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Document Type:Latin Dissertation
Language of Document:English
Record Number:52367
Doc. No:TL22321
Call number:‭3510072‬
Main Entry:Shira Lezer
Title & Author:In-vivo characterization of plant Thioredoxins (Trx)Shira Lezer
College:The Weizmann Institute of Science (Israel)
Date:2009
Degree:Ph.D.
student score:2009
Page No:86
Abstract:Redox regulation via thiol-disulfide exchange reactions appears to be involved in multiple key biological processes in species as diverse as human and bacteria. Yet, the regulatory mechanisms and the physiological function of most of the implicated redox active factors remain unanswered. The subcellular localization, expression pattern and the unique protein-protein interactions as well as redox reaction kinetics of these regulatory proteins are all determinants that could affect their mode of action and facilitate specificity in redox signaling. Thioredoxins (Trx) are considered to be major players in the reversible reduction of regulatory disulfide bonds. Whereas bacterial and mammalian cells contain only two Trx genes, plant cells contain multiple genes encoding six different types of Trxs divided according to their primary structure and subcellular localization. The multiplicity of Trx genes in plants has made it difficult to assign a function to a particular Trx family member. A multitude of in-vitro biochemical and proteomic approaches were used to identify potential protein targets and to study the Trxs specificity towards them. Though these studies have provided unprecedented insight into the multitude of cellular processes regulated by redox, these studies did not reveal targets that are specific to a unique thioredoxin. It is therefore suggested that isolation of Trx target complexes produced in-vivo rather than in-vitro should be key to our understanding of Trx function in plants. Therefore, in this work, functional genomic approaches were used to elucidate the mechanisms of the Trx-mediated redox signal transduction pathways and to study in-vivo the role of unique Trxs. The moss Physcomitrella patens, which exhibits a very high rate of homologous recombination (HR) in its nuclear genome, was used as a plant model. Specifically, HR was used not only to delete genes but also as a tool for targeted tagging and modification of chromosomal genes. First, the complete set of Trx genes was identified in P. patens and isolated. Then, based on the characterization of their expression and in-vivo subcellular localization, two Trxs were selected as model proteins: Trx ƒ as a model for chloroplast Trx, and Trx h3 as a novel endoplasmic reticulum (ER) Trx. The unique biological role of the chosen model Trx ƒ in the chloroplast was explored by several functional genomic approaches. Tagging the chromosomal Trx ƒ gene via HR allowed us to examine the subcellular localization of the protein under the endogenous promoter, and to characterize in-vivo its authentic redox state and unique protein-protein interactions. The lower number of Trx ƒ targets identified in this study in comparison with previous in-vitro studies might suggest that the in-vivo technique is indeed more stringent than the previously described in-vitro methods, and that the targets observed here are more likely to represent the authentic targets of Trx ƒ. Known and novel putative Trx ƒ target proteins were isolated in-vivo and identified by affinity chroromatography and mass spectrometry. Though validation of the identified Trx ƒ targets is required, this study brings an in-vivo evidence that the main role of Trx ƒ is in regulating metabolic processes in the chloroplast. The redox state of this Trx was also examined in-vivo under different physiological conditions. The light dependent reduction of Trx ƒ is consistent with its proposed role in the reductive activation of its target enzymes in a light dependent manner. My studies of Trx h3 indicate for the first time that a Trx with a sequence signature of a reductive-type active site might function in the ER. I established the ER and Golgi localization of Trx h3 by several methods and showed that its first 39 amino acids- long leader, that include a transmembrane domain (TMD), are necessary for ER targeting and for anchoring the protein to membranes. The membrane topology of the protein was examined and suggested that Trx h3 belongs to type II signal anchors proteins with an cyt/Cexo protein orientation, resulting in endofacial luminal position of the Trx domain. My findings that the oxidoreductase activity of this protein depends in-vitro on dithiol reduction and not on glutathione, and that the protein is mostly reduced in-vivo suggest that Trx h3 reduces protein disulfides of ER substrates. The identification of the two main targets isolated in this study, is required for describing its biological function in further detail. Finally, the possible biological role of Trx in an oxidative environment such as the ER lumen is discussed.
Subject:Pure sciences; Biological sciences; Protein-protein interactions; Redox regulation; Thiol-disulfide exchange; Thioredoxins; Plant sciences; Biochemistry; 0487:Biochemistry; 0479:Plant sciences
Added Entry:A. Danon
Added Entry:The Weizmann Institute of Science (Israel)