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

Front Cover 1
Methods in Enzymology: The Unfolded Protein Response and Cellular Stress, Part C 4
Copyright Page 5
Contents 6
Contributors 14
Preface 20
Volume in Series 22
Section 1: New Approaches to Studying UPR and Cell Stress 52
Chapter 1: CFTR Expression Regulation by the Unfolded Protein Response 54
1. Introduction 55
2. Methods 57
Acknowledgments 73
References 73
Chapter 2: GRP78/BiP: Modulation ofGRP78/BiP in Altering Sensitivityto Chemotherapy 76
1. Introduction 77
2. Measurement of ER Stress In Vitro and In Vivo 78
3. Conclusion 87
References 87
Chapter 3: Targeting the Unfolded Protein Response in Cancer Therapy 88
1. Introduction 89
2. The UPR and Cancer 89
3. Targeting the UPR 91
4. Combination of EGF-SubA with Other UPR-Targeting Drugs 101
Acknowledgments 105
References 105
Chapter 4: Large-Scale Analysis of UPR-Mediated Apoptosis in Human Cells 108
1. Introduction 109
2. Monitoring Proliferation and Caspase Activation Following UPR Activation 111
3. Monitoring the Expression of UPR and Cell Death Target Genes 112
4. DNA Fragmentation Analysis 119
Acknowledgments 120
References 120
Chapter 5: Quantitative Analysis of Amino Acid Oxidation Markers by Tandem Mass Spectrometry 124
1. Introduction 126
2. Experimental Procedures 127
3. Results 133
4. Conclusions 137
Acknowledgments 137
References 138
Chapter 6: Animal Models in the Study of the Unfolded Protein Response 142
1. Introduction 143
2. Activating Transcription Factor 6 144
3. IRE1/X-Box-Binding Protein-1 145
4. PKR-Like ER Kinase 148
5. eIF2a 149
6. ATF4 150
7. CHOP 151
8. GADD34 152
9. P58IPK 153
10. Transgenic Mouse Models for Monitoring ER Stress 153
11. UPR and Lipid Metabolism 154
12. UPR, Hypoxia, and Cancer 155
13. UPR and Inflammatory-Mediated Demyelination 156
14. Future Challenges 156
References 157
Chapter 7: Measurement of Fluoride-Induced Endoplasmic Reticulum Stress Using Gaussia Luciferase 162
1. Introduction 163
2. Properties of Gaussia Luciferase 165
3. Materials 166
4. Procedure 169
5. Conclusions 174
References 175
Chapter 8: Analysis of Nelfinavir-Induced Endoplasmic Reticulum Stress 178
1. Introduction 179
2. Obtaining Nelfinavir 180
3. Using Nelfinavir 180
4. Analysis of Nelfinavir-Induced ER Stress 181
5. Immunoblot Analysis 185
6. RT-PCR Analysis 186
7. Conclusions 192
Acknowledgment 193
References 193
Chapter 9: Using Temporal Genetic Switches to Synchronize the Unfolded Protein Response in Cell Populations In Vivo 194
1. Introduction 195
2. Variable Functions of CHOP in the PERK Signaling Pathway 197
3. Mutations in the PLP1 Gene: A Model UPR Disease 198
4. Problems with Current Paradigms Used to Characterize the UPR 200
5. A Novel In Vivo Genetic Switch Solves Many of These Problems 201
6. Generalizing GST to Study Other UPR Diseases 208
Acknowledgments 208
References 209
Chapter 10: Glycoprotein Maturation and the UPR 214
1. Introduction 214
2. N-glycosylation 216
3. O-glycosylation 226
Acknowledgment 232
References 232
Chapter 11: Monitoring and Manipulating Mammalian Unfolded Protein Response 234
1. Introduction 235
2. Monitoring Mammalian UPR 236
3. Chemical-Genetic Manipulation of Mammalian UPR 240
4. Concluding Remarks 245
Acknowledgments 246
References 246
Chapter 12: A Screen for Mutants Requiring Activation of the Unfolded Protein Response for Viability 250
1. Introduction 251
2. Screening for Mutants That Require UPR Activation for Viability 253
3. Cloning and Sequencing per Genes 260
4. Monitoring the UPR Activity 263
5. SGA Database 264
6. Media Recipes 265
7. Closing Remarks 265
Acknowledgment 266
References 266
Chapter 13: Signaling Pathways of Proteostasis Network Unrevealed by Proteomic Approaches on the Understanding of Misfolded Protein Rescue 268
1. Introduction 269
2. 2DE-Based Proteomics Approach Analysis to Study Protein Expression Profile 270
3. Experimental Design 271
4. 2D Gel Electrophoresis 272
5. Protein Identification by MS 274
6. Biochemical Validation 276
7. Data Mining and Publication 277
Acknowledgments 282
References 282
Chapter 14: Decreased Secretion and Unfolded Protein Response Upregulation 286
1. Introduction 287
2. Heterologous Protein Expression 288
3. Quality Control Mechanisms of the Secretory Pathway 289
4. Endoplasmic Reticulum Export and Trafficking 290
5. Experimental Systems to Evaluate Expression, UPR, and Secretory Processing 290
6. S. cerevisiae Strains Used for Optimal Expression 291
7. Plasmid Design 292
8. Evaluation of Heterologous Protein Expression 294
9. Statistical Analysis of Microarray Results 299
Acknowledgment 308
References 308
Chapter 15: Measuring Signaling by the Unfolded Protein Response 312
1. Introduction 313
2. Methods to Induce the UPR 317
3. Measuring Signaling by the UPR in S. cerevisiae 317
4. Measuring Signaling by the UPR in Mammalian Cells 327
Acknowledgments 339
References 340
Chapter 16: Quantitative Measurement of Events in the Mammalian Unfolded Protein Response 344
1. Introduction 345
2. Cell Culture and ER Stress Inducers 346
3. Northern Blots for GRP78/BiP and EDEM 347
4. RT-PCR Analysis of XBP1 mRNA Splicing 350
5. Immunoblotting and Measurement of ATF6 353
6. Measurement of Inhibition of Protein Synthesis 353
7. Measurement of Stimulation of LLO Extension 354
8. Calculations of Transcript Accumulation, Signal Activation, and Correlation 355
9. Conclusion 356
Acknowledgments 357
References 357
Chapter 17: Regulation of Immunoglobulin Synthesis, Modification, and Trafficking by the Unfolded Protein Response: A Quantitative Approach 360
1. Introduction 361
2. Isolation of Splenic B Cells and Infection Thereof by Retroviruses 363
3. Measurement of Protein Synthesis in Plasma Cells 367
4. Measurement of Ig Mislocalization in Primary B Cells 371
Acknowledgments 375
References 375
Chapter 18: Use of Chemical Genomics in Assessment of the UPR 378
1. Introduction 379
2. Assessment of the Activation of UPR Transcriptional Program in Cancer Cell 380
3. Gene Expression Signature-Based Identification of UPR Modulators 385
4. Future Perspective of Chemical Genomics in UPR Research 389
Acknowledgments 390
References 390
Chapter 19: Small GTPase Signaling and the Unfolded Protein Response 394
1. Introduction 395
2. Materials and Methods 397
3. Monitoring the Unfolded Protein Response 399
4. Monitoring Small GTPase Activity 404
5. Pharmacological and Genetic Modulation of UPR and GTPase Signaling 407
Acknowledgments 409
References 409
Chapter 20: Inhibitors of Advanced Glycation and Endoplasmic Reticulum Stress 412
1. Introduction 413
2. Advanced Glycation and Its Pathophysiology 414
3. Measurement of Advanced Glycation 415
4. Link Between Advanced Glycation and ER Stress 420
5. Effects of Advanced Glycation Inhibitor Against ER Stress 424
6. Conclusion 428
Acknowledgments 428
References 428
Author Index 432
Subject Index 450
Color Plates 460