Thiol Redox Transitions in Cell Signaling, Part 2

Engineering of Fluorescent Reporters into Redox Domains to Monitor Electron Transfers\r......Page 38
Introduction\r......Page 39
The Problem: Low Sensitivity and Improperly Rate-Limited Assays for Redox Functions of Bacterial Peroxiredoxin Systems\r......Page 40
The Solution: Engineering of Fluorescent Redox Reporters into the N-Terminal Domain of AhpF and E. coli Grx1\r......Page 41
Engineering of Disulfide-Containing Electron Acceptor Domains to Detect Electron Transfers via Fluorescence Changes; Linkage of Fluorescein to Bacterial\rAhpC via a Reducible Disulfide Bond\r......Page 44
Solutions\r......Page 45
Proteins\r......Page 46
Generation of modified AhpC proteins linked to fluorescein via a disulfide bond\r......Page 47
Characterization of the fluorescence and activity of the S128W mutant of the NTD S. typhimurium AhpF\r......Page 49
Fluorescence-based peroxidase activity assays of S. typhimurium AhpC with S128W NTD using stopped-flow analysis\r......Page 50
Generation and testing of the F6W mutant of E. coli Grx1 as an electron donor to E. coli BCP\r......Page 52
References......Page 56
Blot-Based Detection of Dehydroalanine-Containing Glutathione Peroxidase with the Use of Biotin-Conjugated Cysteamine\r......Page 59
Introduction......Page 60
Oxidative Inactivation of Glutathione Peroxidase and the Conversion of Its Active Site Sec to DHA\r......Page 61
Preparation of Biotin-Conjugated Cysteamine......Page 63
Blot-Based Detection of DHA-GPx1 in RBCs......Page 64
Effects of Oxidative Stress on the Formation of DHA-GPx1 in RBCs......Page 65
Concluding Remarks......Page 66
References......Page 68
Analysis of the Redox Regulation of Protein Tyrosine Phosphatase Superfamily Members Utilizing a Cysteinyl-Labeling Assay\r......Page 71
Introduction......Page 72
Active-Site Structure, Catalysis, and Oxidation......Page 73
Detection Methods......Page 75
General Principle of the Assay......Page 76
Preparation of the Lysis Buffer......Page 78
Preparation of Cell Lysates......Page 79
Acute Stimulus-Induced Reversible Oxidation of PTPs......Page 80
Perspectives......Page 81
References......Page 84
Measuring the Redox State of Cellular Peroxiredoxins by Immunoblotting\r......Page 87
Introduction......Page 88
Principle of the method......Page 90
Alkylation of suspension cells......Page 91
Nonreducing electrophoresis and Prx Western blotting......Page 92
Examples......Page 93
Principle of method......Page 95
General method......Page 96
Discussion......Page 97
References......Page 100
Thiol Redox Transitions by Thioredoxin and Thioredoxin-Binding Protein-2 in Cell Signaling\r......Page 103
Functional Regulation of Redox-Sensitive Proteins by Thiol Modification......Page 104
Thioredoxin......Page 105
Thiol Reduction by the Thioredoxin Redox System......Page 106
Thiol-redox regulation by thioredoxin in kinase-mediated cellular signal transduction\r......Page 107
Thiol-redox regulation by thioredoxin in nuclear receptors and transcription factors-mediated cellular signal transductio\r......Page 108
Thioredoxin Superfamily......Page 109
Reversible Redox and Signal Regulation by Thioredoxin and Thioredoxin-binding Protein-2 (TBP-2)\r......Page 110
References......Page 112
Detection of Protein Thiols in Mitochondrial Oxidative Phosphorylation Complexes and Associated Proteins\r......Page 119
Introduction......Page 120
Preparation of mitochondria from liver......Page 122
Labeling of reduced (i.e., unmodified) mitochondrial protein thiols using IBTP\r......Page 123
Preparation and running of 1D BN-PAGE gels......Page 125
Separation of the protein complexes into their individual protein subunits using 2D BN-PAGE\r......Page 128
Immunoblotting protocol for gels......Page 130
Imaging and analysis of gels and blots......Page 131
Mass spectrometry identification of proteins......Page 133
Other Considerations......Page 140
Conclusion......Page 141
References......Page 142
Mitochondrial Thioredoxin Reductase: Purification, Inhibitor Studies, and Role in Cell Signaling\r......Page 145
Introduction\r......Page 146
Preparation and purification of mitochondria\r......Page 147
Freeze/thaw cycles and disruption of mitochondria\r......Page 148
DEAE-Sephacel chromatography\r......Page 149
Rechromatography on 2,5-ADP-Sepharose 4B\r......Page 150
Purification of TrxR2 from whole organs or cultured cells\r......Page 152
Estimation of Thioredoxin Reductase Activity\r......Page 153
Role in Cell Signaling\r......Page 154
References\r......Page 156
Measuring Mitochondrial Protein Thiol Redox State\r......Page 159
Introduction\r......Page 160
Quantification of Mitochondrial Protein Thiols\r......Page 163
Quantification of Glutathionylation of Mitochondrial Proteins\r......Page 166
Quantification of protein-bound glutathione\r......Page 167
Recycling assay for measurement of mitochondrial GSH, GSSG, and protein-bound GSH\r......Page 168
Identification of glutathionylated proteins and cysteine residues\r......Page 169
Assessment of S-Nitrosated Protein Thiols\r......Page 171
Quantification of protein S-nitrosothiols\r......Page 172
Selective labeling of S-nitrosated mitochondrial protein thiols\r......Page 173
Measurement of the Thioredoxin and Peroxiredoxin Redox States\r......Page 175
Measuring thioredoxin redox poise using the PEGylation assay\r......Page 176
Conclusions\r......Page 179
References......Page 180
Measurement of Extracellular (Exofacial) Versus Intracellular Protein Thiols \r......Page 184
Measurement of Mitochondrial Thiol Status\r......Page 185
Isolation of mitochondria\r......Page 186
Mitochondrial purity, yield, and special considerations for thiol status\r......Page 187
Measuring mitochondrial thiols with common thiol reagents\r......Page 188
Measurement of mitochondrial thiol status in situ using IBTP\r......Page 190
Global intracellular thiol measurement\r......Page 191
Measurement of exofacial thiol status in situ\r......Page 192
Measurement of exofacial thiol status by fractionation\r......Page 193
Miscellaneous other methods to measurement exofacial thiols\r......Page 194
Exofacial Thiol Status and Cancer\r......Page 195
References\r......Page 197
Redox Clamp Model for Study of Extracellular Thiols and Disulfides in Redox Signaling\r......Page 200
Key Concepts for Use\r......Page 201
Principles for Experimental Design\r......Page 202
Summary of Available Redox Clamp Studies\r......Page 206
Perspectives and Conclusion\r......Page 212
References......Page 213
Redox State of Human Serum Albumin in Terms of Cysteine-34 in Health and Disease\r......Page 215
HPLC Analysis\r......Page 216
Sample preparation\r......Page 219
Other species\r......Page 220
Albumin Thiol State and Exercise\r......Page 221
Albumin Oxidation in Disease\r......Page 222
Liver disease\r......Page 223
Albumin Thiol State During Aging\r......Page 225
References......Page 227
Methods for Studying Redox Cycling of Thioredoxin in Mediating Preconditioning-Induced Survival Genes and Proteins\r......Page 230
Hormetic Mechanism: Role of Redox Cycling of Thioredoxin......Page 231
Redox Functioning of Thioredoxin......Page 232
Implications of Preconditioning Protection from Preclinical and Clinical Studies......Page 233
Drugs Mimic Thioredoxin-Medicated Preconditioning-Induced Signaling and Protection in Cells\r......Page 235
The in vitro preconditioning human cell model......Page 236
NOS1 activity: Formation of NO and activation of cGMP\r......Page 237
Lipid peroxidation......Page 238
Molecular biology method for redox-sensitive genes, mRNA, and proteins\r......Page 239
Ex vivo autoradiographic image of nigrostriatal dopamine terminals in the rat brain\r......Page 240
Materials......Page 241
References......Page 242
Oxidative Stress, Thiol Redox Signaling Methods in Epigenetics\r......Page 246
Introduction......Page 249
Histone protein extraction from cells and tissues......Page 252
Histone acetylation assay......Page 253
Histone Acetylation by Immunoblotting......Page 254
HAT activity assay using kits......Page 255
HDAC assay sample preparation......Page 256
HDAC activity assay......Page 257
HDAC assay using commercial kits......Page 258
SIRT1 activity assay......Page 259
HDACs Levels by Immunoblotting......Page 260
Posttranslational Modifications of HDACs and SIRTs (Sirtuins 1-7) by Immunoprecipitation\r......Page 261
From cells......Page 262
Protein carbonylation......Page 263
Protein carbonylation using proteomics approach......Page 264
Biotin-switch assay for HDACs and SIRT1......Page 265
Chromatin Immunoprecipitation (ChIP) Assay......Page 266
Methods for ChIP assay using cells......Page 267
Methods for ChIP assay using tissues......Page 269
General considerations for ChIP assay......Page 270
Conclusions......Page 271
References......Page 272
Characterization of Protein Targets of Mammalian Thioredoxin Reductases\r......Page 278
Introduction......Page 279
Preparation of TR-immobilized Affinity Resins......Page 280
Method......Page 282
Materials......Page 283
Concluding Remarks and Future Perspectives......Page 285
References......Page 286
Alteration of Thioredoxin Reductase 1 Levels in Elucidating Cancer Etiology\r......Page 288
Introduction\r......Page 289
Mammalian cell lines and mice\r......Page 290
Targeted removal and reexpression of TR1\r......Page 291
Northern blot analysis\r......Page 292
Thioredoxin reductase activity assay\r......Page 293
Anchorage-independent growth assay\r......Page 294
In vitro chemotaxis and invasion assays\r......Page 295
Results and Discussion\r......Page 296
Phenotypic changes in siTR1 transfected cells\r......Page 297
Tumorigenesis of TR1-deficient cells\r......Page 299
In vitro and in vivo metastatic analysis of TR1-deficient cells\r......Page 301
Reexpression of TR1 in TR1-deficient cells\r......Page 302
Conclusions and Future Perspectives\r......Page 304
References......Page 305
Regulation of Apoptosis Signal-Regulating Kinase 1 in Redox Signaling\r......Page 309
Overview......Page 310
Cell culture......Page 313
Solutions......Page 314
Method 1: Preparation of glutathione S-transferase-tagged thioredoxin (GST-Trx)\r......Page 315
Method 2: Preparation of ASK1 protein......Page 316
Method 4......Page 317
Comment......Page 318
References......Page 319
Protocols for the Detection of S-Glutathionylated and S-Nitrosylated Proteins In Situ\r......Page 321
Protein S-Glutathionylation......Page 322
In situ detection of S-glutathionylated proteins\r......Page 323
Protein S-Nitrosylation......Page 325
In situ detection of S-nitrosylated proteins......Page 326
References......Page 327
Synthesis, Quantification, Characterization, and Signaling Properties of Glutathionyl Conjugates of Enals\r......Page 329
Introduction\r......Page 330
Synthesis\r......Page 332
Characterization of GS-HNE\r......Page 333
Synthesis and characterization of esterified glutathionyl conjugates\r......Page 335
Cellular metabolism of HNE\r......Page 337
Systemic metabolism of HNE\r......Page 338
Systemic metabolism of acrolein\r......Page 339
Effect of glutathionyl conjugates of HNE on acute peritonitis\r......Page 341
Conclusions\r......Page 342
References......Page 343
Thioredoxin and Redox Signaling in Vasculature-Studies Using Trx2 Endothelium-Specific Transgenic Mice\r......Page 346
Introduction......Page 347
Methods......Page 349
Systemic oxidative stress, total antioxidant, hemodynamic, and echocardiography studies\r......Page 350
Serum NOx, NO release, eNOS phosphorylation, and eNOS enzymatic assay, and vessel function assays\r......Page 351
Mouse EC culture and ROS measurement......Page 352
Caspase-9 activity assay......Page 353
References......Page 354