Cover_Mol Chaperones_MMB_787......Page 1
FrontMatter......Page 2
Molecular Chaperones:
Methods and Protocols......Page 4
Dedication......Page 6
Preface......Page 8
References......Page 10
Acknowledgments......Page 12
Contents......Page 14
Contributors......Page 16
1.1. Mammalian Cells Possess Multiple Hsfs with Diverse Functions......Page 20
2.1. Genomic DNA Identification, Isolation, and Analyses......Page 21
2.2. Plasmids and Phages......Page 22
3.1. Knockout of hsf1 Gene......Page 23
3.2. Knockout of hsf2 Gene......Page 28
3.3. Knockout of hsf4 Gene......Page 31
3.4. Physiological Function of Mice with a Targeted Disruption of the hsf1 , hsf2 , or hsf4 Gene......Page 33
4. Notes......Page 36
References......Page 38
1. Introduction......Page 40
2.5. Buffers......Page 42
3.1. Purification of
Heat Shock Factors
and In Vitro EMSA
......Page 43
3.1.2. Purification of HSF2 Proteins......Page 44
3.1.3. Nuclear Extraction from Tissue Culture Cells and EMSA......Page 45
3.2. Measuring HSF1 Binding to HSP Promoters In Vivo by the Chromatin Immunoprecipitation Assay......Page 46
3.3.1. Luciferase Reporter Assays for HSF Activity......Page 47
4. Notes......Page 48
References......Page 49
1. Introduction......Page 52
2. Steroid Hormone Receptor Hsp90 Clients......Page 55
3. Kinase Clients of Hsp90......Page 56
4. Nonsignal Transduction Hsp90 Clients......Page 58
References......Page 60
1. Hsp90 Molecular Chaperone......Page 64
2. Cytosolic Hsp90 Function is Dependent on Co-chaperone Proteins......Page 67
2.1. Co-chaperones that Bind the Amino-Terminal ATPase Domain of Hsp90 p23......Page 68
2.5. Co-chaperones that Bind the Middle Domain of Hsp90: Aha1......Page 69
2.6.3. Immunophilins......Page 70
2.6.5. FKBP51 and FKBP52......Page 71
2.7.5. CHIP......Page 72
2.8. Other Co-chaperones that Do Not Appear to Bind Hsp90 Directly but Are Known to Affect Hsp90 Client Protein Activity......Page 73
3. Assembly of Hsp90 Client Protein Complexes......Page 74
3.2. Intermediate Stage: Adaptor Proteins Recruit Hsp90 to Client Complexes......Page 75
3.3. Late Stage: Interaction with p23 and Immunophilins......Page 76
3.4. Evidence for Direct Interaction of Co-chaperones with Client Proteins......Page 78
4. Co-chaperones as Drug Targets......Page 79
References......Page 80
1. Introduction......Page 86
2. Materials......Page 88
3. Methods......Page 89
4. Notes......Page 91
References......Page 92
1. Introduction......Page 94
2.3. Buffers and Solutions......Page 96
3. Methods......Page 97
4. Notes......Page 98
References......Page 100
1. Introduction......Page 102
3.1. NEF Effects on Hsp70 Steady-State ATPase Activity......Page 104
3.2. Determination of Nucleotide Exchange Using Radiolabeled Nucleotide......Page 105
3.3. Determination of Nucleotide Release Rates by Stopped-Flow Instrumentation......Page 107
4. Notes......Page 108
References......Page 109
1. Introduction......Page 112
2.1. Protein Purifications......Page 113
3. Methods......Page 114
3.1.1. Purification of GST–NBD1–R Fusion Protein from E. coli Strain BL21(DE3)......Page 115
3.1.2. Purification of Recombinant Human Hsp70, Hdj-2, or CHIP Protein from E. coli Strain BL21(DE3)......Page 116
3.1.4. In Vitro Reconstitution of the Hsp70/Hsp40-Dependent U......Page 117
3.1.5. In Vitro Reconstitution of the Hsp70- and Hsp40-Independent CHIP Ubiquitin Ligase Activity......Page 119
4. Notes......Page 120
References......Page 121
1. Introduction......Page 124
2. Materials......Page 127
3.1. Cell Culture......Page 129
3.2.1. Cell Fractionation......Page 130
3.2.2. Native Size Analysis......Page 131
3.3. Immunoprecipitation, Interaction with Partners, or Client Proteins......Page 132
3.6. Modulation of HspB1 Level of Expression......Page 134
3.7. Expression of HspB1 Dominant-Negative Mutants Devoid of Holdase Activity......Page 135
References......Page 136
1. Introduction......Page 140
2.2. Cells and Vectors......Page 143
Phagemid Particles......Page 144
Simian Virus 40......Page 145
Lentivirus......Page 146
3.1.2. Nonviral Vectors......Page 149
Cationic Lipids......Page 150
Nanoparticles......Page 151
References......Page 152
1. Introduction......Page 156
2.4.1. Chemical Aggregation Test......Page 158
3.3. Cell Lysis......Page 159
4. Notes......Page 160
References......Page 162
1. Introduction......Page 164
2.2. Preparation of Cell/Tissue Extracts......Page 167
3. Methods......Page 168
4. Notes......Page 170
References......Page 171
1.1. The Flow Cytometer......Page 174
1.2. Flow Cytometry in HSP Research......Page 175
2.4. Detection of Intracellular Heat-Shock Proteins......Page 176
3.1. Isolation of Leukocytes Using the LWB Method......Page 177
3.3. Detection of Surface Heat-Shock Proteins......Page 178
3.4. Detection of Intracellular Heat-Shock Proteins......Page 180
4. Notes......Page 181
References......Page 182
1. Introduction......Page 184
2.2. Buffers and Reagents......Page 187
3.1. Sample Collection and Preparation......Page 188
3.1.1. Off-Line Preparation......Page 189
3.1.3. Immunodepletion......Page 190
3.1.4. In-Gel Digestion......Page 191
3.2.1. Standard Curves and Quality Control Standards......Page 192
3.2.2. Selectivity......Page 193
3.2.3. Matrix Effects......Page 194
3.2.4. HPLC-Chip/Mass Spectrometry Analysis......Page 195
Cell Lysate......Page 198
Small Molecules in Dried Blood Spot......Page 199
Steroids in Human Hair......Page 200
Heat-Shock Proteins......Page 201
4. Notes......Page 202
References......Page 204
1. Introduction......Page 208
2. Programs and Data Sources......Page 210
3.1.2. Enrichment of Known Interactions Using BioGRID......Page 211
3.2.1. Determination of the Number of Shared Interactors Among a Group of Chaperones......Page 212
3.2.2. Using Z -Score for Chaperone Module Identification......Page 213
3.2.3. Further Validation of Chaperone Modules: Retrieving Functional Modules from a Consensus Chaperone Network......Page 214
3.3.2. KEGG Pathway Relationship of Proteins......Page 216
3.3.4. Determining Probability of Pathway Relationship Between Two Chaperones in a Two-Component Functional Module......Page 217
4. Note......Page 220
References......Page 222
1. Introduction......Page 224
2.1. Hsp70 Is a Molecular Chaperone......Page 225
2.2.1. Cell Signaling and Apoptosis......Page 227
Hsp70 and Mitochondrial-Dependent Apoptosis......Page 228
Hsp70 and Alternatives, Caspase-Independent, Apoptosis-Like Pathways......Page 231
In Conclusion, Hsp70 Can Be Considered as the Quintessential Inhibitor of Apoptosis......Page 232
3.1. Hsp70: Tumorigenicity and Cancer Cell Resistance......Page 233
Targeting the PBD......Page 234
Targeting Hsp70 Co-chaperones......Page 235
4.1. Extracellular Hsp70 has an Immunological Function......Page 236
4.2. Immuno-Therapeutical Approaches Based on Hsp70......Page 238
5. Concluding Remarks......Page 240
References......Page 241
1. Introduction......Page 250
2.4. Method 4......Page 251
3.1. Determination of Fractions of Apoptotic and Necrotic Cells by Staining with Labeled Annexin V and PI......Page 252
3.2. Visualization of Apoptotic Cells by TUNEL Assay......Page 254
3.3. Detection of Apoptotic and Necrotic Cells by Staining with Hoechst 33342 and PI......Page 256
3.5. Clonogenic Assay......Page 258
4. Notes......Page 260
References......Page 262
1. Introduction......Page 264
3. Methods......Page 267
4. Notes......Page 270
References......Page 272
1. Introduction......Page 274
2.3. Preparation of DC–Tumor Fusions......Page 276
3.1. Generation of DC from Human Peripheral Blood Monocytes......Page 277
3.3. Cell Fusion (see Note 1)......Page 278
3.4. Extraction of Hsp70 Peptide Complexes from DC–Tumor Fusion Cell Products (see Note 2)......Page 279
4. Notes......Page 280
References......Page 281
1. Introduction......Page 286
2.3. Buffers and Solutions......Page 288
3.1.2. Tissue Processing......Page 289
3.4. Vaccine Preparation......Page 290
3.6.2. Coating Procedure......Page 292
4. Notes......Page 293
References......Page 294
1. Introduction......Page 296
2.1. Preparation of Recombinant Large HSPs and Protein Antigen......Page 297
2.4. Measuring the Immunogenicity of Chaperone Complex Vaccines In Vivo......Page 298
3.1. Preparation of Recombinant Large Heat-Shock Protein and Protein Antigen......Page 299
3.2. Complex Formation of Large Heat-Shock Protein and Protein Antigen......Page 300
3.3. Measuring the Chaperone Complex-Facilitated Antigen Cross-Presentation In Vitro......Page 302
3.4. Measuring the Chaperone Complex-Induced T-Cell Activation In Vivo......Page 303
4. Notes......Page 304
References......Page 305
1. Introduction......Page 308
2.2. Cell Lines......Page 309
3.1. Screening for HSP Receptors......Page 310
3.1.1. Alexa 488-Labeled Purified HSP70 Preparation......Page 311
3.1.2. Alexa 488-Labeled Purified Hsp90 Preparation......Page 312
3.1.3. HSP Binding Assay......Page 313
3.2. Studying HSP–SREC-I Interaction In Vivo......Page 317
4. Notes......Page 318
References......Page 319
1. Introduction......Page 322
2. Materials......Page 324
3.1. Assay Preparation Overview......Page 325
3.2.2. Background Migration Controls......Page 326
3.4.2. Harvesting of Cells......Page 327
3.5.2. Chambers......Page 328
3.5.4. Assembling Filter, Silicone Gasket, and Top Chamber......Page 329
3.8.1. Disassembly of the Chamber......Page 331
3.10. Washing the Chambers......Page 332
4. Notes......Page 333
References......Page 335
Index......Page 338