Chemokines, Part B

Series Editor Page......Page 1
Copyright-Page.pdf......Page 2
Contributors-to-Volume-461.pdf......Page 3
Preface.pdf......Page 7
Methods in Enzymology......Page 8
Isolation, Identification, and Production of Posttranslationally Modified Chemokines......Page 35
Introduction......Page 36
Isolation and Stimulation of Peripheral Blood Mononuclear Cells (PBMC)......Page 37
Concentration of Isolated Proteins......Page 38
Specific Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)......Page 39
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)......Page 40
Ion Exchange Chromatography......Page 41
Ion Trap Mass Spectrometry......Page 42
Edman Degradation......Page 43
Total Protein Quantification Methods......Page 44
Illustration: Isolation and Identification of Natural Posttranslationally Modified CXCL8 Isoforms......Page 45
Solid-Phase Peptide Synthesis......Page 46
Synthesis of the peptide chain......Page 47
Detailed synthesis protocol......Page 49
Folding of the raw protein......Page 51
Citrullination of Chemokines......Page 53
Identification of Enzymes Generating the Natural Posttranslationally Modified Chemokines, as Exemplified by Aminopeptidase N(APN)/CD13......Page 55
Comparison of the Heparin-Binding Properties of Chemokine Isoforms......Page 56
References......Page 57
Homo- and Hetero-Oligomerization of Chemokines
......Page 62
Introduction......Page 63
Analytical ultracentrifugation (AUC); sedimentation equilibrium......Page 66
Pulsed-field gradient diffusion by NMR......Page 70
Dynamic light scattering......Page 71
Fluorescence polarization......Page 72
Size exclusion chromatography......Page 73
NMR: Heteronuclear single quantum correlation (HSQC) spectroscopy......Page 74
NMR: Detection of nuclei in close proximity by means of the nuclear Overhauser effect (NOE)......Page 75
Filtered NOE experiments......Page 76
References......Page 78
Lymphotactin Structural Dynamics......Page 82
Introduction......Page 83
Production of Biologically Active Lymphotactin......Page 85
Ltn Folds into Two Unrelated Native State Structures......Page 87
Kinetics of Interconversion in the Ltn Conformational Equilibrium......Page 88
Engineering Conformationally Restricted Lymphotactin Variants......Page 91
Functional Analysis of Distinct Ltn Native State Conformations......Page 92
GAG Binding Residues are Linked to the Ltn Conformational Equilibrium......Page 96
Conclusions......Page 98
References......Page 99
Interactions of Chemokines with Glycosaminoglycans
......Page 102
Introduction......Page 103
Heparin affinity chromatography......Page 111
Equilibrium competition binding......Page 112
Materials and methods......Page 113
Tritiated heparin binding assay......Page 114
Enzyme-linked immunosorbent saturation binding assay......Page 115
In vivo cellular recruitment......Page 116
Materials and methods......Page 117
Materials and methods......Page 118
Surface plasmon resonance (SPR)......Page 119
Materials and methods......Page 120
Sedimentation equilibrium analytical ultracentrifugation......Page 121
NMR: Heteronuclear single quantum correlation (HSQC) spectroscopy......Page 122
Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS)......Page 124
Materials and methods......Page 126
Emerging mass spectrometry methods for characterizing protein:GAG interactions: Hydrogen deuterium exchange and radiolytic oxidation......Page 127
Summary......Page 128
References......Page 129
Multiple Approaches to the Study of Chemokine Receptor Homo- and Heterodimerization......Page 134
Introduction......Page 135
Biochemical Techniques to Measure Chemokine Receptor Oligomerization......Page 136
Western blot and immunoprecipitation......Page 137
Colocalization assays......Page 138
Fluorescence labeling of antibodies......Page 139
Resonance Energy Transfer (RET) Techniques......Page 140
Bioluminiscence resonance energy transfer (BRET) techniques......Page 141
Fluorescent resonance energy transfer (FRET) techniques......Page 142
Sequential BRET-FRET (SRET) Technology......Page 147
Acknowledgments......Page 148
References......Page 149
Plasmon Resonance Methods in Membrane Protein Biology: Applications to GPCR Signaling
......Page 152
Introduction......Page 153
Description of surface plasmons......Page 154
Conventional surface plasmon resonance (SPR)......Page 155
Plasmon-waveguide resonance (PWR)......Page 157
Sensor Construction and Sample Deposition......Page 158
Lipid Bilayer Deposition......Page 160
Spectral Data Analysis......Page 161
Membrane Protein Insertion......Page 165
Ligand and G-Protein Binding by GPCRs......Page 167
Conclusions......Page 170
References......Page 173
Tyrosine Sulfation of HIV-1 Coreceptors and Other Chemokine Receptors......Page 176
Disovery of tyrosine-sulfated peptides and proteins......Page 177
Tyrosine sulfation of HIV-1 coreceptors......Page 178
Tyrosine sulfation of other chemokine and related receptors......Page 179
Tyrosine sulfation of coreceptor-binding site antibodies......Page 182
Useful properties of sulfotyrosine......Page 183
Chemically synthesized tyrosine-sulfated peptides......Page 184
Production of sulfated peptides in mammalian cells......Page 185
Modulation of peptide sulfation......Page 187
Uses of tyrosine-sulfated peptides......Page 188
Mutagenesis of candidate sulfotyrosines......Page 189
A system to introduce non-native amino acids into proteins......Page 190
Advantages and uses of the system......Page 191
Radiolabeled reagents and labeling media......Page 192
General considerations......Page 193
General considerations......Page 194
General considerations......Page 195
Conclusions......Page 196
References......Page 197
Activation Mechanisms of Chemokine Receptors......Page 200
Introduction......Page 201
Current Models for 7TM Receptor Activation......Page 204
Constitutive Activity of 7TM Receptors......Page 205
CCR8 small molecules-semiclassic pharmacophores for CC-chemokine receptors......Page 206
CXCR3-small-molecule anchorage......Page 207
CCR1-allosteric modulators......Page 210
Experimental Procedures......Page 212
Day two: Seeding and incubation with 3H-myoinositol......Page 214
References......Page 215
The Duffy Antigen Receptor for Chemokines......Page 220
Introduction......Page 221
Methods for the Study of DARC as a Malarial Receptor......Page 223
Methods for the Study of DARC as a Chemokine Sink......Page 227
Isolation of Erythrocytes and Measurement of Chemokines......Page 228
DARC as a Chemokine Transcytosis Receptor......Page 229
The Assay of Chemokine Transcytosis by DARC......Page 230
Conclusions......Page 231
References......Page 232
Introduction......Page 236
Coimmunoprecipitation......Page 237
Coimmunoprecipitation protocol following heterologous expression of epitope-tagged chemokine receptors......Page 240
Resonance Energy Transfer Techniques......Page 241
Single-point BRET2 protocol......Page 242
Saturation BRET2 protocol......Page 243
Data analysis......Page 244
Time-resolved FRET......Page 245
Protocol......Page 246
FRET imaging in living cells......Page 247
Data analysis......Page 249
Developing Techniques......Page 250
References......Page 251
Subsecond Analyses of G-Protein Coupled-Receptor......Page 255
GPCR biology......Page 256
Introduction......Page 257
Standardization of flow cytometry data with fluorescence calibration beads......Page 258
General considerations......Page 259
Materials......Page 260
Materials......Page 262
Modular assembly of ternary complexes on beads......Page 263
Preparation of M2 beads......Page 264
Preparation of G (alphabetagamma) on M2 beads......Page 265
Assembly of LRG (alphabetagamma) on beads......Page 266
Analysis of Modular Dissassembly of LRG Modules......Page 267
Real-time spectrofluorometric measurement of the dissociation of L from R......Page 268
Rapid mix measurement of guanine nucleotide-induced disassembly of ternary complexes......Page 270
Rapid mix measurement of the dissociation of R from Galphabetagamma: disassembly of modules in Fig. 11.2A,D......Page 271
Rapid-mix measurement of the dissociation of LF from RGalphabetagamma: disassembly of module in Fig. 11.2C......Page 272
References......Page 273
The Use of Receptor Homology Modeling to Facilitate the Design of Selective Chemokine Receptor Antagonists......Page 276
Introduction......Page 277
Protocol development......Page 287
Application of the balloon expansion approach to model CCR2 antagonists......Page 292
Comparison of our beta2- and rhodopsin-derived models of CCR2 antagonist docking......Page 294
Application of the beta2 template to develop a new model of CCR1: Comparison to rhodopsin and MembStruk models......Page 296
Application of the beta2 template to develop a new model of CCR5......Page 299
Conclusions......Page 301
References......Page 303
Characterizing Proteolytic Processing of Chemokines by Mass Spectrometry, Biochemistry, Neo-Epitope Antibod......Page 307
Introduction......Page 308
In vitro cleavage assays......Page 309
Endogenous protease cleavage of chemokine......Page 312
Detecting chemokine cleavage by TRIS-tricine PAGE......Page 313
Identifying cleavage sites by mass spectrometry......Page 315
Detection of processed chemokines by neo-epitope antibodies......Page 320
In vitro functional characterization of proteolysis......Page 321
Transwell migration as an indication of chemotactic potential......Page 323
Intracellular calcium mobilization to evaluate receptor activation......Page 326
Binding to glycosaminoglycans......Page 327
Chemotaxis in air pouch and peritonitis models......Page 329
Acknowledgments......Page 330
References......Page 331
Real-Time In Vitro Assays
For Studying the Role of
Chemokines in Lymphocyte
Transendothelial Migration Under
Physiologic Flow Conditions......Page 334
Introduction......Page 335
Live imaging microscopy of human T-cell crawling and TEM......Page 337
Equipment......Page 339
Preparation of effector T-cells......Page 340
Measurements of lymphocyte crawling and transendothelial migration under shear flow......Page 341
Analysis of lymphocyte crawling and TEM......Page 342
Isolation and preparation of murine T-splenocytes......Page 345
Live fluorescence imaging of T-cell crawling and transendothelial migration......Page 346
Immunofluorescent staining of integrins, integrin ligands, and cytoskeletal adaptors in crawling and transmigrating T-c......Page 347
Procedure......Page 348
Tracking transiently expressed fluorescent-tagged proteins on endothelial cells in real time......Page 350
Procedure......Page 351
References......Page 353
A Microfluidics-Based Method for Analyzing Leukocyte Migration to Chemoattractant Gradients......Page 356
Chemoattractants, chemoattractant receptors, and leukocyte trafficking......Page 357
Microfluidic devices for cell migration research......Page 358
Design of microfluidic devices......Page 359
Substrate preparation......Page 360
Principle of microfluidic gradient generation......Page 361
Isolation of human blood leukocytes......Page 362
Assembly of microfluidic system......Page 363
Time-lapse optical microscopy......Page 364
Characterizations of cell motility......Page 366
Subregion analysis......Page 367
Conclusion......Page 368
References......Page 369
Two-Photon Microscopy and Multidimensional Analysis of Cell Dynamics......Page 371
Introduction......Page 372
Scan heads and femtosecond lasers......Page 374
Signal detection and optical filters......Page 377
Laser attenuation and fast shuttering......Page 378
Pulse compression......Page 379
Dye-labeling protocol for leukocytes......Page 380
Genetically encoded fluorescent proteins......Page 381
Explant imaging......Page 382
Explant imaging protocol......Page 383
Intravital imaging protocol......Page 385
Imaging peripheral tissues......Page 386
Image Acquisition......Page 387
Laser power and PMT gain......Page 388
Z-series acquisition and time resolution......Page 389
Cell detection......Page 390
Cell and tissue morphology......Page 391
Analysis of cell migration......Page 392
Motility coefficient......Page 393
Turning angle......Page 394
2-D images......Page 395
Cell tracks......Page 396
References......Page 397
Zymosan-Induced Peritonitis as a Simple Experimental System for the Study of Inflammation......Page 401
Acute and chronic inflammation......Page 402
Peritoneal models of acute inflammation......Page 403
Applications of the ZIP model......Page 404
Materials......Page 406
Lavaging the peritoneal cavity......Page 407
Cell counting and FACS staining to determine cellular composition......Page 408
Data calculations......Page 409
Leukocyte recruitment......Page 410
Inflammatory mediators......Page 412
Dose of zymosan......Page 414
Gr-1 or Ly-6G?......Page 415
References......Page 416
A Chemokine-Mediated In Vivo T-Cell Recruitment Assay......Page 419
Introduction......Page 420
Purification of CD8 T lymphocytes and preparation of antigen-presenting cells......Page 421
Culturing of CD8 T lymphocytes......Page 423
In vitro characterization of CD8 T lymphocytes......Page 424
Adoptive transfer of T lymphocytes......Page 426
Intratracheal instillation of chemokines......Page 427
Bronchial alveolar lavage and T lymphocyte analysis......Page 428
Use of In Vivo Recruitment Assay for Chemokine Studies......Page 429
Chemokine mutant analysis......Page 430
Chemokine receptor mutant analysis......Page 431
In vivo testing of inhibitory antibodies and small molecule antagonists......Page 432
Acknowledgments......Page 433
References......Page 434
B......Page 435
C......Page 437
D......Page 438
F......Page 439
G......Page 440
H......Page 441
J......Page 442
K......Page 443
L......Page 444
M......Page 445
O......Page 446
P......Page 447
S......Page 448
T......Page 450
V......Page 451
Y......Page 452
Z......Page 453
Subject-Index.pdf......Page 454