extracts by standard biochemical techniques such as coimmunoprecipitation. This is most likely due to the transient nature of such interactions. The mammalian hypoxia-inducible factors HIF-1a and HIF-like factor (HLF) are two highly related basic helix–loop–helix/Per–Arnt–Sim homology (bHLH/PAS) transcription factors that are rapidly activated by oxygen deprivation to induce a network of genes responsible for maintaining oxygen homeostasis (reviewed by Semenza3). The molecular mechanisms of oxygen sensing and signaling that lead to the activation of HIF-1a and HLF are poorly understood. However, evidence suggests the involvement of an oxygen-regulated redox signal that may activate a kinase cascade or modify the HIFs directly.4 In support of this, over-expression of redox factors Ref-1 and thioredoxin enhances the hypoxic response of HIF-1a and HLF.5–7 We have shown that Ref-1 can specifically enhance the in vitro DNA-binding activity of HLF by reducing a cysteine residue in the basic DNA-binding domain of HLF.8 Here we describe a cell-based mammalian two-hybrid assay that is capable of detecting the intracellular interaction of Ref-1 with the basic DNA-binding domain of HLF. By using this assay we are able to demonstrate that the interaction between Ref-1 and HLF is dependent on a redox-sensitive cysteine in the basic region of HLF. Methods and Procedures
Principle
The mammalian two-hybrid system is a simple and inexpensive, yet powerful technique capable of assaying for protein–protein interactions within a cellular environment. Like the yeast two-hybrid system originally described by Fields and Song,9 this is an in vivo assay based on the functional reconstitution of a transcription activator. Typically, in this system, one protein of interest is expressed as a fusion protein with the yeast Gal4 DNA-binding domain (DBD), and the other protein is expressed as a fusion to the activation domain of the VP16 protein of the herpes simplex virus. The vectors that encode these fusion proteins are then cotransfected along with a luciferase reporter gene into a mammalian cell line. The expression of the luciferase gene within the reporter plasmid is under the control of Gal4-binding elements. If the two fusion proteins interact, this will bring together the Gal DBD and VP16 domains, forming a hybrid transcription activator capable of binding the Gal4 response elements and increasing the expression of the luciferase reporter gene. The increase in luciferase can then be quantitated with a luminometer. Plasmids
The mammalian expression vectors we use to analyze Ref-1 interaction with HLF and HIF-1a were constructed from pCMX/GalDBD and pCMX/VP16 vectors described previously8,10 and schematically outlined in Fig. 1. A description of general molecular biology techniques such as cloning, mutagenesis, and plasmid propagation and isolation can be found elsewhere.11 Briefly, the Gal4DBD chimeras contain the yeast DBD of Gal4 (amino acids 1–147) fused in frame with either full-length Ref-1 (GalDBD/Ref-1), HLF amino acids 1–265 (GalDBD/HLF 1–265), or HIF-1a amino acids 1–245 (GalDBD/HIF-1a 1–245). The VP16 chimeras contain the 78-amino acid activation domain from the herpes simplex virus fused in frame with either full-length Ref-1 (VP16/Ref-1), HLF amino acids 1–265 (VP16/HLF 1–265), or HLF 1–265 cysteine (amino acid residue 25)-to-serine point mutant (VP16/HLF 1–265 C25S). The luciferase reporter construct G5E1b-Luc contains five copies of the Gal4-binding site upstream of the minimal E1b promoter-driven firefly luciferase gene. Fig. 1 Schematic representation of GalDBD (A) and VP16 (B) fusion constructs used to analyze the interaction of Ref-1 with HLF and HIF-1a. The amino acid compositions of the basic regions for HLF and HIF-1a are included. bHLH, basic helix–loop–helix domain. Two-Hybrid Assay
Several methods to transfect plasmid DNA into mammalian cells are available, including calcium phosphate, DEAE-dextran, and liposome-mediated transfection. To analyze the in vivo interaction of Ref-1 with HLF we use the monkey kidney COS-1 cell line in conjunction with the liposomal transfection reagent LipofectAMINE 2000 (Life Technologies, Rockville, MD) and the Dual-Luciferase reporter assay system (Promega, Madison, WI). Although we have obtained similar results with other transfection reagents (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP; Boehringer Mannheim, Germany) and cell lines (human embryonic kidney, HEK293T), in our hands COS-1 cells and the LipofectAMINE 2000 reagent routinely give high transfection efficiencies, resulting in consistent two-hybrid results. A description of general mammalian cell tissue culture techniques is beyond the scope of this chapter; readers who require further information on these techniques are therefore referred to methods and protocols given elsewhere.11 For two-hybrid assays COS-1 cells are cultured in a humidified incubator (95% air/5% CO2) at 37° in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO-BRL, Gaithersburg, MD) supplemented with 10% (v/v) fetal calf serum (FCS; GIBCO-BRL). Before transfection, cells are trypsinized and plated onto 24-well tissue culture plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) at a density of 5 × 104 viable cells per well in a final volume of 450 µ1 of DMEM–FCS. After 24 hr (50–60% cell confluency), transfections are carried out with LipofectAMINE 2000 reagent as follows. 1. For each transfection (or well of cells), 425 ng of plasmid DNA consisting of 300 ng of G5Elb-Luc reporter, 50 ng of GalDBD or GalDBD chimera, 50 ng of VP16 or VP16 chimera, and 25 ng of Renilla internal control pRL-TK plasmid (Promega) is diluted into DMEM without FCS to a final volume of 25 µl. The pRL-TK control plasmid provides constitutive expression of Renilla luciferase from the herpes simplex virus thymidine kinase (TK) promoter and its addition serves as an internal control of transfection efficiency. 2. For each well of cells to be transfected, dilute 1 µ1 of LipofectAMINE 2000 reagent into 25 µ1 of DMEM without FCS. 3. Combine the 25 µ1 of plasmid-DMEM mix (step 1) with 25 µ1 of diluted LipofectAMINE 2000 reagent (step 2). Incubate the mixture at room temperature for 20 min to allow plasmid allow plasmid DNA–LipofectAMINE 2000 reagent complexes to form. Note: Diluted LipofectAMINE 2000 from step 2 must be combined with plasmid DNA mix within 10 min of preparation. 4. After incubation add the plasmid DNA–LipofectAMINE 2000 reagent complexes (50 µ1) directly to each well. To obtain consistency and high transfection efficiencies it is important to disperse the plasmid DNA–LipofectAMINE 2000 reagent complexes thoroughly and evenly throughout each well. This is best done by gently rocking the plate back and forth three or four times. 5. Return the transfected cells to the incubator and incubate for 24 to 48 hr. We have found that it is not necessary to remove the complexes or change the medium during the assay, as prolonged cell exposure to LipofectAMINE 2000 does not affect the transfection activity. 6. After transfection wash all wells once with 500 µ1 of phosphate-buffered saline solution and harvest with Dual-Luciferase lysis buffer (100 µ1/well; Promega). Lysed extracts (3 µ1) are then analyzed for luciferase activity in a Turner Designs (Sunnyvale, CA) T20/20 luminometer, using the Dual-Luciferase reporter assay system, and measured G5E1b-Luc reporter activity is normalized to the activity of the internal control (pRL-TK). Results and Discussion
Typical results of a two-hybrid assay showing Ref-1 interaction with HLF are...