Ubiquitin and Protein Degradation, Part A

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Ubiquitin and Protein Degradation, Part A will cover high level purification, bioinformatics, and analysis of major protein involved in protein degradation. The chapters are highly methodological and focused on purification and analysis.

Topics include:
E1 Enzymes
E2 Enzymes
E3 Enzymes
Proteasomes
Isopeptidases

Section I - E1 Enzymes
Ch1 High level expression and purification of recombinant E1 enzyme
Ch 2 Expression, purification and characterization of E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex
Ch 3 A FRET-based assay to study SUMO modification in solution
Ch 4 Properties of the ISG15 E1 enzyme UbE1L

Section II - E2 Enzyme
Ch 5 Purification and properties of Ubiquitin Conjugating Enzymes Cdc34 and Ubc13-Mms2
Ch 6 Eexpression, purification, and properties of Ubc4 / 5 family E2 enzymes
Ch 7 In vitro systems for Nedd8 conjugation by Ubc12
Ch 8 Purification and activity assays for Ubc9, the ubiquitin conjugating enzyme for the small ubiquitin-like modifier SUMO
Ch 9 High-througput assay to monitor Formation of the E2-Ubiquitin thioester intermediate

Section III - E3 Enzyme
Ch 10 Expression and Evaluation of RING Finger proteins
Ch 11 Expression and assay of HECT domain ligases
Ch 12 High Level Expression and Purification of Recombinant SCF Ubiquitin Ligase
Ch 13 In vitro reconstitution of SCF substrate ubiquitination with purified proteins
Ch 14 Expression and assay of glycoprotein-specific ubiquitin ligases
Ch 15 Affinity purification and assay of APC / C on p13 Suc1
Ch 16 Large-Scale Purification of the Vertebrate Anaphase-Promoting Complex / Cyclosome
Ch 17 Purification and assay of the budding yeast Anaphase Promoting Complex
Ch 18 Enzymology of the Anaphase-Promoting Complex
Ch 19 Identification of Cell Cycle Dependent Phosphorylation Sites on the Anaphase-Promoting Complex / Cyclosome by Mass Spectrometry
Ch 20 Purification and assay of Mad2: A Two-state Inhibitor of APC / C
Ch 21 Identification, expression and assay of an oxidative-specific ubiquitin ligase, HOIL-1
Ch 22 (In revision) Purification and assay of chaperone-dependent ubiquitin ligase CHIP
Ch 23 Methods for the functional genomic analysis of ubiquitin ligases

Section IV - Proteasome
Ch 24 Purification of PA700, the 19S regulators complex of the 26S Proteasome
Ch 25 Purification and analysis of recombinant 11S acti vators of the 20S proteasome: Trypanosoma brucei PA26 and human PA28a, PA28B, and PA28y
Ch 26 Purification and assay of proteasome activator PA200
Ch 27 Purification, crystallization and X-ray analysis of the yeast 20S proteasome
Ch 28 (In revision due to figures only) Preparation of Hybrid (19S-20S-PA28) Proteasome Complexes and Analysis of Peptides Generated During Protein Degradation
Ch 29 Characterization of the Proteasome using Native Gel Electrophoresis
Monitoring and inhibition of 26S proteasomes with fluorogenic peptide substrates
Ch 31 The Synthesis and proteasomal degradation of a model substrate Ub5DHFR
Ch 32 Assaying Degradation and Deubiquitination of a ubiquitinated substrate by purified 26S proteasomes
Ch 33 Probing the Ubiquitin / Proteasome System with Ornithine Decarboxylase, and Ubiquitin-Independent Substrate
Ch 34 Atomic force microscopy of the proteasome
Ch 35 Characterization of noncompetitive regulators of the proteasome activity
Ch 36 Development and use of anti-proteasome monoclonal antibodies

Section V - Isopeptidases
Ch 37 Preparation and characterization of yeast and human desumoylating enzymes
Ch 38 Purification of the COP9 signalosome from porcine spleen, human cell lines and from Arabidopsis thaliana plants
Ch 39 Purification method of COP9 signalosome from human erythrocytes
Ch 40 UBP43, an ISG15-specific deconjugating enzyme: Expression, purification and enzymatic assays
Ch 41 Strategies for assaying deubiquitinating enzymes
Ch 42 In vitro cleavage of Nedd8 from cullin 1 by COP9 Signalosome and Deneddylase 1
Ch 43 Ubiquitin-ovomucoid fusion proteins as a model substrate for monitoring degradation and deubiquitination by proteasomes
Ch 44 Using deubiquitinating enzymes as research tools
Ch 45 Functional annotation of deubiquitinating eyzymes using RNA interference