DNA sensors and inflammasomes / edited by Jungsan Sohn.

Format:

Book

Contributor:

Sohn, Jungsan, editor.

Series:

Methods in enzymology ; Volume 625.

Methods in Enzymology ; Volume 625

Language:

English

Subjects (All):

DNA.

Physical Description:

1 online resource (370 pages).

Place of Publication:

Cambridge, MA : Academic Press an imprint of Elsevier, [2019]

Summary:

DNA Sensors and Inflammasomes, Volume 625, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. New sections in this release include Phosphorylation and dimerization of STING and IRF3, cGAS enzymology, Synthesis and identification of immuno-stimulatory CDNs, Tracking cGAS activity/ cGAMP formation using SPR/NMR, Using an enzyme coupled assay to track cGAS activity under steady states, Tracking the polymerization of DNA sensors, inflammasome receptors, and downstream signaling partners using FRET, NLRC4 structure, Tracking TREX1 activity, DNA association and dissociation kinetics of PARP1, and more.- Provides the authority and expertise of leading contributors from an international board of authors- Presents the latest release in the Methods in Enzymology series- Includes the latest information on DNA sensors and inflammasomes

Contents:

Front Cover

DNA Sensors and Inflammasomes

Copyright

Contents

Contributors

Preface

Chapter One: Intercellular communication in the innate immune system through the cGAS-STING pathway

1. Introduction

2. cGAS-STING signaling

2.1. Inter-cellular communication

2.2. Type 1 IFN

3. Immuno-signaling to neighboring cells through gap junctions

3.1. cGAMP in virus particles

3.2. Extracellular vesicles

4. Exosomes and immune responses to virus infections

5. Exosomes and immune responses to bacterial infections

6. Concluding remarks

References

Chapter Two: Analysis of human cGAS activity and structure

2. A genetic assay for screening cGAS activity using bacteria

2.1. Assay setup and considerations

2.2. Protocol

2.3. Future applications

3. Crystallization and structural determination of the human cGAS-DNA complex

3.1. Preparation of human cGAS protein and DNA

3.1.1. Equipment

3.1.2. Reagents

3.1.3. Protocol

3.2. Assays to assess human cGAS-DNA complex formation

3.2.1. Equipment

3.2.2. Reagents

3.2.3. Protocol

3.3. Human cGAS-DNA crystallization protocol

3.3.1. Equipment

3.3.2. Reagents

3.3.3. Protocol

3.4. Human cGAS-DNA structure determination

3.4.1. Equipment

3.4.2. Reagents

3.4.3. Protocol

4. Crystallization of a human cGAS-DNA-ligand complex

4.1. Crystal soaking and human cGAS-DNA-ligand crystallization

4.2. Protocol for ligand soaking and crystal harvest

4.2.1. Reagents

4.2.2. Protocol

5. Summary

Acknowledgments

Chapter Three: Chemical synthesis, purification, and characterization of 3-5-linked canonical cyclic dinucleotides (CDNs)

2. Synthesis of crude CDNs

2.1. Chemicals

2.2. Equipment

2.3. Synthesis procedure for crude c-AMP-CMP.

3. Purification of crude CDNs

3.1. Preparation of HPLC buffers

3.2. Purification protocol for crude c-AMP-CMP

3.3. HPLC purification of crude c-AMP-CMP

4. Characterization of CDNs

4.1. Materials and equipment

4.2. MS analysis of c-AMP-CMP fractions

4.3. NMR analysis of c-AMP-CMP

5. Summary and prospects

Chapter Four: An SPR-based analysis of cGAS substrate KD and steady-state KM values

2. Consideration for the immobilization of proteins, multimers, and the cGASds-DNA complex

3. Considerations for SPR-based enzymology

4. Protocols

4.1. Protein reagents

4.2. Chemical reagents

4.3. Equipment

4.4. Chip preparation and protein capture

4.5. cGAS-ligand binding studies

4.5.1. Preparation of samples

4.5.2. Details of injections and data collection

4.5.3. Data analysis

4.6. cGAS activity assays

4.6.1. Preparation of samples

4.6.2. Details of injections and data collection

4.6.3. Data analysis

Chapter Five: A pyrophosphatase-coupled assay to monitor the NTase activity of cGAS

2. Materials

2.1. Chemicals, materials and enzymes

2.2. Buffers and assay reagents

2.2.1. Solution stocks

2.2.2. Buffers and assay solutions

2.2.3. Recombinant enzymes

2.2.4. Equipment

3. Implementing the PPiase-coupled NTase activity assay

3.1. Measuring steady-state NTase activity

3.1.1. Protocol

3.2. Data processing and analysis

3.3. Additional experimental notes

3.3.1. Measuring the specific activity of PPiase

3.3.2. Steady-state activity

3.3.3. Measuring the effectiveness of allosteric ligands

4. Summary and prospects

Chapter Six: Tracking the polymerization of DNA sensors and inflammasomes using FRET

2. Protocols.

2.1. Preparing PYDs for labeling

2.2. Labeling strategy

2.3. FRET-based assays

Acknowledgment

Reference

Chapter Seven: Monitoring gasdermin pore formation in vitro

1. Introduction to the gasdermin family

1.1. Pyroptosis, inflammasomes, and gasdermins

1.2. Pore-forming mechanism of gasdermins

2. Generation of recombinant gasdermins and proteases

2.1. Molecular cloning, expression, and purification of gasdermins from E. coli

2.2. Overproduction of caspase-11 using insect cells

3. Liposome leakage assay

3.1. Liposome formulation

3.2. In vitro pore formation, assay protocol, and data interpretation

4. Conclusions and discussion

Chapter Eight: Measuring TREX1 and TREX2 exonuclease activities

2. Considerations when purifying TREX1 enzymes

2.1. Preparation of truncated enzyme

2.2. Preparation of heterodimers

3. Purifying recombinant TREX1 enzymes

3.1. Equipment

3.2. Materials

4. Measuring TREX1 activity on ssDNA

4.1. Equipment

4.2. Materials

5. Measuring TREX1 activity on dsDNA with fluorescence

5.1. Equipment

5.2. Materials

6. Visualizing TREX1 activity on dsDNA with agarose gel electrophoresis

6.1. Equipment

6.2. Materials

7. Protocols

7.1. Preparation of phosphate cellulose resin

7.2. Overexpression and affinity chromatography purification of TREX1

7.3. Measuring TREX1 activity on ssDNA

7.4. Measuring TREX1 activity on dsDNA with fluorescence

7.5. Visualizing TREX1 activity on dsDNA with agarose gel electrophoresis

8. Calculating TREX1 exonuclease activities

8.1. Quantifying TREX1 activity on ssDNA

8.2. Quantifying TREX1 activity on dsDNA with fluorescence

9. Exonuclease activities of TREX1 mutant enzymes

10. Summary

References.

Chapter Nine: Kinetics of DNA-protein association and dissociation by stopped-flow spectroscopy

2. Motivation

3. Theoretical considerations

4. Practical considerations

5. Sample preparation

6. Instrument operation and data collection

7. Experimental troubleshooting

8. Data analysis

9. Conclusions

Chapter Ten: Characterization of DNA bound cyclic GMP-AMP synthase using atomic force microscopy imaging

2. Equipment

3. Materials

4. Protocol

4.1. Protein preparation

4.2. Mica surface modification with 1-(3-aminopropyl)silatrane

4.3. Preparation of DNA-cGAS complexes

4.4. Deposition of DNA-cGAS protein complexes on APS-modified mica surface

5. Atomic force microscopy imaging

5.1. Brief overview

5.2. Atomic force microscopy imaging

5.3. AFM image analysis

6. Summary

Chapter Eleven: Preparation of filamentous proteins for electron microscopy visualization and reconstruction

2. Construct design

2.1. Controlling oligomerization

2.2. Filament separation (bundling)

2.3. Buffer components

3. Negative stain electron microscopy

3.1. Staining protocol

3.1.1. Equipment required

3.1.2. Protocol (Fig. 2)

3.1.3. Variations

4. Cryo-EM sample preparation

4.1. Grid type

4.2. Sample concentration and distribution

4.3. Other parameters and tips

Chapter Twelve: Cryo-EM studies of NAIP-NLRC4 inflammasomes

2. Preparing NAIP-NLRC4 inflammasomes for EM

2.1. Heterogeneity-related obstacles

2.2. Sensitivity to the air-water interface and preferred orientations

2.3. Low particle number

3. Biochemical preparation

3.1. Preventing constitutional heterogeneity

3.2. Preventing conformational heterogeneity.

3.3. Protocol for biochemical preparation of NAIP5-NLRC4 inflammasomes

3.3.2. Materials

4. Preparation of cryo-EM grids

4.1. Minimizing preferred orientations and increasing particle density

4.2. Protocol for NAIP5-NLRC4 inflammasome cryo-EM grid preparation

4.2.1. Equipment

4.2.2. Materials

4.2.3. Protocol

5. Cryo-EM data collection and analysis

5.1. Data collection

5.2. Micrograph selection and particle picking

5.3. Sorting particles in 2D and 3D

5.4. Refinement and modeling

Chapter Thirteen: An integrative protocol for the structure determination of the mouse ASC-PYD filament

2. Experimental data

2.1. Sample preparation and characterization

2.2. Solid-state NMR spectroscopy

2.3. Cryo-electron microscopy

3. Structure determination protocol

3.1. Structure of the ASC-PYD monomer

3.2. Refinement of the ASC-PYD filament structure

3.3. Automatic interpretation of CHHC and PAR solid-state data

3.3.1. Automatic peak list interpretation

3.3.2. Validation of the peak interpretation procedure

3.4. Second-stage full structure refinement of the ASC-PYD filament

4. Effect of the cryo-EM electron density resolution on the structure quality

Chapter Fourteen: Protein interactions of the inflammasome adapter ASC by solution NMR

2. Overexpression and purification of ASC, ASC in minimal media

2.1. Equipment

2.2. Chemicals

2.3. Protein expression in minimal media and preparation for purification

2.4. Purification protocol

3. Aspects to consider in the design of solution NMR studies of ASC, ASC

3.1. Factors related to protein solubility (pH, protein and salt concentration, temperature).

3.2. Protein NMR chemical shift assignment for binding studies: Standard 2D and 3D protein NMR experiments and fast acqui ...

Notes:

Description based on print version record.

ISBN:

0-12-818360-8

0-12-818359-4