Nuclear Receptors

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Preface......Page 1
Table of Contents......Page 3
Contributors to Volume 353......Page 6
Introduction......Page 12
General Considerations......Page 14
Solid-Phase Extraction......Page 16
Reversed-Phase HPLC......Page 19
How Much Material is Required for Chemical Characterization?......Page 22
Activity-Guided Fractionation......Page 23
Transfection Protocol......Page 25
Luciferase and beta-Galactosidase Assays......Page 27
Structure Determination by Mass Spectrometry......Page 29
Exact Mass Measurement by ES-MS......Page 30
Electron-Impact Mass Spectrometry......Page 31
Concluding Remarks......Page 32
Biological Significance and Available Methods......Page 34
Content of Oxysterols in Tissues......Page 35
Methodology. Internal Standards......Page 37
Synthesis of [2H6]4beta-Hydroxycholesterol......Page 38
Synthesis of [2H6]7beta-Hydroxycholesterol and [2H6]7-Oxocholesterol......Page 39
Sample Preparation......Page 40
Determination of Oxysterols in Human PlasmaDetermination of 3β-Hydroxy-5-cholestenoic Acid in Plasma......Page 42
Common Procedures for the Quantitative Determination of Different Oxysterols......Page 43
Pitfalls......Page 45
Conclusions......Page 46
Introduction......Page 48
Engineering of Single Reactive-Cysteine ER Constructs......Page 49
Expression of Single Reactive-Cysteine ER Constructs......Page 50
Affinity Purification and Site-Specific Labeling of ER with a Single Fluorophore......Page 51
Characterization of the Native Activity of Fluorescent ER......Page 52
Choice of Fluorophores and Fluorescent Techniques......Page 53
Ligand Effects on the Thermodynamic Stability of ERalpha-LBD Dimers......Page 56
Ligand Effects on the Kinetic Stability of ERalpha-LBD Dimers......Page 58
A Functional Assay to Identify Ligand Character based on Selective Stabilization......Page 62
Conclusion......Page 64
Introduction......Page 65
Gel Filtration......Page 67
Scintillation Proximity Assay......Page 71
SPA Saturation Assay for Estrogen Receptor Alpha LBD......Page 72
Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET)......Page 74
Fluorescence Polarization (FP)......Page 78
ALPHAScreen Assay for Estrogen Receptor Beta (ERbeta)......Page 81
Summary......Page 82
Nuclear Receptor Ligands......Page 84
Biosynthesis......Page 85
Structural Studies of Ligand-Receptor Interactions......Page 86
Thyroid Hormone Receptor as Model System for Ligand Development......Page 87
Design and Synthesis of Thyromimetic GC-1......Page 89
Design of T3 Antagonists: Extension Hypothesis......Page 92
Design......Page 93
Properties......Page 96
Design......Page 97
Synthesis......Page 99
Properties......Page 100
Conclusions......Page 104
Introduction......Page 107
Basic Use of BLAST......Page 109
Data Mining in Nonannotated Sequences......Page 112
Basic Animal Phylogeny......Page 114
Alignment and Phylogeny......Page 117
Evolution of Nuclear Receptors......Page 121
Classification of Nuclear Receptors......Page 122
Dedicated Databases......Page 123
Nuclear Receptors in Other Databases......Page 124
Exploiting the Structure......Page 126
Promoter Prediction......Page 127
Data-Mining in Functional Genomics......Page 128
Conclusion......Page 129
Introduction......Page 131
Construction of Random Peptide Libraries in Bacteriophage M13......Page 137
Materials......Page 138
Methods......Page 139
Materials......Page 144
Methods......Page 145
Validation of Receptor-Interacting Peptides in Mammalian Cells......Page 151
Materials......Page 152
Use Receptor-Interacting Peptides to Inhibit Receptor Transcriptional Activity......Page 153
Methods for Detecting Domain Interactions in Nuclear Receptors......Page 156
Methods for Determining Nuclear Receptor Domain Interactions......Page 159
Procedure......Page 161
Procedure......Page 163
Procedure......Page 165
Introduction......Page 167
A Three-Hybrid Assay for SHP Interference......Page 169
Transfection......Page 171
Results......Page 172
Summary......Page 173
The Five-protein hsp90 Heterocomplex Assembly System......Page 175
Essential versus Nonessential Chaperones......Page 178
The Mechanism of Cleft Opening......Page 179
Immunoadsorption of GR......Page 182
Assay of Steroid-Binding Capacity......Page 183
Protein Purification......Page 184
Purification of hsp70 and hsp90......Page 185
Purification of Hop......Page 187
Purification of p23......Page 188
Conclusion......Page 189
Introduction......Page 190
Types of Phosphorylation Studies......Page 191
In Vitro versus In Vivo Phosphorylation......Page 194
Methods for Identification of Phosphorylation Sites......Page 195
Mass Spectrometry......Page 196
Approach for Use with Low Protein Levels......Page 198
In Vivo Labeling of Cells and Tissue Minces......Page 199
In Vitro Labeling of Nuclear Receptors......Page 201
Purification of Nuclear Receptors......Page 205
Procedure 3. Trypsin Digestion of Nuclear Receptor in a Gel Slice......Page 206
Comments and Critical Points......Page 207
Procedure 4. Reverse Phase HPLC......Page 208
Procedure 5. Alkaline Polyacrylamide Gel Electrophoresis......Page 210
Procedure 6. Two-Dimensional Phosphopeptide Mapping......Page 211
Secondary Proteinase Digestion......Page 214
Modified Manual Edman Degradation......Page 215
Procedure 8. Modified Manual Edman Degradation......Page 216
Procedure 9......Page 217
Comments and Critical Points......Page 219
Introduction......Page 222
Triton Acid Urea Gel Electrophoresis......Page 224
In Vivo Acetylation......Page 226
Protein Arginine Methyltransferases (PRMTs)......Page 229
Specificity of PRMTs......Page 231
Inter-Correlation Between Acetylation and Methylation of Histones......Page 233
Analysis of Oligomerization of PRMTs......Page 234
In Vitro Chromatin Assembly and Transcription Reactions......Page 235
Summary......Page 239
Introduction......Page 241
Background......Page 242
Design of Peptidomimetic Libraries......Page 245
CombiDOCK Method......Page 246
Synthesis of the Peptidomimetic Libraries......Page 250
Preparation of Fmoc Protected Amino Acids......Page 251
Preparation of Peptidomimetics......Page 253
Assay of the Peptidomimetic Efficacy and Selectivity......Page 256
In Vitro Assay Conditions......Page 257
Equilibrium Binding of the Probe to the Receptor......Page 259
FP Competition Procedure......Page 260
Data Analysis......Page 261
Conclusion......Page 262
Introduction......Page 265
Establishing a HDAC Assay......Page 267
Preparation of HDAC Substrate......Page 268
Purification of a Core SMRT Corepressor Complex from HeLa Cells......Page 269
Purification of SMRT Corepressor Complex......Page 271
Reconstitution of Enzymatically Active SMRT Corepressor Complex in Rabbit Reticulocyte Lysate......Page 272
Isolation of a Purified SMRT-HDAC 3 Complex from Baculovirus......Page 274
Isolation and Functional Characterization of the TRAP/Mediator Complex......Page 277
Isolation of the TRAP/Mediator complex......Page 278
Establishment and Propagation of Stable Cell Lines Expressing an Epitope-Tagged TRAP/Mediator Subunit......Page 279
Purification of TRAP/Mediator Complex from Nuclear Extract of the f:NUT2 Cell Line......Page 284
Functional Analysis of TRAP/Mediator Complex......Page 285
Purification of Pol II and GTFs......Page 286
In Vitro Transcription Assay for TRAP/Mediator Function on DNA Templates......Page 295
Alternative Assay System for TRAP/Mediator......Page 298
Introduction......Page 305
Cell Growth and Hormone Treatment......Page 308
Cell Harvest and Nuclear Isolation......Page 309
Chromatin Fragmentation......Page 310
Materials and Reagents......Page 311
Immunoprecipitation......Page 312
Optional Secondary Immunoprecipitation......Page 313
PCR Primer Design......Page 314
PCR Analysis, Gel Electrophoresis, and Data Analysis......Page 315
Introduction......Page 320
Two Approaches to Modulate the Transcriptional Activity of Nuclear Receptors......Page 321
Isolation of Nuclear Receptor Target Genes Using SAGE Technology......Page 322
The Underlying Principle of the SAGE Technology......Page 323
The Limitations of the SAGE Technology......Page 324
SAGE Data Analysis......Page 325
SAGE Target Identification and Analysis......Page 327
Generalities About Gene Trapping......Page 328
Inducible Over-Expression of the Nuclear Receptor......Page 329
Gene Trapping Strategy......Page 330
Production of the Trapping Library......Page 332
Analysis of the Trapping Library......Page 333
Future Perspectives......Page 335
Transporters for Nuclear Receptor Ligands......Page 344
General Approaches to Expression Cloning......Page 345
Strategy and General Concerns......Page 346
Isolation of Total RNA......Page 347
Isolation of Poly(A)+ RNA......Page 348
cDNA Synthesis......Page 349
cDNA Synthesis Using the SuperScript cDNA System (Invitrogen)......Page 350
Plasmid Expression Vectors......Page 351
Transformation of E. coli......Page 352
Preparation of Electroporation Competent Cells......Page 353
Selection of the Library Pool Size and Positive Controls......Page 354
Preparation of Library Pools......Page 356
Sibling Selection......Page 357
Cell Recipient......Page 358
Assay Development......Page 359
Library Screening......Page 361
Introduction......Page 362
Strategy......Page 363
Cell Culture and Chromatin Preparation......Page 364
Chromatin Immunoprecipitation......Page 366
Cloning of Fragments Isolated by ChIP......Page 367
Criteria for the Analysis of the Isolated Sequences......Page 368
Validation of the ChIP Experiment (Positive Control)......Page 369
Validation of a Newly Isolated and Cloned Sequence......Page 370
Changes in Gene Expression Levels Induced by Nuclear Receptors......Page 371
Conclusion......Page 372
Introduction......Page 374
Pathways of Eukaryotic mRNA Degradation......Page 375
Estrogen-Mediated Stabilization of Vitellogenin mRNA......Page 376
Preparation of Polysome Salt Extracts from X. laevis......Page 377
RNA Gel Mobility Shift Assays......Page 378
Preparation of the 32P-UTP Labeled RNA Probe......Page 379
Materials and Solutions......Page 382
Materials and Solutions......Page 383
Setting up a New RNA Gel Shift Assay......Page 384
Introduction......Page 386
Preparation of Mammalian cDNA Library from Kidney of VDR KO Mice......Page 389
Isolation of 1alpha(OH)ase cDNA by Expression Cloning......Page 393
Application......Page 400
Introduction......Page 403
Selective Expression of Cre-ERT2 in Basal Keratinocytes: K14-Cre-ERT2 Transgenic Mice......Page 408
Generation of a Floxed RXRalpha Mouse Line......Page 412
Temporally controlled Selective Disruption of RXRalpha in Basal Keratinocytes......Page 414
Phenotypic Analysis of Mice Lacking RXRalpha in Epidermal Keratinocytes......Page 415
Temporally controlled Ablation of both RARalpha and RARgamma in Adult Mouse Epidermis does not Impair Homeostasic Keratinocyte......Page 417
Role of RARs in RA-induced Epidermal Hyperproliferation......Page 420
Selective Disruption of the RXRalpha gene in Suprabasal Keratinocytes does not Result in RXRalpha Protein Depletion......Page 426
Conclusion and Future Directions......Page 428
Introduction......Page 433
Zebrafish Genetics and Vertebrate Physiology......Page 434
Zebrafish as a Model to Study Human Physiology and Disease......Page 435
Lipid Metabolism in Fish......Page 436
Optical Biosensors to Visualize Lipid Metabolism in Live Larvae......Page 437
Screening Strategies......Page 442
Prostanoid Synthesis and Signaling......Page 446
Functional Analyses of Vertebrate COX Proteins......Page 447
Molecular, Biochemical, Pharmacological and Functional Analyses of Zebrafish COXs......Page 448
Future Directions......Page 450
Summary......Page 451
Introduction......Page 452
Analysis of Auditory Function: the Auditory-Evoked Brainstem Response (ABR)......Page 453
Testing procedure......Page 454
Data Recording......Page 456
Age and Background Strain Considerations......Page 457
Materials......Page 458
Fixation and Decalcification......Page 459
Analysis of Gene Expression......Page 461
Materials......Page 463
Post-fixation......Page 464
Color Spot Test......Page 465
Posthybridization Washes......Page 466
Materials......Page 467
Pre-treatments/deproteination......Page 468
Histochemistry......Page 469
Materials......Page 470
Cochlear Explant Cultures......Page 471
Cochlear Dissection......Page 472
Cochlear RNA Analysis......Page 473
Introduction......Page 475
Western Blotting......Page 483
Identification of ER by Protein Sequencing......Page 484
Tissue Specific Problems and Special Retrieval and Fixation Techniques......Page 485
Immunohistochemistry with Paraffin-Imbedded Sections......Page 486
Double Immunohistochemical Staining Using Fluorescence Conjugated Secondary Antibody......Page 487
Western Blotting Analysis......Page 488
Sucrose Density Gradient Centrifugation and ER-Binding Assay......Page 489
Introduction......Page 491
Effector-Reporter Systems......Page 492
Protocol for an In Vitro Coculture Assay......Page 496
Conditioned Medium from Tissue or Primary Cell Cultures......Page 498
Isolation of DHA, A Novel RXR Ligand......Page 499
Conclusions......Page 502
Introduction......Page 504
The GAL4-LBD System in Drosophila......Page 505
The Constructs......Page 506
Histochemical Detection of beta-Galactosidase and Choice of the Reporter......Page 508
Detection of GAL4-LBD Activation In Vivo......Page 509
Strengths and Weaknesses of the Method......Page 511
The Construct......Page 513
The Promoter......Page 514
The Spacer......Page 516
Interpreting Phenotypic Effects......Page 517
Strengths and Weaknesses of the Method......Page 518
Conclusions......Page 519
Author Index......Page 520
Subject Index......Page 553