Index. Index 371. Aggresome, cystic fibrosis transmembrane conductance regulator,

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1 Index 371 Index Aggresome, cystic fibrosis transmembrane conductance regulator, centrosome-associated inclusions, characterization of soluble and insoluble fractions, 319 formation induction, 318, 320, 321 immunocytochemistry, 319, 321 centrosome-associated proteins, immunocytochemistry, 319, 320 Western blot, 320 materials for study, overview, 305, 310 subcellular localization, 312 definition, 305 functional significance, 313, 316 protective functions, 305, 306 Alacinomycin A, proteasome inhibition, 11 α-factor, see Endoplasmic reticulumassociated degradation Amino-terminal ubiquitination, see N- terminal ubiquitination, Androgen receptor, aggresome formation, 313 Bortezomib, see VELCADE CFTR, see Cystic fibrosis transmembrane conductance regulator Clasto lactacystin β-lactone, proteasome inhibition, 9, 10, 15, 16 Cystic fibrosis transmembrane conductance regulator (CFTR), aggresomes, centrosome-associated inclusions, characterization of soluble and insoluble fractions, 319 formation induction, 318, 320, 321 immunocytochemistry, 319, 321 centrosome-associated proteins, immunocytochemistry, 319, 320 Western blot, 320 materials for study, overview, 305, 310 subcellular localization, 312 degradation in rabbit reticulocyte lysate system, 186 endoplasmic reticulum-associated degradation assay, autoradiography of gels, 301, 302 HEK-293 cells, competent cell preparation, 296 harvesting of transfected cells, 297 transfection, 296, 297, 302 kinetic analysis of D508 mutant degradation, materials, , 301, 302 overview, 293, 294 solubility analysis, cell fractionation, 299, 300 solubilization of detergentinsoluble material, 300 Western blot of expression, quality control of folding, 309,

2 372 Index Defective ribosomal proteins (DRiPs), overview, 271, 272 quantitative analysis, cell fractionation, 275 denaturing gel electrophoresis, 277, 280 half-life calculation, materials, principles, 272 proteasome inhibitors, 272, 273, 279 radiolabeling of cells, 274, 275, 279 trichloroacetic acid precipitation and scintillation counting, 275, 277, 279, 280 Deubiquinating enzymes (DUBs), classes, 207 fluorescence assay, , 215 identification 209 isopeptidase T, see Isopeptidase T substrates, 207, 208 yeast enzymes, 208 DRiPs, see Defective ribosomal proteins, DUBs, see Deubiquinating enzymes, E1, adenylate assay, 30, 32 substrate preparation, 30, 31, 33 thiol ester assay, 30, 31, 33, 34 function, 23, 24, 255 purification, fast protein liquid chromatography, 29, 30 human erythrocyte fractionation, 27, 28, 32, 33 materials, 25, 26 overview, 24, 25 ubiquitin affinity column, chromatography, 28, 29, 33 preparation, 26, 27, 32 reconstitution in vitro, 24 thermosensitive E1 mutant cell line studies of ornithine decarboxylase degradation, 87, 88, 94 E2, function, 24, 224, 255 polyubiquitin chain synthesis, see Polyubiquitin, recombinant protein expression in Escherichia coli, 24 E3, auto-ubiquitination assay using purified recombinant protein, 40, 41, 44 coupled immunoprecipitation and auto-ubiquitination assay, 41 materials, 39, 40 MDM2-mediated p53 ubiquitination assay, 43, 44 SCF b-trcp assay, enzyme preparation, 42 incubation conditions and immunoblotting, 42, 44 overview, 41, 42 substrate preparation, 42 function, 24, 224, 255 parkin activity, see Parkin RING domain proteins, substrate identification, 39, 256 types, 255 Edman degradation, proteasomal cleavage product analysis, 109 Electrophoretic mobility shift assay (EMSA), sumoylated transcription factors, 333, 335, 336 Electrospray ionization mass spectrometry, see Mass spectrometry; Protein identification

3 Index 373 EMSA, see Electrophoretic mobility shift assay Endoplasmic reticulum-associated degradation (ERAD), assays using reticulocyte lysate, advantages, 186, 187 affinity adsorption of protein components, affinity depletion, 197, 198, 203 histidine-tagged recombinant protein purification, 195, 197, 203 degradation quantification, calculation of percentage of protein degraded, 193, 194, 202 polyacrylamide gel electrophoresis, 193 scintillation counting, 193, 202 integral membrane protein release assay, 194, 195, 202 materials, microsomes, collection and resuspension, 192, 193 preparation, 191 rabbit reticulocyte lysate preparation, 190, 191, 201 transcription/translation in vitro, 192, 201 translocation activity inactivation and reconstitution, alkylation, 198, 199 proteolysis, 198, 199, 202 SRP receptor α-subunit repopulation of inactivated membranes, 199, 201 assays with yeast membranes, cytosol, data collection and analysis, 181 materials, prepro α-factor substrate translocation into microsomes, 178, 179, 182 pro α-factor degradation cytosol assay, 179, 182, 183 purified proteasome reconstitution assay, 179, 180, 183 pro α-factor export cytosol assay, 180, 181, 183 purified proteasome reconstitution assay, 181, 183 cystic fibrosis transmembrane conductance regulator as model substrate, autoradiography of gels, 301, 302 HEK-293 cells, competent cell preparation, 296 harvesting of transfected cells, 297 transfection, 296, 297, 302 kinetic analysis of Δ508 mutant degradation, materials, , 301, 302 overview, 293, 294 solubility analysis, cell fractionation, 299, 300 solubilization of detergentinsoluble material, 300 Western blot of expression, overview, 175, 176, 186 quality control of protein folding, 309, 310 substrates, 293 yeast mutant screening, cloning of functional wild-type genes, 287 colony immunoblotting for detection of mutated cell clones, cycloheximide decay experiment,

4 374 Index ethylmethane sulfonate mutagenesis, 284, 285, 287 genome-wide screen, materials, 290, 292 multitransformation of yeast, 291, 292 overview, 289, 290 materials, 284 overview, 283, 284, 287 Epoxomicin, proteasome inhibition, 9 ERAD, see Endoplasmic reticulumassociated degradation Ethylmethane sulfonate mutagenesis, see Endoplasmic reticulumassociated degradation Fast protein liquid chromatography (FPLC), E1, 29, 30 human core proteasome particle, 100, 101 FPLC, see Fast protein liquid chromatography Gel filtration, isopeptidase T, proteasome assembly and maturation, 246, 247, 253 Gliotoxin, proteasome inhibition, 11 High-performance liquid chromatography (HPLC), see Protein identification; Reversedphase high-performance liquid chromatography HPLC, see High-performance liquid chromatography Huntingtin, aggresome formation, 313 Id2, ubiquitination, 256, 257, 259 IFN-γ, see Interferon-γ Immunoprecipitation, E3, 41 parkin, 358, 360, 368 pulse chase analysis of proteasome maturation, 252, 253 sumoylated proteins, 330, 331, 336 ubiquitination assay, 227, 228 Interferon-γ (IFN-γ), antigen presentation enhancement mechanisms, 307 Isopeptidase T (USP5), fluorescence assay, 211, 215 high-performance liquid chromatography assay, 211, 212 materials, 210 purification, affinity chromatography, 215 anion-exchange chromatography, bacteria growth and induction, 215, 217 cell pellet lysis and sonication, 215 gel filtration, 215, 216 materials, 210, 211 yeast homolog, 208 Lactacystin, proteasome inhibition, 9, 15, 16 LMP1, ubiquitination, 256, 257, 259 LMP2A, ubiquitination, 256, 257, 259 MALDI-directed nanoesi-ms/ms, protein identification, 142 Mass spectrometry (MS), proteasomal cleavage product analysis, 108, 109 protein identification, see Protein identification synthetic peptide purity analysis for digestion assay, 104 ubiquitination enrichment of tagged proteins, affinity tags, 166 elution from beads, 166

5 Index 375 on-bead digestions, 166, 168, 171, 172 solution digestions, 168, 171, 172 literature searches, 160, 162 materials, 154 matrix-assisted laser desorption ionization mass spectrometry of proteins, 156, 171 model peptides, gluc fragmentation, 162 synthesis, 160 tryptic peptide fragmentation, 162, 166 overview, 153, 154 peptide identification, 168, 169, 171, 172 tandem mass spectrometry of peptides, 157, 160 ubiquitination site identification, 168, 169, 171, 172 Matrix-assisted laser desorption ionization mass spectrometry, see Mass spectrometry; Protein identification MDM2, p53 ubiquitination assay, 43, 44 MG132, proteasome inhibition, 6, 7 MG262, proteasome inhibition, 8 Microsome, see Endoplasmic reticulum-associated degradation MS, see Mass spectrometry Multiple myeloma, see VELCADE, MyoD, ubiquitination, 256, 257, 259 NF-κB, see Nuclear factor-κb NIP-LLL-VS, proteasome inhibition, 7 N-terminal ubiquitination, evidence, 258 overview, pathology, protein conjugation and degradation cultured cell extracts, fractionation, preparation, 262 degradation intact cell studies, 267, 268 in vitro, 266, 267 proteasome inhibitor effects, 267, 268 protein half-life determination, 267, 268 labeling of protein substrates, biosynthetic labeling, 264, 265 overview, 263 radioiodination, 264 materials, reticulocyte lysate preparation, 261, 262 ubiquitination in vitro, 265, 266 protein stability effects, 256, 257 Nuclear factor-κb (NF-κB), proteasome regulation, 308 ODC, see Ornithine decarboxylase Ornithine decarboxylase (ODC), function, 83 ubiquitin-independent proteasomal degradation, materials for characterization, 84 overview, 83, 84 purified reconstitution system, degradation conditions and analysis, 92, 94 expression plasmids, 88, 89, 94 proteasome purification, 92 purification of recombinant proteins, 90, 91 recombinant protein expression in Escherichia coli, 90 reticulocyte lysate system, degradation reaction, 85, 86

6 376 Index lysate preparation and fractionation, 85 recombinant protein expression, 85 thermosensitive E1 mutant cell line studies, 87, 88, 94 p21, ubiquitination, PA28, functions, 307 pathology, 312, 313 subunits, 307 PA700, chaperone-like activity assay, citrate synthase thermal aggregation, 79 insulin reductive aggregation, 79 materials, 76 substrates, 78, 79 conformational remodeling of Ub 5 - dihydrofolate reductase, 77, 79 functions, ATPase activity, 74, 307 chaperone-like activity, 74, 75, 307 isopeptidase activity, 73, 74 polyubiquitin chain binding, 73 protein remodeling activity, 75 orientation within proteasome, 71, 72 proteasome core particle association assay, 76, 78, 79 purification from bovine red cells, chromatography, 78 extraction, materials, 76 structure, 306 PAGE, see Polyacrylamide gel electrophoresis Parkin, antibodies for analysis, dot blot screening, 355, 356 production, 354, 355 Western blot characterization, 356, 357, 368 brain immunohistochemistry, 361, 363 immunofluorescence microscopy in neurons, 357, 358 materials for study, 353, 354 protein protein interactions, 352, 353 RING domain, 352 structure, 352 ubiquitin ligase activity, brain extracts, 363, 364, 369 neuron lysates, 360, 361, 368 parkin transfected cell activity against brain substrates, , immunoprecipitation, 358, 360, 368 substrates, overview, 353 neural parkin, 366 Parkinson disease (PD), gene mutations, 352 parkin studies, see Parkin pathophysiology, 351 ubiquitination in Lewy bodies, 352 PD, see Parkinson disease Peptide mass fingerprinting, protein identification, 120, 148 Peripheral myelin protein-22 (PMP22), aggresome formation, 313 PMP22, see Peripheral myelin protein- 22 Polyacrylamide gel electrophoresis (PAGE), see also Electrophoretic mobility shift assay; Western blot, defective ribosomal proteins, 277, 280 endoplasmic reticulum-associated degradation 181, 193 human proteasome core particle, 102

7 Index 377 proteasome assembly and maturation nondenaturing polyacrylamide gel electrophoresis, electrophoresis conditions, 250 gel preparation, 249, 250, 253 in-gel peptidase assay, 250 Western blot, 250 pulse chase analysis of maturation, protein identification techniques with mass spectrometry, see Protein identification ubiquitination assay, 154 unconjugated ubiquitin assay, 236, 237 yeast proteasomes, denaturing gel electrophoresis, 68 native gel electrophoresis, Polyubiquitin, synthesis of chains linked through K48 and K63, deblocking reactions, 51, 52, 54 K48-Ub 2 synthesis and purification, 49 51, 54 K48-Ub 4 synthesis and purification, 52, 54 K48-Ub x synthesis and purification, 52 K63-Ub 3 synthesis and purification, 53 materials, 48, 49, 54 overview, 47, 48 yield and recovery, 53, 54 PR-11, proteasome inhibition, 10, 11, 16 PR-39, proteasome inhibition, 10, 11, 16 Presenelin-1, aggresome formation, 313 Proteasome, assembly and maturation, see Proteasome assembly and maturation core particle, peptidases, 72, 73 structure, 72 functions, cell cycle regulation, 307, 308 nuclear factor-κb regulation, 308 protein degradation, human core particle, cleavage product analysis, Edman degradation, 109 examples, mass spectrometry, 108, 109 reversed-phase highperformance liquid chromatography, 107, 113 denaturing gel electrophoresis, 102 fluorogenic substrates for peptidase assay, 103, 113 proteasome inhibitor testing, 103 purification, ammonium sulfate precipitation, 100 batch adsorption, 99, 100, 112, 113 buffer exchange and concentration, 102 erythrocyte preparation and hypotonic lysis, 99, 112 fast protein liquid chromatography, 100, 101 glycerol gradient centrifugation, 101, 113 materials, 98, 99 synthetic peptide digestion, incubation conditions, 104, 106 preparative digestion, 106, 113 purity requirements and analysis, 104 rationale for assay, 103 solvation, 104, 113 Western blot, 102, 103 whole protein digestion, incubation conditions, 106, 107

8 378 Index preparative digestion, 107 immunoproteasome subunits, 72 ornithine decarboxylase degradation, see Ornithine decarboxylase protein identification in complexes, see Protein identification regulatory particles, see PA28; PA700 structural overview, 4, 97, 98, 243, 244, 306 yeast proteasome, denaturing gel electrophoresis, 68 native gel electrophoresis, peptidase activity assay, 67, 69 purification, affinity purification, conventional purification, 61 64, 68, 69 core particle, 63, 64 66, 69 holoenzyme, 62, 63, 65, 68, 69 lid and base, 66, 67, 69 materials, 60, 61 overview, 59, 60 regulatory particle, 64 66, 69 structure, subunits, 58, 59 Proteasome assembly and maturation, gel filtration, 246, 247, 253 materials, nondenaturing polyacrylamide gel electrophoresis, electrophoresis conditions, 250 gel preparation, 249, 250, 253 in-gel peptidase assay, 250 Western blot, 250 peptidase assay, 247 pulse chase analysis of maturation, gel electrophoresis and autoradiography, immunoprecipitation, 252, 253 in vivo pulse chase, 251, 253 overview, 251 protein extraction and trichloroacetic acidprecipitable counts, 252, 253 Western blot, 247, 249, 250, 253 yeast culture and protein extraction, 246, 253 model, 243, 244 Proteasome inhibitors, characterization, IC50 determination, 11, 12, 17 kinetic parameters and type of inhibition, 13, 14 proteasome preparation, 11, 16 reversibility assay, 12, 13, 17, 18 clinical applications, 3, 4 defective ribosomal protein quantification, 272, 273, 279 pure proteasome testing, 103 small molecule inhibitors, lactone derivatives, clasto lactacystin β-lactone, 9, 10, 15, 16 lactacystin, 9, 15, 16 overview, 9, 15 noncompetitive inhibitors, alacinomycin A, 11 gliotoxin, 11 PR-11, 10, 11, 16 PR-39, 10, 11, 16 nonspecific inhibitors, 6 peptide aldehydes, MG132, 6, 7 overview, 6, 15 PSI, 7 peptide boronic acids, MG262, 8 overview, 8, 15 PS-341, 8, Z-LLL-boronate, 8 peptide epoxyketones, epoxomicin, 9

9 Index 379 overview, 8, 15 YU101, 9 YU102, 9 peptide vinyl sulfones, aminohexanoic acid derivatives of trileucine vinyl sulfone, 7, 8 NIP-LLL-VS, 7 overview, 7, 15 Z-LLL-VS, 7 research applications, 5, 14 ritonavir, 10 stock solution preparation, 5, 6, 14, 15 VELCADE treatment of multiple myeloma, see VELCADE Protein identification, mass spectrometry, database searching, 144, 145, 149 liquid chromatography tandem mass spectrometry, in-gel digested samples, 139, 140 in-solution digested samples, 140, 149 on-bead digestion, 142, 149 principles, 127 lyophilization and reconstitution of samples, 137 MALDI-directed nanoesi-ms/ MS, 142 materials, peptide mass fingerprinting, 120, 148 sample introduction, nano-electrospray ionization, 137, 139 spotting techniques for matrixassisted laser desorption, 137, 149 tandem mass spectrometry, 137 sample preparation with gels, automated in-gel digestion, band excision, 132 Coomassie staining of gels, extraction from unstained gels, 142, 144 manual in-gel digestion, 132, 134 polyacrylamide gel electrophoresis, 127, 129, 148 silver staining of gels, 131, 132, 149 Spyro Ruby staining of gels, 131, 148 sequence-tag approach, 121, 124, 127, 148 success rates, 142 top-down protein identification, 144 validation of search results, 148 overview of techniques, 117, 148 PS-341, see VELCADE PSI, proteasome inhibition, 7 Pulse chase analysis, see Defective ribosomal proteins; Proteasome assembly and maturation Reticulocyte lysate, see also Endoplasmic reticulumassociated degradation, cystic fibrosis transmembrane conductance regulator degradation, 186 N-terminal ubiquitination see N-terminal ubiquitination ornithine decarboxylase and ubiquitin-independent proteasomal degradation, degradation reaction, 85, 86 lysate preparation and fractionation, 85 recombinant protein expression, 85 sumoylated protein analysis, 333

10 380 Index Reversed-phase high-performance liquid chromatography, proteasomal cleavage product analysis, 107, 113 synthetic peptide purity analysis for digestion assay, 104 Rhodopsin, aggresome formation, 313 RING domain proteins, see E3; Parkin Ritonavir, proteasome inhibition, 10 SCF b-trcp, see E3 Sequence-tag approach, protein identification, 121, 124, 127, 148 Small ubiquitin-related modifier (SUMO), sumoylated protein analysis, gel mobility shift assay of transcription factors, 333, 335, 336 immunoprecipitation, 330, 331, 336 in vitro HeLa cytosolic extract preparation, 332, 333 rabbit reticulocyte lysate translated proteins, 333 recombinant SUMO-1 and Ubc-9 expression, 332 materials, 330 overview, 329 sumoylating enzymes, 329 SOD-1, see Superoxide dismutase-1 SUMO, see Small ubiquitin-related modifier Sumoylation, see Small ubiquitinrelated modifier Superoxide dismutase-1 (SOD-1), aggresome formation, 313 Tandem mass spectrometry, see Mass spectrometry; Protein identification Ubiquitin, amino-terminal ubiquitination, see N-terminal ubiquitination chain synthesis see E2; Polyubiquitin functions, 223, 237, 306 mass spectrometry see Mass spectrometry, pathology, 224, 231, 232 site identification on proteins, 168, 169, 171, 172 Western blot see Western blot, Ubiquitin-activating enzyme, see E1 Ubiquitin-conjugating enzyme, see E2 Ubiquitin ligase, see E3 USP5, see Isopeptidase T VELCADE, antitumor activity, 340 multiple myeloma management, dosage and administration, 343, 344 drug storage and preparation, 343, 348, 349 Food and Drug Administration approval, 340 infusion-related event management, 344, 345 materials, 340 monitoring, 348 patient selection, 340, 343 toxicity management, asthenia, 345 cytopenias, 346 gastrointestinal, 345, 346 neuropathies, proteasome inhibition, 8, 339, 340 structure, 341 Western blot, centrosome-associated proteins, 320 cystic fibrosis transmembrane conductance regulator expression,

11 Index 381 human proteasome core particle, 102, 103 parkin antibody characterization, 356, 357, 368 ubiquitination assay, blocking and probing of blots, 230, 231 gel electrophoresis, 228, 229 heat activation of proteins on blots, 230 immunoprecipitation, 227, 228 materials, , 231 membrane transfer, 229, 230 overview, 154, 224, 226, 227 sample preparation, 227, 231 verification of ubiquitination YU101, proteasome inhibition, 9 YU102, proteasome inhibition, 9 Z-LLL-boronate, proteasome inhibition, 8 Z-LLL-VS, proteasome inhibition, 7

The Nobel Prize in Chemistry 2004

The Nobel Prize in Chemistry 2004 Ubiquitous Quality Control of Life C S Karigar and K R Siddalinga Murthy The Nobel Prize in Chemistry for 2004 is shared by Aaron Ciechanover, Avram Hershko and Irwin