In Vivo Measurement of Redox-Regulated TG2 Activity

Arek V Melkonian; Nielson Weng; Brad A Palanski; Chaitan Khosla

doi:10.1007/978-1-4939-9187-7_16

In Vivo Measurement of Redox-Regulated TG2 Activity

Methods Mol Biol. 2019:1967:263-274. doi: 10.1007/978-1-4939-9187-7_16.

Authors

Arek V Melkonian^{1

2}, Nielson Weng^{2

3}, Brad A Palanski³, Chaitan Khosla^{4

5

6}

Affiliations

¹ Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
² School of Medicine, Stanford University, Stanford, CA, USA.
³ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁴ Department of Chemical Engineering, Stanford University, Stanford, CA, USA. [email protected].
⁵ Department of Chemistry, Stanford University, Stanford, CA, USA. [email protected].
⁶ Stanford ChEM-H, Stanford University, Stanford, CA, USA. [email protected].

Abstract

Transglutaminase 2 (TG2) is a ubiquitous mammalian enzyme that is implicated in a variety of physiological processes and human diseases. Normally, extracellular TG2 is catalytically dormant due to formation of an allosteric disulphide bond between Cys370 and 371 of the enzyme. In this protocol, we describe a method to reduce this disulphide bond in living mice and to monitor the resulting in vivo TG2 activity. Briefly, exogenous thioredoxin-1 protein (TRX) is prepared and administered as a specific, physiologically relevant reductant of the Cys370-371 disulphide along with the small molecule 5-biotinamidopentylamine (5-BP) as a TG2 activity probe. Tissue cryosections are then analyzed by immunohistochemistry to ascertain the extent of 5-BP incorporation, which serves as a record of the redox state of TG2 in vivo. This protocol focuses on the modulation and measurement of TG2 in the small intestine, but we encourage investigators to evaluate it in their organ(s) of interest.

Keywords: Activation; Disulphide; In vivo; Inhibition; Mouse study; Probe; Redox; Small intestine; Thioredoxin; Transglutaminase.

MeSH terms

Amines / chemistry
Animals
Biotin / analogs & derivatives
Biotin / chemistry
Disulfides / chemistry*
GTP-Binding Proteins / chemistry
GTP-Binding Proteins / isolation & purification*
Humans
Mice
Molecular Biology / methods*
Oxidation-Reduction
Protein Glutamine gamma Glutamyltransferase 2
Thioredoxins / chemistry
Transglutaminases / chemistry
Transglutaminases / isolation & purification*

Substances

Amines
Disulfides
5-(biotinamido)pentylamine
Thioredoxins
Biotin
Protein Glutamine gamma Glutamyltransferase 2
Transglutaminases
GTP-Binding Proteins

Abstract

MeSH terms

Substances

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