Modification of thiol residues on proteins via redox signals is an important mechanism by which redox signal transduction occurs in cells. Sensing of redox signals by these residues cause changes in the structure and hence function of the protein, thereby initiating a series of signaling cascades (1). Identification of such proteins is therefore key towards understanding how redox signaling occurs. To identify such proteins, different approaches can be used, such

John T. Hancock (ed.), Methods in Molecular Biology, Redox-Mediated Signal Transduction, vol. 476 © 2008 Humana Press, a part of Springer Science + Business Media, Totowa, NJ DOI: 10.1007/978-1-59745-129-1_7

as tagging thiol residues with detectable groups such that the tagged protein can be subsequently identified or using proteins mutated in thiol residues, either as a pure protein or in its native organism, to analyze its function. Examples of these will be presented in this chapter.

1.1. Outline to the 5'-iodoacetamide fluorescein (IAF) Procedure

Fluorescent covalently modified iodoacetamide can react with free thiol groups on proteins, such that when the protein is preoxi-dized by reactive oxygen species (ROS), the iodoacetamide group cannot react with the thiol group. Therefore, we can assess the effect of ROS on thiol modification by conducting experiments in the presence and absence of ROS, along with the iodoacetamide dye, subsequently followed by protein analysis. Using such an approach, it has recently been shown that the plant enzyme cytosolic glyceradehyde 3-phosphate dehydrogenase (GAPDH) is a target of ROS modification (2). In the first tool described in this chapter, the iodoacetamide labeling procedure followed by proteomics analysis of the labeled proteins is described. It is important to note that the concentrations of ROS that are used in these experiments are physiological to the system that is being used here, which in this case are cell cultures of Arabidopsis thaliana.

1.2. Outline to the Use The advantage of using an organism whose genome has been of Mutants sequenced, such as Homo sapiens or A. thaliana, is that a number of mutants are readily available. A huge international community has contributed to a wealth of knowledge in the model plant A. thaliana, which has also led to development of various public resources, such as the availability of seed stocks of different mutants via stock centers. Using this approach, a family of genes called histidine kinases were screened to test their ability to act as ROS sensors. One group of proteins in this family belongs to the class of ethylene receptors. Ethylene is a plant hormone involved in a number of physiological processes, such as fruit ripening, the falling of leaves, and the formation of root hairs. A group of five receptors of the histidine kinase family act as ethylene receptors. A large amount of research into ethylene signaling has involved the use of Arabidopsis plants mutated in these receptors. One such mutant is mutated in a Cys residue at the ethylene binding site. Therefore, the role of this Cys, and hence this protein, in ROS signaling in plants has been explored by studying different physiological responses to ROS (3). Some of these include the effect of hydrogen peroxide on stomatal closure, gene expression and root growth. By comparison of wild-type and mutant plants grown under exactly the same conditions, analysis of the requirement for a functional protein in ROS signalling can be achieved.

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