The Ubiquitin Code

Preface
Contents
Contributors
Part I: Chain Diversity
Chapter 1: Thioester and Oxyester Linkages in the Ubiquitin System
1 The Ubiquitin Code
2 Stability of Ester Bonds
2.1 Thioester Bonds in the Ub System
2.2 Oxyester Bonds in the Ub System
3 Experimental Strategies to Identify Ester-Linked Ubiquitylation
4 Cellular Context of CST Ubiquitylation
4.1 Examples of OCE
4.1.1 Endoplasmic Reticulum-Associated Protein Degradation
4.1.2 CST Ubiquitylation in Regulated Proteasomal Degradation
4.1.3 Linear Ubiquitin Assembly Complex and Cytokine Production
4.2 Examples for ACE
4.2.1 The PEX System
4.2.2 Ubiquitylation in the N-Terminal Region of Bid
4.2.3 The MYC-Binding Protein 2 E3 Enzyme
5 Ester Linkages Within Poly-Ubiquitin Chains
6 Outlook
References
Chapter 2: Getting to the Root of Branched Ubiquitin Chains: A Review of Current Methods and Functions
1 Introduction
2 Function of Branched Ubiquitin Chains
3 Methods for Studying Branched Chains
3.1 Detection
3.1.1 Ub Mutants
3.1.2 UbiCRest
3.1.3 Antibody and Pull-Down Techniques
3.1.4 Mass Spectrometry
3.2 Synthesis
3.2.1 Enzymatic
3.2.2 Synthetic
4 Future Perspectives
4.1 Chain Architecture
4.2 Branch-Specific UBPs
4.3 Regulation of Branched Chain Formation
4.4 Therapeutic Applications
5 Conclusion
5.1 Concluding Remarks
5.2 Unanswered Questions
Bibliography
Part II: Ub and UbL Chemical Tools
Chapter 3: Chemical Synthesis of Non-hydrolyzable Ubiquitin(-Like) Hybrid Chains
1 Introduction
2 Materials
2.1 Synthesis
2.2 Analysis
3 Methods
3.1 Synthesis
3.1.1 Preparation of Reagents for CuAAC Reaction Mixture
3.1.2 Copper-Catalyzed Azide Alkyne Cycloaddition Reaction for 1 Mg of Bio-UbL-PA
3.2 Data Analysis
3.2.1 High-Resolution (HR) LC-MS Analysis
3.2.2 SDS-Page Analysis
4 Notes
References
Chapter 4: Total Linear Chemical Synthesis of LC3A and LC3B
1 Introduction
2 Materials
2.1 Solid-Phase Peptide Synthesis
2.2 Global Deprotection
2.3 Preparative High-Performance Liquid Chromatography (HPLC)
2.4 Folding and Analysis
3 Methods
3.1 Solid-Phase Peptide Synthesis
3.2 Global Deprotection
3.3 Purification
3.4 Folding and Analysis
4 Notes
References
Part III: Methods to Study Ubiquitin Conjugating Enzymes (E3s)
Chapter 5: A Microbead-Based Flow Cytometry Assay to Assess the Activity of Ubiquitin and Ubiquitin-Like Conjugating Enzymes
1 Introduction
2 Materials
2.1 Equipment
2.2 Readout Protein Expression and Purification
2.3 Coupling of the Readout Proteins to Magnetic Beads
2.4 Cell Extract Preparation and Activity Assay
3 Methods
3.1 Readout Protein Expression
3.2 Readout Protein Purification
3.3 Coupling of Readout Proteins to the Magnetic Beads
3.3.1 Activation of the Beads
3.3.2 Coupling of Readout Proteins
3.3.3 Beads Quantification and Analysis of the Coupling Efficiency
3.4 Cell Extracts
3.4.1 Spleen Preparation
3.4.2 Cell Lysis
3.5 Assay of UbL Conjugation Activity
3.5.1 Cell Extracts
3.5.2 Recombinant Enzymes (for SUMOylation) (See Note 9)
3.5.3 Reaction Mix
3.6 Flow-Cytometry Analysis of UbL Conjugation
4 Notes
References
Chapter 6: Monitoring HECT Ubiquitination Activity In Vitro
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Protein Expression Vectors and Competent Cells
2.3 Reagents
2.4 Equipment
3 Methods
3.1 His-UBA1 Protein Production and Purification
3.2 His-UBE2D3 Protein Production and Purification
3.3 E3 Enzyme Protein Production
3.4 Substrate Protein Production
3.5 Ub-TR-FRET Assay for Enzyme Self-Ubiquitination and Polyubiquitin Chains Detection
3.6 Ub-TR-FRET Assay for Substrate Ubiquitination Detection
3.7 Anti-Ub Immunoblot
3.8 Anti-GST Immunoblot
4 Notes
References
Part IV: Methods to Study DUBs
Chapter 7: RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs
1 Introduction
1.1 The GFP Pull-Down Strategy
1.2 Immunoblotting-Based Identification of Substrate/DUB Pairs
2 Materials
2.1 DUB Silencing and GFP-Tagged Protein and FLAG-Tagged Ubiquitin Overexpression
2.2 GFP Pull-Down Strategy
2.3 Western Blot
3 Methods
3.1 DUB Silencing and GFP-Tagged Protein and FLAG-Tagged Ubiquitin Overexpression
3.2 GFP Pull-Down Strategy
3.3 Western Blot
3.4 Data Analysis
4 Notes
References
Chapter 8: Strategies for Monitoring ``Ubiquitin C-Terminal Hydrolase 1´´ (Yuh1) Activity
1 Introduction
2 Materials
2.1 Growth Media for Bacteria
2.2 Growth Media for Saccharomyces cerevisiae
2.3 Yeast Transformation
2.4 Buffers and Solutions
2.5 Reagents for the Readout Assay
2.6 Equipment
2.7 Disposables
2.8 Plasmids
2.9 Competent Bacteria Strains
2.10 Yeast Strains
3 Methods
3.1 Activity Assay of the Bacterially Expressed Yuh1
3.1.1 Expression and Induction of Bacterial Proteins
3.1.2 Extraction of Recombinant Proteins
3.1.3 Evaluation of Recombinant Yuh1 Activity by Immunoblotting
3.2 Direct Readout for Enzymatic Activity of Yuh1
3.2.1 Transformation of Plasmids to the Δyuh1 Mutant Strain
3.2.2 Production of Total Cell Extracts
3.2.3 Assay and Analysis of the Results by Plate Reader
4 Notes
Bibliography
Part V: Methods to Identify New Targets and Regulators
Chapter 9: Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies
1 Introduction
2 Materials
2.1 Nanobodies Purification
2.2 Nanobody-Magnetic Beads Cross-Linking
2.3 Isolation of Ubiquitylated Proteins from Mammalian Cells
2.4 Mass Spectrometry
3 Methods
3.1 Purification of Ub Chain-Specific Nanobodies
3.2 Nickel Magnetic Beads Cross-Linking Protocol
3.3 Isolation of Ubiquitylated Proteins from Mammalian Cells
3.4 Sample Preparation for Mass Spectrometry Analysis
4 Notes
References
Chapter 10: A Mass Spectrometry-Based Strategy for Mapping Modification Sites for the Ubiquitin-Like Modifier NEDD8
1 Introduction
2 Materials
2.1 Common Solutions and Equipment
2.2 Cell Culture
2.3 Cell Treatment and Lysis
2.4 Protein Precipitation and Digestion
2.5 Peptide Purification
2.6 Di-Glycine-Modified Peptide Immuno-Enrichment
2.7 Micro Tip-Based Strong Cation Exchange Chromatography (Micro-SCX)
2.8 Desalting and Concentration of Peptides
2.9 Analysis of Peptides by LC-MS/MS
3 Methods
3.1 Cell Treatment and Lysis
3.2 Protein Precipitation and Digestion
3.3 Peptide Purification
3.4 Di-Glycine-Modified Peptide Immuno-Enrichment
3.5 Micro Tip-Based Strong Cation Exchange Chromatography (Micro-SCX)
3.6 Desalting and Concentration of Peptides
3.7 Analysis of Peptides by LC-MS/MS
3.8 Data Analysis
4 Notes
References
Chapter 11: Isolation of the Hepatic Ubiquitome/NEDDylome by Streptavidin Pull-Down Assay in the Biotinylated Ubiquitin (bioUb...
1 Introduction
2 Materials
2.1 Materials
2.2 Equipment
2.3 Reactives
2.4 bioUb and bioNEDD8 Mice
2.5 Preparation of Buffers
3 Methods
3.1 Tissue (Liver) Collection
3.2 Homogenization of Liver Tissue Samples
3.3 Preparation of PD-10 Desalting Columns and Loading of Sample
3.4 Preparation of Standard-Capacity Beaded Agarose Resin of Immobilized Streptavidin or NeutrAvidin Protein
3.5 Desalting of Samples and Binding of Standard-Capacity Beaded Agarose Resin of Immobilized Streptavidin or NeutrAvidin Prot...
3.6 Pull-Down Assay
3.7 Sample Final Elution
4 Notes
References
Chapter 12: The Lambda Display Technology: A Useful Tool for the Identification of Ubiquitin-and Ubiquitin-Like-Binding Domains
1 Introduction
2 Materials
2.1 Selection of Recombinant λ Phage Particles
2.2 Plaque Assay
2.3 Preparation of λ DNA Suitable for Sequencing
3 Methods
3.1 Affinity Selection on Sepharose Beads
3.2 Plaque Assay
3.3 Preparation of λ DNA Suitable for Sequencing
4 Notes
References
Chapter 13: SUMO-ID: A Strategy for the Identification of SUMO-Dependent Proximal Interactors
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cultured Cells and Treatments
2.3 Lentiviral Packaging and Transduction
2.4 Cell Lysis and Streptavidin Pull-Down
3 Methods
3.1 SUMO-ID Cell Line Generation
3.2 SUMO-ID Treatments and Cell Lyses
3.3 Streptavidin Pull-Down
4 Notes
References
Chapter 14: Analysis of ATG8 Family Members Using LC3-Interacting Regions (LIR)-Based Molecular Traps
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 Affinity Chromatography
2.3 Dialysis
3 Methods
3.1 Purification of Recombinant LC3 Traps
3.2 Pull Down of Recombinant ATG8 Proteins
3.3 Capture and Identification of ATG8 Proteins from Distinct Biologic Models
3.3.1 Lysis of Mammalian Cells
3.3.2 Lysis of Nematodes
3.3.3 Lysis of Yeast
3.3.4 Pull Down and Analysis of Distinct ATG8 Family Members
4 Notes
References
Chapter 15: A Computational Tool for Analysis of Mass Spectrometry Data of Ubiquitin-Enriched Samples
1 Introduction
2 Materials
3 Methods
3.1 Software Setup and Installation of R Packages
3.2 Perform UPS Protein Expression Analysis
3.3 Visualize Quantitative UPS Data in R
3.4 Comparative Average Branch Topology for Ubiquitin
4 Notes
References
Part VI: Methods to Measure Proteasome Activity
Chapter 16: In-Plate and In-Gel Assays for the Assessment of Proteasome Activity in Caenorhabditis elegans
1 Introduction
2 Materials
2.1 In-Plate Proteasome Activities
2.1.1 Reagents and Equipment for Protein Extraction
2.1.2 Reagents for In-Plate Activities Measurement
2.2 In-Gel Proteasome Activities Assay
2.2.1 Reagents for Protein Extraction from Nematode Samples
2.2.2 Reagents Needed for the Native Gel Electrophoresis Buffers
2.2.3 Proteasome Activity Buffer Reagents
2.2.4 Reagents and Equipment for Immunoblotting and Detection of Native Gels
3 Methods
3.1 In-Plate Activities
3.1.1 Protein Extraction from Nematode Samples
3.1.2 Measurement of In-Plate Proteasome Activities
3.2 In-Gel Activities
3.2.1 Protein Extraction and Preparation of Nematode Samples
3.2.2 Native Gel Electrophoresis
3.2.3 Measurement of In-Gel Activities
3.2.4 Proteasome Levels and Assembly
4 Notes
Bibliography
Chapter 17: Isolation of Proteasome-Trapped Peptides (PTPs) for Degradome Analysis
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Native Cell Lysate (Mammalian Cells)
3.2 Preparation of Native Cell Lysate (Yeasts)
3.3 Proteasome Separation
3.4 Native Gel Preparation and Electrophoresis
3.5 Proteasome In-Gel Activity Assay
3.6 Peptide Isolation
3.7 Peptide Desalting
3.8 Peptide Extraction
4 Notes
References
Index