Selenoprotein structure and function / edited by Eranthie Weerapana.

Format:

Book

Contributor:

Weerapana, Eranthie, editor.

Series:

Methods in enzymology ; Volume 662.

Methods in Enzymology ; Volume 662

Language:

English

Subjects (All):

Organoselenium compounds.

Physical Description:

1 online resource (xvi, 399 pages) : illustrations.

Edition:

1st ed.

Place of Publication:

Cambridge, MA : Academic Press, [2022]

Summary:

Selenoprotein Structure and Function, Volume 662 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters written by an international board of authors.

Contents:

Intro

Selenoprotein Structure and Function

Copyright

Contents

Contributors

Preface

Chapter One: Examining xCT-mediated selenium uptake and selenoprotein production capacity in cells

1. Introduction

2. Measurement of selenium uptake and selenoprotein production as mediated by xCT transporter

2.1. Evaluation of xCT expression of SLC7A11 and SLC3A2

2.1.1. Technical tips for the non-heat-denaturing Western blot of xCT

2.2. Measurement of extracellular thiols as readout of xCT function leading to uptake

2.2.1. Protocol for Ellman´s test

2.2.1.1. Ellman´s test for measuring xCT activity on extracellular reduction

2.2.1.2. Materials and equipment

2.2.1.3. Step-by-step method details

2.2.1.4. Expected outcomes

2.2.1.5. Quantification and statistical analysis

2.3. Measurement of selenium uptake

2.3.1. Protocol for fluorometric assay

2.3.1.1. Measurement of cellular selenium uptake capacity using fluorometric assay

2.3.1.2. Materials and equipment

2.3.1.3. Step-by-step method details

2.3.1.4. Expected outcomes

2.3.1.5. Quantification and statistical analysis

2.3.1.6. Advantages

2.3.1.7. Safety considerations and standards

2.3.2. Protocol for sample prep for ICP-MS analysis

2.3.2.1. Measurement of cellular selenium uptake capacity using ICP-MS

2.3.2.2. Materials and equipment

2.3.2.3. Step-by-step method details

2.3.2.4. Example condition for ICP-MS run

2.3.2.5. Expected outcomes

2.3.2.6. Quantification and statistical analysis

2.3.2.7. Advantages

2.3.2.8. Limitations

2.3.2.9. Safety considerations and standards

2.4. Western blotting of selenoproteins as final readout

2.4.1. Technical tips for the Western blotting of selenoproteins

3. Summary

Acknowledgment

References.

Chapter Two: Methods for accurate and reproducible studies of pharmacological effects of selenium in cancer

2. Selenium cytotoxicity in cancer cell lines

2.1. Effects of cell culture media composition on IC50 determinations of selenium compounds in cancer cell lines

2.2. Phenotype expression depending on cell culture media

2.3. Protocol for adherent cells

2.3.1. Materials

2.3.2. Procedure

2.4. Protocol for cells in suspension

2.4.1. Preparation of cell blocks for suspension cells

2.4.2. Materials and equipment

2.4.3. Procedure

2.5. The activity of the selenoprotein thioredoxin reductase (TXNRD) in cells cultured in different media

2.6. The extracellular redox milieu

2.7. Thiol measurements

2.7.1. Materials

2.7.2. Procedure

2.7.3. Quantification

2.8. Alternative method-Thiol measurement by DTDP

2.8.1. Buffers and chemical preparation

2.8.2. Materials and equipment

2.8.3. Procedure

2.8.4. Expected outcomes

2.8.5. Quantification

2.8.6. Advantage

2.9. Interference of selenium compounds with cell viability tests and mycoplasma infection

3. Metabolites and selenoproteins in plasma and serum after administration of pharmacological high doses of selenite IV

4. Mammalian thioredoxin reductase (TrxR, TR, TXNRD) assay

4.1. Buffers and chemical preparation

4.2. Materials and equipment

4.3. Procedure

4.4. Quantification and expected outcomes

5. KYAT determination

5.1. Se-methylselenocysteine

5.2. Simple spectrophotometric protocol for KYAT1 transamination activity

5.3. Buffers and chemical preparation

5.3.1. Phosphate EDTA buffer pH 7.4

5.3.2. Ammediol-HCL buffer

5.3.3. Materials and equipment

5.3.4. Procedure

5.3.5. Expected outcomes

5.3.6. Quantification

5.3.7. Advantage

5.4. Alternative method.

5.4.1. Materials and equipment

5.4.2. Procedure

5.4.3. Expected outcomes

5.4.4. Quantification

5.4.5. Advantage

5.5. Simple spectrophotometric protocol for KYAT1 β-elimination activity

5.5.1. Buffers and chemical preparation

5.5.2. Materials and equipment

5.5.3. Procedure

5.5.4. Expected outcomes

5.5.5. Quantification

5.5.6. Advantage

5.6. Alternative and preferred method

5.6.1. Materials and equipment

5.6.2. Procedure

5.6.3. Expected outcomes

5.6.4. Quantification

5.6.5. Advantage

6. Selenium treatment and microRNA expression

6.1. Determination of microRNA expression

6.2. qPCR-based method for measurement of microRNA expression

6.3. Materials

6.4. Equipment

6.5. Procedure

6.6. qPCR

7. Selenium and the immune system

7.1. Effects of selenium on antitumor immunity

7.2. Cytokine assays: Multiplex immunoassays

8. Concluding remarks

Acknowledgments

Conflicts of interest

References

Chapter Three: Using selenocysteine-specific reporters to screen for efficient tRNA variants

2. Natural selenocysteine translation

3. Recombinant selenocysteine translation

4. Evolving tRNA variants for increased Sec incorporation

4.1. Equipment

4.2. Reagents and materials

5. Protocol

5.1. Electrocompetent cells

5.2. Primer design to generate a targeted tRNA library

5.3. Making the tRNA library

5.4. Screening the tRNA library using Sec-intein reporters

5.5. Confirm efficiency of screened tRNA for Sec incorporation

6. Expected outcomes

7. Advantages

8. Limitations

9. Optimization and troubleshooting

Funding

Chapter Four: Expressing recombinant selenoproteins using redefinition of a single UAG codon in an RF1-depleted E. coli h ...

2. Selenoprotein production.

2.1. Alternative methods to produce selenoproteins: Pros and cons

2.2. Expressing recombinant selenoproteins in E. coli: Prior developments and alternative methods

3. Using UAG redefinition as a sec codon in an RF1-depleted E. coli host strain

4. Materials

4.2. Chemicals

4.3. Molecular biology reagents

4.4. Plasmids

4.5. Sequencing primers

4.6. Strains

4.7. Medium, solutions and buffers

5.1. Plasmid construction for selenoprotein expression in an RF1-depleted E. coli host strain

5.2. Culture and induction conditions

5.3. Selenoprotein purification

6. Selenoprotein analysis and quality assessment

6.1. Protein concentration measurements

6.2. Apparent protein purity assessed by gel electrophoresis

6.3. Enzyme activity measurements of sec-dependent activities of certain selenoproteins

6.4. Sec contents validation by direct methods

6.4.1. ICP-MS

6.4.2. LC-MS/MS

7. Concluding remarks

Conflict of interests

Chapter Five: Selenocysteine substitutions in thiyl radical enzymes

2. Recombinant expression of selenoproteins by non-sense suppression

2.1. Notes on choosing the right vector

3. Assembling pCm-based vectors and optimizing selenoprotein expression

3.1. Materials

3.2. Gibson assembly (2-3days)

3.3. Expression and amber codon suppression optimization (6days)

3.4. Cell lysis and solubility analysis (6days)

4. Milligram scale selenoprotein preparation

4.1. Materials

4.2. 1.5L protein expression (2-3days)

4.3. Protein purification (1day)

4.4. (Optional) fusion protein or His-tag removal (1-3h)

4.5. Selenium quantification by ICP-OES (2days)

5. Evaluating the effect of cysteine-to-selenocysteine substitution

Chapter Six: Expression of selenoproteins via genetic code expansion in mammalian cells

2. Methods

2.1. Before you begin

2.2. Key resources table

2.3. Materials and equipment

2.4. Step-by-step method details

2.4.1. Expression of SelenoPOI or eGFP containing DMNB-Sec

2.4.2. Uncaging of DMNB-Sec and harvesting of cells

2.4.3. Evaluation of incorporation and uncaging

2.5. Applications

3. Conclusions and outlooks

Chapter Seven: Applying selenocysteine-mediated expressed protein ligation to prepare the membrane enzyme selenoprotein S

2. Considerations for Sec-mediated EPL using selenopeptides

2.1. Selenopeptide preparation

2.2. Preparation of cselenos- and selenos-thioester

2.3. Selecting the ligation conditions

3. Materials

3.1. Reagents for molecular cloning

3.2. Reagents for protein expression and purification

3.3. Materials for selenopeptide synthesis, purification, and quantification

3.4. Reagents for expressed protein ligation

3.5. Materials for characterization by mass spectrometry

3.6. Materials for characterization by circular dichroism spectroscopy

4. Protocol

4.1. Cloning and expression of MBP-TEV-cselenos (52-187)-GyrA and MBP-TEV-selenos (1-187)-GyrA

4.2. Purification of cselenos (52-187) thioester and selenos (1-187) thioester

4.3. Synthesis and purification of the UG selenopeptide

4.4. NMR quantification of selenopeptide purity and concentration

4.5. Ligating the protein thioester with the UG selenopeptide

5. Analysis of the product

5.1. Intact protein mass spectrometry

5.2. Protein sequencing by tandem mass spectroscopy

5.3. Circular dichroism spectroscopy

6. Summary

Chapter Eight: Chemoproteomic interrogation of selenocysteine by low-pH isoTOP-ABPP.

1. Introduction.

Notes:

Includes bibliographical references.

Description based on print version of record.

Description based on publisher supplied metadata and other sources.

ISBN:

0-323-90735-0

OCLC:

1294317155