Unfolded Protein Response and Cellular Stress, Part B (eBook)

Front Cover 1
Methods in Enzymology: The Unfolded Protein Response and Cellular Stress, Part B 4
Copyright 5
Contents 6
Contributors 12
Preface 18
Methods in Enzymology 20
Chapter 1: Methods for Investigating the UPR in Filamentous Fungi 50
1. Introduction 51
2. Conditions to Study the UPR in Filamentous Fungi 54
3. Analysis of Expression During the ER Stress 60
4. Modifying the UPR Signaling by Targeted Gene Replacement 63
Acknowledgments 74
References 75
Chapter 2: Assays for Detecting the Unfolded Protein Response 80
1. Introduction 82
2. Experimental Approaches for the Detection of ER Stress 85
3. Concluding Remarks 96
Acknowledgments 98
References 98
Chapter 3: Analysis of the Role of Nerve Growth Factor in Promoting Cell Survival During Endoplasmic Reticulum Stress in PC12 102
1. Introduction 103
2. Induction of ER Stress in PC12 Cells 104
3. Measurement of Caspase-3 Activity 111
4. Fragmentation of Caspase-12 112
5. Hyperinduction of GRP78 by NGF 114
Acknowledgments 117
References 117
Chapter 4: Measuring ER Stress and the Unfolded Protein Response Using Mammalian Tissue Culture System 120
1. Introduction 121
2. ER Stress and the UPR 122
3. Cell Culture System and ER Stress Induction 125
4. Measuring ER Stress 127
5. Studying the UPR Activation 128
6. Studying UPR Downstream Markers and Responses 133
Acknowledgments 137
References 137
Chapter 5: Real-Time Monitoring of ER Stress in Living Cells and Animals Using ESTRAP Assay 142
1. Introduction 143
2. SEAP Reporter System 144
3. Monitoring of ER Stress in Culture Cells by ESTRAP 145
4. Monitoring of ER Stress In Vivo by ESTRAP 150
5. Conclusion 153
References 154
Chapter 6: HIV Protease Inhibitors Induce Endoplasmic Reticulum Stress and Disrupt Barrier Integrity in Intestinal Epithelial Cells 156
1. The Pathophysiological Relevance of HIV PI-Induced ER Stress and UPR Activation in IECs 157
2. Analysis of HIV PI-Induced ER Stress and UPR Activation in Cultured IECs 158
3. Analysis of HIV PI-Induced ER Stress and Disruption of Barrier Integrity in the Intestine 163
4. Summary 167
Acknowledgments 167
References 167
Chapter 7: Dexamethasone Induction of a Heat Stress Response 170
1. Introduction 171
2. Evaluation of the Heat Stress Response in Patients Treated with Dexamethasone 173
3. Heat Stress Response and UPR in Mice and Cellular Models 175
References 183
Chapter 8: Detecting and Quantitating Physiological Endoplasmic Reticulum Stress 186
1. Introduction 187
2. Detecting UPR at the Level of UPR Sensors 188
3. Quantitating ER Stress at the Level of UPR Sensors 190
4. Detecting Levels of Other Common UPR Targets 191
5. Important Tips 192
6. Concluding Remarks 193
Acknowledgments 194
References 194
Chapter 9: PI 3-Kinase Regulatory Subunits as Regulators of the Unfolded Protein Response 196
1. The Unfolded Protein Response 197
2. PI 3-Kinase Regulatory Subunits as Modulators of the UPR 199
3. Assessing UPR Pathway Activation 200
References 206
Chapter 10: The Emerging Role of Histone Deacetylases (HDACs) in UPR Regulation 208
1. HDAC Enzymes and Cancer 208
2. HDAC Inhibitors 209
3. HDAC Inhibition and the UPR 211
4. Future Directions 221
References 222
Chapter 11: Immunohistochemical Detection of Activating Transcription Factor 3, a Hub of the Cellular Adaptive-Response Network 224
1. Introduction 225
2. An IHC Protocol for ATF3 230
Acknowledgments 241
References 241
Chapter 12: Experimental Approaches for Elucidation of Stress-Sensing Mechanisms of the Ire1 Family Proteins 244
1. Introduction 245
2. Monitoring In Vivo Activity of Mutated Ire1 Family Proteins 248
3. Interaction Between BiP and the Ire1 Family Proteins 255
4. Direct Interaction of Unfolded Proteins with Yeast Ire1 but not with Mammalian IRE1a 259
5. Conclusion and perspective 262
Acknowledgments 263
References 263
Chapter 13: Measurement and Modification of the Expression Level of the Chaperone Protein and Signaling Regulator GRP78/BiP... 266
1. Introduction 267
2. Detection of Total GRP78 269
3. Detection of Cytosolic GRP78 Isoform 273
4. Detection of Cell Surface GRP78 277
Acknowledgments 280
References 281
Chapter 14: The Endoplasmic Reticulum-Associated Degradation and Disulfide Reductase ERdj5 284
1. Introduction 285
2. Productive Folding of Newly Synthesized Proteins in the ER 287
3. ERAD: A Strategy for ERQC Mechanism 291
4. Conclusion 298
References 301
Chapter 15: Structural Insight into the Protective Role of P58(IPK) during Unfolded Protein Response 308
1. Endoplasmic Reticulum Stress and Unfolded Protein Response 309
2. P58(IPK) Might be a Dual-Function Protein 310
3. Knocking Out P58(IPK) in Mouse Models 310
4. P58(IPK) is an ER-Resident Hsp40 311
5. Crystal Structure of Mouse P58(IPK) TPR Domain 312
6. P58(IPK) Functions as a Molecular Chaperone to Bind Unfolded Proteins Using Subdomain I 313
7. Structural Basis for P58(IPK) J Domain-BiP Interaction 316
8. Working Model of P58(IPK) During UPR 316
9. Future Research 316
References 317
Chapter 16: Principles of IRE1 Modulation Using Chemical Tools 320
1. Introduction: The Unfolded Protein Response 321
2. Structural Biology of IRE1-XBP1 Signaling 325
3. Chemical Approaches to Modulate IRE1 Function 331
References 341
Chapter 17: Methods to Study Stromal-Cell Derived Factor 2 in the Context of ER Stress and the Unfolded Protein Response... 344
1. Introduction 345
2. Arabidopsis sdf2 T-DNA Insertion Lines 346
3. Inducing and Monitoring ER Stress in Arabidopsis sdf2 Mutants 348
4. Analyzing the ER Stress-Induced Expression Pattern of SDF2 in Arabidopsis 353
5. The Subcellular Localization of SDF2 358
6. Purification of Recombinant SDF2 Protein from E. coli 362
Acknowledgments 365
References 366
Chapter 18: Nitrosative Stress-Induced S-Glutathionylation of Protein Disulfide Isomerase 370
1. Introduction 371
2. Identification and Confirmation of S-Glutathionylated Proteins in Cells 373
3. Identification of Target Cysteine Residues 376
4. Characterization of Structural and Functional Consequences of S-Glutathionylated PDI 378
5. Summary 380
Acknowledgments 380
References 380
Chapter 19: Methods for Analyzing eIF2 Kinases and Translational Control in the Unfolded Protein Response 382
1. Introduction to Translational Control in the UPR 383
2. Measuring Activation of the eIF2 Kinase Pathway During ER Stress 387
3. Investigating General and Gene-Specific Translation Control 394
Acknowledgments 402
References 402
Author Index 406
Subject Index 424
Colour Plate 441