SUMO: Methods and Protocols (Methods in Molecular Biology, 1475)

✍ Scribed by Manuel S. Rodriguez (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 304
• Category: Library

✦ Synopsis

This volume explores various methodologies to study biochemical, molecular, and cellular biology aspects of some processes regulated by protein SUMOylation. SUMO: Methods and Protocols is organized into four parts, and starts with an historical overview on protein SUMOylation and a presentation of the methods included in the book. The first part also includes a review on chromatin regulation by dynamic SUMO modifications. The second part focuses on in vitro techniques, including biochemical methods to study mechanistic aspects of protein SUMOylation. The third part includes protocols to be used with cell cultures, which often are the first approaches used in most laboratories. The final part includes methodologies adapted for the analysis in vivo using distinct model organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include a brief introduction to the subject, a list of necessary materials and reagents, a step-by-step reproducible laboratory protocol ending with a Notes section on troubleshooting tips, and tips and strategies to avoid known pitfalls.

Unique and cutting-edge, SUMO: Methods and Protocols provides a comprehensive source of protocols for specialists and researchers not familiar with this vital system.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Introduction
Chapter 1: Concepts and Methodologies to Study Protein SUMOylation: An Overview
1 A Brief History of the Discovery of Small Ubiquitin-Related Modifiers
1.1 First Discoveries of Genes and cDNAs Coding for SUMO Proteins
1.2 Early Discoveries of SUMO Proteins as Posttranslational Protein Modifications
1.3 Initial Characterization of SUMO Paralogs
2 The Complexity of Protein SUMOylation
2.1 SUMO Conjugation Machinery
2.2 SUMO Consensus and Interacting Sequences
2.3 Diversity of SUMO E3s
2.4 Regulation of SUMOylation by Specific Proteases
3 Methodologies to Study Protein SUMOylation
4 Conclusions and Future Directions
References
Chapter 2: The Regulation of Chromatin by Dynamic SUMO Modifications
1 The SUMO Pathway
2 SUMO and Transcription: The Example of the Yeast Tup1-Ssn6 Corepressor
3 SUMO Localization Across the Genome
4 SUMO Across the Mammalian Genome
5 SUMO Across the Yeast Genome
6 SUMO-Chromatin Interactions During Heat Shock
7 SUMO and Histones
8 Histone Sumoylation Regulates Transcription
9 SUMO and Chromatin-Modifying Enzymes
10 Conclusions and Future Directions
References
Part II: Procedures to Study Protein SUMOylation In Vitro
Chapter 3: Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex
1 Introduction
2 Materials
2.1 Purification of Complex Components
2.1.1 General Expression and Purification Supply
2.1.2 Expression Constructs
2.1.3 Purification of SUMO1
2.1.4 Purification of Ubc9
2.1.5 Purification of RanGAP1
2.1.6 Purification of RanBP2-BD3-4
2.2 Reconstitution and Purification of the RanBP2 Complex
3 Methods
3.1 Purification of the Complex Components
3.1.1 Purification of SUMO1
3.1.2 Purification of Ubc9
3.1.3 Purification of RanGAP1
3.1.4 Purification of RanBP2-BD3-4
3.1.5 Quantitative SUMOylation of RanGAP1
3.2 Reconstitution and Purification of Complex
4 Notes
References
Chapter 4: Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria
1 Introduction
2 Materials
2.1 Expression Constructs
2.2 Components
2.3 Buffers
3 Methods
3.1 Application of the Protocol to the Production of SUMOylated c-Jun
4 Notes
References
Chapter 5: A Fluorescent In Vitro Assay to Investigate Paralog-­Specific SUMO Conjugation
1 Introduction
2 Materials
2.1 Purification of SUMO Species
2.2 Labeling of SUMO Proteins
2.3 In Vitro SUMO Chain Formation Assay
2.4 In Vitro Substrate Modification Using Fluorescent SUMO
3 Methods
3.1 Purification of SUMO Species
3.2 Labeling of SUMO Proteins
3.3 Paralog-Specific In Vitro SUMO Chain Formation Assay
3.4 In Vitro Substrate Modification Using Fluorescent SUMO
4 Notes
References
Chapter 6: Identification and Characterization of SUMO-SIM Interactions
1 Introduction
2 Materials
2.1 Media, Buffers, and Other Material Needed for the Y2H
2.1.1 Yeast Strains (See Note 1)
2.1.2 Plates for Cultivating Yeast
2.1.3 Yeast Media
2.1.4 Buffers, Antibiotics, and Other Solutions
2.1.5 Mini-prep
2.1.6 Bait Proteins
2.2 Buffers and Materials for Investigating SUMO-SIM Interactions by Reconstituted In Vitro Binding Experiments
2.2.1 Buffers
3 Methods
3.1 Screening a  Yeast cDNA Library for SUMO-­Interacting Proteins
3.1.1 Transformation of Yeast with the Bait Construct
3.1.2 Verification of Protein Expression
3.1.3 Testing of Bait Autoactivation (See Note 4)
3.1.4 Transformation of Bait-Containing Yeast with cDNA Library
3.1.5 Determination of Transformation and Mating Efficiency
3.1.6 Restreaking of Yeast on QDO/X/A Plates and Isolation of Plasmids
3.1.7 Sequencing and Analysis of Sequencing Results
3.1.8 Analyze the Obtained Sequencing Results by Using BLAST Software (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
3.2 Validation of SUMO-SIM Interactions by GST Affinity Interaction Studies
3.2.1 Expression of His-SUMO and Acetyl-His-­SUMO Variants
3.2.2 Expression and Purification of GST-Fusion Proteins Used as Affinity Baits
3.2.3 GST Pull-Down Experiments Using GST-SIM Proteins
4 Notes
References
Chapter 7: Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties
1 Introduction
2 Materials
2.1 Preparation of Ligands and Analytes
2.2 Chip Surface Preparation for Non-covalent Capture of Ligands
2.3 SPR Experiment
3 Methods
3.1 Chip Surface Preparation for Non-covalent Ligand Capture
3.2 SPR Experiment
3.3 Analysis of Data
4 Notes
References
Chapter 8: Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins
1 Introduction
2 Material
2.1 Cloning
2.2 Protein Expression and Purification Materials and Reagents
2.3 Protein Purification Buffers
2.4 In Vitro Modification Assay and Pull-­Down Reagents
2.5 Western-Blot Analysis Reagents
3 Methods
3.1 Protein Expression and Purification
3.1.1 Purification of GST-BirA
3.1.2 Preparation of Recombinant bioSUBEs
3.2 In Vitro Biotinylation Assay
3.3 In Vitro SUMOylation Assay
3.4 Protein Pull-
3.5 Western Blot Analysis
4 Notes
References
Chapter 9: In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases
1 Introduction
2 Materials
2.1 Expression of Ulp2 and Substrate Chains
2.2 Substrate Chain Purification
2.3 Preparation of Cell Extract Containing Ulp2
2.4 Ulp2 Activity Assay
2.5 Ulp2 Activity Assay
3 Methods
3.1 Cloning
3.2 Protein Overexpression in E. coli
3.3 Substrate Chain Purification
3.4 Preparation of Cell Extract Containing Ulp2
3.5 Ulp2 Activity Assay
3.6 Assay Analysis
4 Notes
References
Part III: Analysis of Protein SUMOylation Using Cell Lines
Chapter 10: Detection of Protein SUMOylation In Situ by Proximity Ligation Assays
1 Introduction
2 Materials
2.1 Culturing of HeLa cells, Arsenic Treatment, Harvesting, and Fixation
2.2 Proximity Ligation Assays and Detection of PLA Signals
3 Methods
3.1 Treatment and Harvesting of Cells
3.2 Proximity Ligation Assay: Antibody Incubations and Enzymatic Reactions
3.3 Immuno-fluorescence Analysis and Quantification
4 Notes
References
Chapter 11: In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells
1 Introduction
2 Materials
2.1 Cell Culture and Permeabilization
2.2 In Situ SUMOylation Reaction
2.3 In Situ deSUMOylation Reaction
2.4 Microscope Observations
3 Methods
3.1 Cell Culture and Cell Permeabilization
3.2 In Situ SUMOylation Reaction
3.3 In Situ deSUMOylation Reaction
3.4 Microscope Observations
4 Notes
References
Chapter 12: Analysis of SUMOylated Proteins in Cells and In Vivo Using the bioSUMO Strategy
1 Introduction
2 Materials
3 Methods
3.1 Cell Transfection and Biotin Supplementation
3.2 Collection of Samples
3.3 Lysis and Binding
3.4 Washes
3.5 Elution
4 Notes
References
Chapter 13: Label-Free Identification and Quantification of SUMO Target Proteins
1 Introduction
2 Materials
2.1 Cell Growth and Lysis
2.2 Enrichment of SUMO Target Proteins
2.3 Concentration and In-Solution Digestion of SUMO Target Proteins
2.4 LC-MS/MS Analysis of SUMO Target Proteins
3 Methods
3.1 Cell Growth and Lysis
3.2 Enrichment of SUMO Target Proteins
3.3 Concentration and In-Solution Digestion of SUMO Target Proteins
3.4 LC-MS/MS Analysis of SUMO Target Proteins
4 Notes
References
Chapter 14: The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins
1 Introduction
2 Materials
2.1 Bacterial Growth
2.2 Protein Purification
2.3 Pull-Down
3 Methods
3.1 Protein Expression in Bacteria
3.2 GST-Fusion Protein Purification from Escherichia coli
3.2.1 Preparation of the Beads
3.2.2 GST-Fusion Protein Binding and Washing
3.3 GST Pull-Down Assay
4 Notes
References
Chapter 15: Isolation of In Vivo SUMOylated Chromatin-Bound Proteins
1 Introduction
2 Materials
2.1 Cell Growth and Transfection
2.2 Harvest Cell Samples
2.3 Immuno-precipitation of Proteins
2.4 Sodium Dodecyl Sulfate-
2.5 Immunoblot for Protein Detection
2.6 Strip and Re-probe Membrane
3 Methods
3.1 Cell Growth and Transfection
3.2 Harvest Cell Samples
3.3 Immuno-precipitation of Protein
3.4 SDS-PAGE
3.5 Immunoblot for Protein Detection
3.6 Strip and Re-probe Membrane
4 Notes
References
Part IV: Procedures to Study Protein SUMOylation In Vivo
Chapter 16: Identification of Substrates of Protein-Group SUMOylation
1 Introduction
2 Materials
2.1 Yeast Strains
2.2 Yeast Culture Media
2.3 Cell Lysis and Denaturing Ni-NTA Pull-­Down of HISSUMO Conjugates
2.4 SDS-
3 Methods
3.1 Growing Yeast Cultures for SILAC Labeling
3.2 Cell Lysis and Denaturing Ni-NTA Pull-­Down of HISSUMO Conjugates
3.3 SDS-­Polyacrylamide Gel Electrophoresis and MS Analysis
4 Notes
References
Chapter 17: Tools to Study SUMO Conjugation in Caenorhabditis elegans
1 Introduction
2 Materials
2.1 Strains
2.1.1 C. elegans Strains
2.1.2 Bacterial Strains
2.2 Plasmids
2.3 Antibody Methods
2.4 Recombinant Proteins
2.5 Live Imaging
2.5.1 In Utero Live Imaging
2.5.2 Ex Utero Live Imaging
2.6 Proximity Ligation Assay
2.7 Purification of 6×His-­CeSUMO Conjugates
3 Methods
3.1 Polyclonal Antibody Preparation
3.1.1 Test Sera with Specific and Nonspecific Antigens in Dot-Blot Assays
3.1.2 Coupling of Proteins/Peptides/Bacterial Lysate to NHS Beads
3.1.3 Affinity Purification
3.2 Monoclonal Antibody Preparation
3.2.1 Hybridoma Supernatant Preparation
3.2.2 Protein G Purification
3.3 Ex Utero Live Imaging
3.3.1 Preparing the Worms
3.3.2 Meiotic Recording
3.3.3 Mitotic Recording
3.4 In Utero Live Imaging
3.5 Proximity Ligation Assay
3.5.1 Making PLA Probes with Duolink® In Situ Probemaker
3.5.2 PLA
3.6 Purification of 6×His-­CeSUMO Conjugates
3.7 Protein Purification
3.7.1 CeSUMO, UBC-9, GEI-17, and ULP-4
3.7.2 Expression and Purification of TEV Protease
3.8 SUMO Conjugation
3.9 Processing of SUMO and Chain Editing
4 Notes
References
Chapter 18: Purification of SUMO Conjugates from Arabidopsis for Mass Spectrometry Analysis
1 Introduction
2 Materials
2.1 Purification of Recombinant A. thaliana SUMO1
2.2 Affinity Purification of Anti-SUMO Antibodies
2.3 Protein Extraction
2.4 First Ni-NTA Affinity Purification
2.5 Anti-SUMO Immunoprecipitation
2.6 Second Ni-NTA Affinity Purification
3 Methods
3.1 Purification of Recombinant A. thaliana SUMO1
3.2 Affinity Purification of Anti-SUMO Antibodies
3.3 Protein Extraction
3.4 First Ni-NTA Affinity Purification
3.5 Anti-SUMO Immunoaffinity Chromatography
3.6 Second Ni-NTA Affinity Purification
3.7 Analysis Methods for SUMOylated Proteins
3.7.1 SDS-PAGE and Immunoblotting
3.7.2 Mass Spectrometric Identification
3.7.3 Quantitative Mass Spectrometric Analysis of SUMO Conjugates
4 Notes
References
Chapter 19: Detection of SUMOylation in Plasmodium falciparum
1 Introduction
2 Materials
2.1 P. falciparum Culturing
2.2 Parasite-
2.3 Parasite Lysis Components
2.4 SDS Polyacrylamide Gel Components
2.5 Immunoblot Components
2.6 Antigens and Conjugates
3 Methods
3.1 Preparation of Protein Samples from P. falciparum
3.2 Parasite Lysis
3.3 12.5 % SDS-
3.4 Immunoblot
4 Notes
References
Chapter 20: Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1
1 Introduction
2 Materials
2.1 Immuno-precipitation
2.2 Immunostaining
3 Methods
3.1 Immuno-precipitation
3.1.1 Preparation of Brain Lysate and Chromatography Column
3.1.2 Immunoaffinity Binding and Washing of the Column
3.1.3 Elution
3.1.4 Precipitation of Proteins from Eluates
3.1.5 Bead Recovery
3.1.6 Analysis of Purified Proteins
3.2 Immunostaining
3.2.1 Sample Preparation
3.2.2 Immuno-labeling HA
3.2.3 Mounting and Imaging
3.2.4 Imaging
4 Notes
References
ERRATUM
Index