Biology of Serpins

Front matter
......Page 1
Copyright
......Page 2
Contributors
......Page 3
Preface
......Page 10
Content......Page 12
Analysis of Serpin Secretion, Misfolding, and Surveillance in the Endoplasmic Reticulum......Page 42
Introduction......Page 43
Transfection of mammalian cell lines......Page 44
Metabolic pulse-chase radiolabeling......Page 45
Identification of the intracellular degradation system......Page 46
Identification of physically interacting proteins......Page 47
Alternative methodologies......Page 48
Assessing the time course of detectable protein interactions......Page 49
RNAi technology as a means to validate participation in serpin processing......Page 50
Assessment of total N-glycan number and role......Page 51
Establishment of N-glycan-mediated events......Page 53
References......Page 55
Serpin-Enzyme Receptors: LDL Receptor-Related Protein 1......Page 58
Purification of α2M and preparation of methylamine-reacted α2M-Sepharose
......Page 59
Purification of LRP1 from human placenta......Page 60
Expression of LRP1 fragments in cells......Page 62
Expression of functional LRP1 ``minireceptors´´......Page 63
Determinants on LRP1 that bind to ligands......Page 64
Using an ELISA to measure ligand binding to LRP1......Page 65
Surface Plasmon resonance measurements......Page 66
Determinants on serpins that are responsible for binding to LRP1......Page 67
Clearance of 125I-labeled serpin-enzyme complexes in mice......Page 68
References......Page 69
The Role of Autophagy in Alpha-1-Antitrypsin Deficiency......Page 73
Introduction......Page 74
Autophagy in AT deficiency......Page 75
Plastic-embedded EM......Page 76
Immuno EM......Page 77
LC3 Conversion Assay......Page 78
Gel electrophoresis......Page 79
Use of lysosomal inhibitors to enhance the information provided by LC3 immunoblots......Page 80
Pulse-chase metabolic labeling for long-lived proteins......Page 81
Atg5-/-......Page 82
Pulse-chase protocol......Page 83
Immunoprecipitation to detect specific proteins......Page 84
Analysis of hepatic autophagy in vivo by EM......Page 85
Analysis of autophagy by fluorescence microscopy using the GFP-LC3 mouse......Page 86
Use of autophagy-deficient mouse models......Page 87
PiZ mouse......Page 88
Z mouse......Page 89
OHP measurement......Page 90
Assessment of injury/regenerative activity by BrdU labeling......Page 91
References......Page 92
Serpins and the Complement System......Page 95
Introduction......Page 96
Human Plasma C1-Inhibitor......Page 97
Purification of C1-inhibitor from human plasma......Page 98
Stoichiometry of inhibition analyzed using enzyme assay......Page 102
Measurement of association rate constant for C1-inhibitor with C1s......Page 103
Production of Recombinant C1-Inhibitor in Yeast......Page 104
Cloning......Page 106
Isolation of recombinant P. pastoris clones......Page 107
Expression of C1-inhibitor in flasks......Page 108
Fermentor-based C1-inhibitor expression......Page 109
Purification of recombinant C1-inhibitor......Page 111
Acknowledgments......Page 113
References......Page 114
Use of Mouse Models to Study Plasminogen Activator Inhibitor-1......Page 116
Introduction......Page 117
PAI-1 in cardiovascular disease......Page 119
PAI-1 and cancer......Page 120
Mouse Models to Study the Role of PAI-1......Page 121
Cardiovascular disease......Page 122
Genetically Modified Mice to Study the Role of PAI-1 in Cardiovascular Disease and Cancer......Page 126
Thrombosis......Page 127
Atherosclerosis-(Re)stenosis......Page 129
Cancer......Page 131
Conclusions......Page 132
References......Page 133
Plasminogen Activator Inhibitor Type 2......Page 144
Introduction......Page 145
PAI-2 and the Plasminogen-Activating System......Page 146
Structural considerations......Page 147
Clearance receptors......Page 148
Role of PAI-2 in skin......Page 149
The role of PAI-2 in metastatic cancer......Page 150
Association of PAI-2 with retinoblastoma protein......Page 151
Apoptosis and the innate immune response......Page 152
PAI-2 expression in the brain and its role as a neuroprotective agent......Page 153
PAI-2 Gene Expression and Regulation......Page 154
Transcriptional regulation of PAI-2......Page 155
Epigenetics......Page 156
mRNA stability: General principals......Page 157
Posttranscriptional regulation of PAI-2 expression......Page 158
Assessment of PAI-2 mRNA decay using tetracycline-regulated expression systems......Page 159
The role of AU-rich instability elements in the 3-UTR of PAI-2......Page 160
Conclusions......Page 161
Methodology: Rapid Run-On Transcription Assay Protocol......Page 162
References......Page 165
The SerpinB1 Knockout Mouse......Page 174
Background......Page 175
Plasmid construct design and embryonic stem cell targeting......Page 176
Neutrophil proteases and inhibitors in the lungs......Page 178
Intranasal inoculation......Page 179
Assay buffers......Page 180
Role of serpinB1 in neutrophil homeostasis in the bone marrow......Page 181
Cell surface markers for mouse neutrophils and monocytes......Page 182
Sample preparation and measurements......Page 184
References......Page 185
Investigating Maspin in Breast Cancer Progression Using Mouse Models......Page 188
Introduction......Page 189
Generation of WAP-maspin transgene......Page 190
Isolation and purification of WAP-mapsin construct......Page 191
Generation of WAP-maspin transgenic mice......Page 192
Rationale of the experimental approach......Page 193
Tumor progression......Page 194
Limitations for transgenic mouse models......Page 195
Establishing TM40D maspin transfectants......Page 196
Implantation of modified TM40D cells into BALB/c mice......Page 197
Liposome preparation......Page 198
Maspin gene delivery and analysis of tumor......Page 199
Conclusion......Page 200
References......Page 201
Hsp47 as a Collagen-Specific Molecular Chaperone......Page 205
Introduction......Page 206
Hsp47 as a Collagen-Binding Protein in the ER......Page 207
Interaction and Recognition of Collagen by Hsp47......Page 208
A Phenotype and Abnormal Collagen Maturation in Hsp47 Knockout Mice......Page 209
Possible Roles of Hsp47 in Procollagen Maturation in the ER......Page 210
Hsp47 Null Cells: A Tool for Studying the Fate of Misfolded Collagen......Page 212
Regulation of Hsp47 Expression and Its Clinical Importance......Page 214
References......Page 216
Assays for the Antiangiogenic and Neurotrophic Serpin Pigment Epithelium-Derived Factor......Page 221
Introduction......Page 222
Interphotoreceptor matrix......Page 223
Plasma......Page 224
Ammonium sulfate fractionation......Page 225
Immunoblot reaction with polyclonal antibody Ab-rPEDF......Page 226
Cetylpyridinium chloride precipitation......Page 228
Solution binding assays......Page 229
Surface plasmon resonance assays......Page 230
Determination of bound PEDF......Page 231
Neurotrophic Assays......Page 232
Neurite-outgrowth analyses......Page 233
Protection against oxidative damage......Page 234
Chick embryo aortic arch assay......Page 235
Cell migration......Page 236
Laser-induced CNV......Page 237
Flatmount technique......Page 238
Corneal pocket......Page 239
References......Page 240
The Drosophila Serpins: Multiple Functions in Immunity and Morphogenesis
......Page 243
Introduction......Page 244
Comparison with mammalian serpins......Page 245
Macroglobulins and tight-binding protease inhibitors......Page 247
Terminal peptides and size-conservation of the serpin core......Page 248
The innate immune response......Page 249
Seminal fluid proteins......Page 250
Identification of target proteases and the degradation of serpin/protease complexes in the hemolymph
......Page 251
Inducers......Page 252
Factors to consider when making an immune challenge......Page 253
Direct injection of pathogens......Page 254
Hemolymph collection......Page 255
Wolbachia......Page 256
Nora virus......Page 257
References......Page 258
Modeling Serpin Conformational Diseases in Drosophila melanogaster......Page 264
Introduction......Page 265
Screening for Polymerogenic Mutations in Physiologically Important Fly Serpins
......Page 266
First Steps in the Generation of a Human Serpinopathy Model in Drosophila......Page 267
Longevity Assays in Flies Expressing Human Serpins......Page 270
Behavioral Assays in Flies Expressing Human Serpins......Page 271
Microscopic Phenotyping of Flies Expressing Human Serpins......Page 272
Using Genetic Screens to Identify Genes That Have a Role in Generating, or Suppressing Serpin-Induced Phenotypes.........Page 273
Method 1: Generating Transgenic Drosophila......Page 274
Microinjection method......Page 276
Crossing scheme to identify transformants......Page 278
Method 3: Chemical Mutagenesis and X-ray Mutagenesis......Page 279
EMS mutagenesis......Page 280
Method 4: Screening for Mutations Caused by Chemical and X-ray Mutagenesis......Page 282
Method 6: Examination of the Eye Imaginal Disc......Page 283
Method 7: Pseudopupil Assay......Page 285
Method 8: Protein Extraction from Flies......Page 287
Method 9: Genetic Backcrossing......Page 288
Method 10: Longevity Assays......Page 289
Method 12: Immunostaining of Fly Brains......Page 290
Method 13: P-element and RNAi Screening......Page 291
References......Page 292
Using Caenorhabditis elegans to Study Serpinopathies......Page 296
Introduction......Page 297
Considerations for Transgenesis......Page 298
C. elegans expression vectors......Page 301
Transgenesis methods......Page 302
Coinjection markers......Page 303
Microinjection equipment and reagents......Page 305
Preparation of plasmid DNA for injections......Page 306
Microinjection method......Page 307
Stable integration of transgenes......Page 308
High-Content Drug Screening......Page 310
OP50 food preparation for drug screening......Page 311
Animal preparation and sorting using the COPAS BIOSORT......Page 312
Automatic image acquisition......Page 314
Considerations for HCS......Page 315
References......Page 316
Using C. elegans to Identify the Protease Targets of Serpins In Vivo......Page 319
Introduction......Page 320
Preparation of OP50 for nematode food stocks......Page 321
Methods for Identifying the Targets of Intracellular Serpins in C. elegans......Page 322
Identifying serpin targets using genetics......Page 323
Development assays......Page 324
The use of RNAi by feeding to study protease targets......Page 325
Identifying serpin targets using biochemistry......Page 327
Generation of TAP plasmids for use in C. elegans......Page 328
Large-scale C. elegans growth
......Page 329
Coupling antibody to CnBr beads......Page 330
First and second round of purifications......Page 331
References......Page 333
Viral Serpin Therapeutics: From Concept to Clinic......Page 336
Serpins......Page 337
Viral serpins......Page 339
Serp-1 and viral pathogenesis-Identification of a secreted anti-inflammatory viral serpin......Page 340
Serp-2-Discovery of viral cross-class anti-inflammatory and antiapoptotic serpins......Page 341
Mouse model of arterial angioplasty injury-induced atherosclerosis and aneurysm......Page 342
Preoperative preparation......Page 343
Operation......Page 344
Postoperative care......Page 345
Analysis of cellular mechanisms of anti-inflammatory activity......Page 346
Preparation of single splenocyte suspension......Page 349
Labeling of cells with fluorescent-conjugated antibodies......Page 350
Isolation of PBMCs......Page 351
In vitro adhesion assay after serpin treatment......Page 352
RNA isolation......Page 353
Rationale......Page 354
Stages for proceeding into clinic......Page 355
Clinical trial findings......Page 356
References......Page 361
Human SCCA Serpins Inhibit Staphylococcal Cysteine Proteases by Forming Classic ``Serpin-Like´´ Covalent Complexe......Page 365
Introduction......Page 366
Purification of GST-SCCA1 and GST-SCCA2 Fusion Proteins......Page 367
General inhibition assay......Page 368
Determination of the stoichiometry of inhibitory complex formation......Page 369
Determination of the rate of stable complex formation (kass)......Page 371
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis......Page 372
Western blot analysis......Page 374
Determination of an Interaction Site......Page 375
Summary......Page 377
References......Page 378
Plants and the Study of Serpin Biology......Page 380
Introduction......Page 381
Detection using biotinylated proteases......Page 382
Subcellular localization of Arabidopsis serpins......Page 383
Generation of GFP-serpin fusion constructs......Page 384
Transformation of the constructs and detection of GFP-serpin fusion proteins......Page 385
Purification of serpins from mature seeds......Page 386
Thiol extraction......Page 387
Purification from oat grain......Page 389
Purification from apple seed......Page 390
Separation and visualization of plant serpins using native-PAGE......Page 392
Analysis of Plant Serpin-Protease Interactions......Page 393
Immunoprecipitation using anti-serpin antibodies......Page 394
Use of a mechanism-based probe that targets cysteine proteases......Page 396
References......Page 397
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