Cadenas / Packer Thiol Redox Transitions in Cell Signaling, Part B

1. Auflage 2010
ISBN: 978-0-12-381004-5
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 392 Seiten

This volume, along with its companion (volume 474), presents methods and protocols dealing with thiol oxidation-reduction reactions and their implications as they relate to cell signaling. The critically acclaimed laboratory standard for 40 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. Over 450 volumes have been published to date, and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences.
*Along with companion volume, provides a full overview of techniques necessary to the study of thiol redox in relation to cell signaling
* Gathers tried and tested techniques from global labs, offering both new and tried-and-true methods
* Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

Autoren/Hrsg.

Cadenas, Enrique

Packer, Lester

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;14
6;Preface;20
7;Volumes in Series;22
8;Chapter 1: Engineering of Fluorescent Reporters into Redox Domains to Monitor Electron Transfers;50
8.1;1. Introduction;51
8.2;2. The Problem: Low Sensitivity and Improperly Rate-Limited Assays for Redox Functions of Bacterial Peroxiredoxin Systems;52
8.3;3. The Solution: Engineering of Fluorescent Redox Reporters into the N-Terminal Domain of AhpF and E. coli Grx1;53
8.4;4. Engineering of Disulfide-Containing Electron Acceptor Domains to Detect Electron Transfers via Fluorescence Changes; Linkage of Fluorescein to Bacterial AhpC via a Reducible Disulfide Bond;56
8.5;5. Materials;57
8.5.1;5.1. Solutions;57
8.5.2;5.2. Chemical modification agents;58
8.5.3;5.3. Proteins;58
8.6;6. Methods;59
8.6.1;6.1. Generation of modified AhpC proteins linked to fluorescein via a disulfide bond;59
8.6.2;6.2. Characterization of the fluorescence and activity of the S128W mutant of the NTD S. typhimurium AhpF;61
8.6.3;6.3. Fluorescence-based peroxidase activity assays of S. typhimurium AhpC with S128W NTD using stopped-flow analysis;62
8.6.4;6.4. Generation and testing of the F6W mutant of E. coli Grx1 as an electron donor to E. coli BCP;64
8.7;7. Summary;68
8.8;References;68
9;Chapter 2: Blot-Based Detection of Dehydroalanine-Containing Glutathione Peroxidase with the Use of Biotin-Conjugated Cysteam;72
9.1;1. Introduction;73
9.2;2. Oxidative Inactivation of Glutathione Peroxidase and the Conversion of Its Active Site Sec to DHA;74
9.3;3. Preparation of Biotin-Conjugated Cysteamine;76
9.4;4. Blot-Based Detection of DHA-GPx1 in RBCs;77
9.5;5. Effects of Oxidative Stress on the Formation of DHA-GPx1 in RBCs;78
9.6;6. Concluding Remarks;79
9.7;Acknowledgments;81
9.8;References;81
10;Chapter 3: Analysis of the Redox Regulation of Protein Tyrosine Phosphatase Superfamily Members Utilizing a Cysteinyl-Labeling Assay;84
10.1;1. Introduction;85
10.2;2. Active-Site Structure, Catalysis, and Oxidation;86
10.3;3. Detection Methods;88
10.4;4. General Principle of the Assay;89
10.5;5. Solutions;91
10.6;6. Preparation of the Lysis Buffer;91
10.7;7. Preparation of the Hypoxic Glove Box;92
10.8;8. Preparation of Cell Lysates;92
10.9;9. Cysteinyl-Labeling Assay;93
10.10;10. Acute Stimulus-Induced Reversible Oxidation of PTPs;93
10.11;11. Perspectives;94
10.12;12. Conclusion;97
10.13;References;97
11;Chapter 4: Measuring the Redox State of Cellular Peroxiredoxins by Immunoblotting;100
11.1;1. Introduction;101
11.2;2. Measurement of Prx Dimerization;103
11.2.1;2.1. Principle of the method;103
11.2.2;2.2. General method;104
11.2.3;2.3. Examples;106
11.3;3. Measurement of Prx Hyperoxidation;108
11.3.1;3.1. Principle of method;108
11.3.2;3.2. General method;109
11.3.3;3.3. Examples;110
11.4;4. Discussion;110
11.5;Acknowledgments;113
11.6;References;113
12;Chapter 5: Thiol Redox Transitions by Thioredoxin and Thioredoxin-Binding Protein-2 in Cell Signaling;116
12.1;1. Functional Regulation of Redox-Sensitive Proteins by Thiol Modification;117
12.1.1;1.1. Thioredoxin;118
12.2;2. Thiol Reduction by the Thioredoxin Redox System;119
12.2.1;2.1. Thiol-redox regulation by thioredoxin in kinase-mediated cellular signal transduction;120
12.2.2;2.2. Thiol-redox regulation by thioredoxin in nuclear receptors and transcription factors-mediated cellular signal transduction;121
12.3;3. Thioredoxin Superfamily;122
12.4;4. Reversible Redox and Signal Regulation by Thioredoxin and Thioredoxin-binding Protein-2 (TBP-2);123
12.5;5. Conclusion;125
12.6;References;125
13;Chapter 6: Detection of Protein Thiols in Mitochondrial Oxidative Phosphorylation Complexes and Associated Proteins;132
13.1;1. Introduction;133
13.2;2. Mitochondria Isolation and Protein Thiol Labeling;135
13.2.1;2.1. Preparation of mitochondria from liver;135
13.2.2;2.2. Labeling of reduced (i.e., unmodified) mitochondrial protein thiols using IBTP;136
13.3;3. Application of Blue Native-PAGE for the Isolation of Oxidative Phosphorylation Protein Subunits and Other Proteins Associated with the Complexes;138
13.3.1;3.1. Preparation and running of 1D BN-PAGE gels;138
13.3.2;3.2. Separation of the protein complexes into their individual protein subunits using 2D BN-PAGE;141
13.4;4. Detection of IBTP-Labeled Protein Thiols in Protein Complexes;143
13.4.1;4.1. Immunoblotting protocol for gels;143
13.5;5. Analysis and Mass Spectrometry Identification of Protein;144
13.5.1;5.1. Imaging and analysis of gels and blots;144
13.5.2;5.2. Mass spectrometry identification of proteins;146
13.6;6. Other Considerations;153
13.7;7. Conclusion;154
13.8;Acknowledgments;155
13.9;References;155
14;Chapter 7: Mitochondrial Thioredoxin Reductase: Purification, Inhibitor Studies, and Role in Cell Signaling;158
14.1;1. Introduction;159
14.2;2. Purification of Thioredoxin Reductase from Isolated Mitochondria, Cultured Cells, and Whole Organs;160
14.2.1;2.1. Preparation and purification of mitochondria;160
14.2.2;2.2. Freeze/thaw cycles and disruption of mitochondria;161
14.2.3;2.3. Heat treatment;162
14.2.4;2.4. Ammonium sulfate fractionation;162
14.2.5;2.5. DEAE-Sephacel chromatography;162
14.2.6;2.6. 20,50-ADP-Sepharose 4B affinity chromatography;163
14.2.7;2.7. .-Aminohexyl-Sepharose 4B;163
14.2.8;2.8. Rechromatography on 20,50-ADP-Sepharose 4B;163
14.2.9;2.9. Purification of TrxR2 from whole organs or cultured cells;165
14.3;3. Estimation of Thioredoxin Reductase Activity;166
14.4;4. Inhibitor Studies of Thioredoxin Reductase;167
14.5;5. Role in Cell Signaling;167
14.6;References;169
15;Chapter 8: Measuring Mitochondrial Protein Thiol Redox State;172
15.1;1. Introduction;173
15.2;2. Quantification of Mitochondrial Protein Thiols;176
15.3;3. Quantification of Glutathionylation of Mitochondrial Proteins;179
15.3.1;3.1. Quantification of protein-bound glutathione;180
15.3.2;3.2. Recycling assay for measurement of mitochondrial GSH, GSSG, and protein-bound GSH;181
15.3.3;3.3. Identification of glutathionylated proteins and cysteine residues;182
15.4;4. Assessment of S-Nitrosated Protein Thiols;184
15.4.1;4.1. Quantification of protein S-nitrosothiols;185
15.4.2;4.2. Selective labeling of S-nitrosated mitochondrial protein thi ols;186
15.5;5. Measurement of the Thioredoxin and Peroxiredoxin Redox States;188
15.5.1;5.1. Western blotting to measure reduced and oxidized peroxiredoxin 3;189
15.5.2;5.2. Measuring thioredoxin redox poise using the PEGylation assay;189
15.6;6. Conclusions;192
15.7;Acknowledgments;193
15.8;References;193
16;Chapter 9: Measurement of Extracellular (Exofacial) Versus Intracellular Protein Thiols;198
16.1;1. Measurement of Mitochondrial Thiol Status;199
16.1.1;1.1. Isolation of mitochondria;200
16.1.2;1.2. Mitochondrial purity, yield, and special considerations for thiol status;201
16.1.3;1.3. Measuring mitochondrial thiols with common thiol reagents;202
16.1.4;1.4. Measurement of mitochondrial thiol status in situ using IBTP;204
16.2;2. Measurement of Cytosolic Thiol Status;205
16.2.1;2.1. Global intracellular thiol measurement;205
16.3;3. Measurement of Exofacial Thiols;206
16.3.1;3.1. Measurement of exofacial thiol status in situ;206
16.3.2;3.2. Measurement of exofacial thiol status by fractionation;207
16.3.3;3.3. Miscellaneous other methods to measurement exofacial thiols;208
16.4;4. Exofacial Thiol Status and Cancer;209
16.5;References;211
17;Chapter 10: Redox Clamp Model for Study of Extracellular Thiols and Disulfides in Redox Signaling;214
17.1;1. Introduction;215
17.2;2. Key Concepts for Use;215
17.3;3. Principles for Experimental Design;216
17.4;4. Summary of Available Redox Clamp Studies;220
17.5;5. Perspectives and Conclusion;226
17.6;Acknowledgments;227
17.7;References;227
18;Chapter 11: Redox State of Human Serum Albumin in Terms of Cysteine-34 in Health and Disease;230
18.1;1. Background;231
18.2;2. HPLC Analysis;231
18.2.1;2.1. Sample preparation;234
18.2.2;2.2. Sample storage;235
18.2.3;2.3. Other species;235
18.3;3. Albumin Thiol State and Exercise;236
18.4;4. Influence of Supplementation;237
18.5;5. Albumin Oxidation in Disease;237
18.5.1;5.1. Nonserum albumin;238
18.5.2;5.2. Diabetes;238
18.5.3;5.3. Liver disease;238
18.5.4;5.4. Hemodialysis;240
18.6;6.Albumin Thiol State During Aging;240
18.7;7. Summary;242
18.8;References;242
19;Chapter 12: Methods for Studying Redox Cycling of Thioredoxin in Mediating Preconditioning-Induced Survival Genes and Proteins;246
19.1;1. Hormetic Mechanism: Role of Redox Cycling of Thioredoxin;247
19.2;2. Redox Functioning of Thioredoxin;248
19.3;3. Implications of Preconditioning Protection from Preclinical and Clinical Studies;249
19.4;4. Drugs Mimic Thioredoxin-Medicated Preconditioning-Induced Signaling and Protection in Cells;251
19.5;5. Methods and Materials;252
19.5.1;5.1. The in vitro preconditioning human cell model;252
19.5.2;5.2. Methods for measuring short-lived free radicals;253
19.5.3;5.3. Methods for studying the redox function of endogenous thioredoxin;255
19.5.4;5.4. Molecular biology method for redox-sensitive genes, mRNA, and proteins;255
19.5.5;5.5. Measurement of apoptosis;256
19.5.6;5.6. Ex vivo autoradiographic image of nigrostriatal dopamine terminals in the rat brain;256
19.5.7;5.7. Materials;257
19.5.8;5.8. Statistical analysis;258
19.6;References;258
20;Chapter 13: Oxidative Stress, Thiol Redox Signaling Methods in Epigenetics;262
20.1;1. Introduction;265
20.2;2. Histone Acetylation Assays Using [3H]-Acetate Incorporation;268
20.2.1;2.1. [3H]-Acetate incorporation in cell culture;268
20.2.2;2.2. Histone protein extraction from cells and tissues;268
20.2.3;2.3. Histone acetylation assay;269
20.3;3. Histone Acetylation by Immunoblotting;270
20.4;4. HAT Activity Assay;271
20.4.1;4.1. HAT assay using commercial kits;271
20.4.2;4.2. HAT activity assay using kits;271
20.5;5. HDAC Activity Assay Using [3H]-Labeled Histones;272
20.5.1;5.1. Preparation of [3H]-labeled histones;272
20.5.2;5.2. HDAC assay sample preparation;272
20.5.3;5.3. HDAC activity assay;273
20.6;6. HDAC Activity Assay;274
20.6.1;6.1. HDAC assay using commercial kits;274
20.6.2;6.2. HDAC immunoprecipitation and specific activity assay for various deacetylases using commercial kits;275
20.7;7. HDACs Levels by Immunoblotting;276
20.8;8. Posttranslational Modifications of HDACs and SIRTs (Sirtuins 1-7) by Immunoprecipitation;277
20.9;9. Preparation of Whole Cell Lysate;278
20.9.1;9.1. From cells;278
20.9.2;9.2. From tissues;278
20.10;10. Preparation of Cytoplasmic and Nuclear Proteins;278
20.10.1;10.1. From cells;278
20.10.2;10.2. From tissues;279
20.11;11. Redox-Mediated Posttranslational Modification Assays;279
20.11.1;11.1. Protein carbonylation;279
20.11.2;11.2. Biotin-switch assay;281
20.12;12. Chromatin Immunoprecipitation (ChIP) Assay;282
20.12.1;12.1. Methods for ChIP assay using cells;283
20.12.2;12.2. Methods for ChIP assay using tissues;285
20.12.3;12.3. General considerations for ChIP assay;286
20.13;13. Conclusions;287
20.14;Acknowledgments;288
20.15;References;288
21;Chapter 14: Characterization of Protein Targets of Mammalian Thioredoxin Reductases;294
21.1;1. Introduction;295
21.2;2. Preparation of TR-immobilized Affinity Resins;296
21.2.1;2.1. Materials;298
21.2.2;2.2. Method;298
21.3;3. Identification of Targets of Mammalian TRs in Cell Lysates;299
21.3.1;3.1. Materials;299
21.3.2;3.2. Method;301
21.4;4. Concluding Remarks and Future Perspectives;301
21.5;Acknowledgments;302
21.6;References;302
22;Chapter 15: Alteration of Thioredoxin Reductase 1 Levels in Elucidating Cancer Etiology;304
22.1;1. Introduction;305
22.2;2. Materials and Methods;306
22.2.1;2.1. Mammalian cell lines and mice;306
22.2.2;2.2. Targeted removal and reexpression of TR1;307
22.2.3;2.3. Monitoring TR1 expression;308
22.2.4;2.4. Tumor formation activity of TR1 knockdown cells assessed by in vitro and in vivo assays;310
22.3;3. Results and Discussion;312
22.3.1;3.1. Knockdown of TR1;313
22.3.2;3.2. Phenotypic changes in siTR1 transfected cells;313
22.3.3;3.3. Tumorigenesis of TR1-deficient cells;315
22.3.4;3.4. In vitro and in vivo metastatic analysis of TR1-deficient cells;317
22.3.5;3.5. Reexpression of TR1 in TR1-deficient cells;318
22.4;4. Conclusions and Future Perspectives;320
22.5;Acknowledgments;321
22.6;References;321
23;Chapter 16: Regulation of Apoptosis Signal-Regulating Kinase 1 in Redox Signaling;326
23.1;1. Overview;327
23.2;2. Materials;330
23.2.1;2.1. Cell culture;330
23.2.2;2.2. Reagents;331
23.2.3;2.3. Plasmids;331
23.2.4;2.4. Antibodies;331
23.2.5;2.5. Solutions;331
23.3;3. Methods;332
23.3.1;3.1. In vitro binding assay for ASK1 and Trx;332
23.3.2;3.2. Detection of endogenous ASK1 activation by phospho- ASK antibody;334
23.3.3;3.3. In vitro kinase assay;335
23.4;4. Comment;335
23.5;References;336
24;Chapter 17: Protocols for the Detection of S-Glutathionylated and S-Nitrosylated Proteins In Situ;338
24.1;1. Introduction;339
24.2;2. Protein S-Glutathionylation;339
24.2.1;2.1. In situ detection of S-glutathionylated proteins;340
24.3;3. Protein S-Nitrosylation;342
24.3.1;3.1. In situ detection of S-nitrosylated proteins;343
24.4;4. Summary;344
24.5;References;344
25;Chapter 18: Synthesis, Quantification, Characterization, and Signaling Properties of Glutathionyl Conjugates of Enals;346
25.1;1. Introduction;347
25.2;2. Synthesis, Quantification, and Characterization of Reagent Glutathionyl Conjugates of HNE;349
25.2.1;2.1. GS-HNE;349
25.2.2;2.2. Synthesis, purification, and characterization of reagent GS-DHN;352
25.2.3;2.3. Synthesis and characterization of esterified glutathionyl conjugates;352
25.3;3. Metabolism of HNE;354
25.3.1;3.1. Cellular metabolism of HNE;354
25.3.2;3.2. Systemic metabolism of HNE;355
25.3.3;3.3. Systemic metabolism of acrolein;356
25.4;4. Signaling Properties of Glutathionyl Conjugates of HNE;358
25.4.1;4.1. Effect of glutathionyl conjugates of HNE on acute peritonitis;358
25.5;5. Conclusions;359
25.6;Acknowledgments;360
25.7;References;360
26;Chapter 19: Thioredoxin and Redox Signaling in Vasculature-Studies Using Trx2 Endothelium-Specific Transgenic Mice;364
26.1;1. Introduction;365
26.2;2. Methods;367
26.2.1;2.1. Generation and characterization of the EC-specific transgenic mice expressing Trx2;368
26.2.2;2.2. Systemic oxidative stress, total antioxidant, hemodynamic, and echocardiography studies;368
26.2.3;2.3. Serum NOx, NO release, eNOS phosphorylation, and eNOS enzymatic assay, and vessel function assays;369
26.2.4;2.4. Mouse EC culture and ROS measurement;370
26.2.5;2.5. Localization of Trx2 by confocal immunofluorescence microscopy;371
26.2.6;2.6. Apoptosis assays;371
26.3;References;372
27;Author Index;374
28;Subject Index;382