Searching the function of Escherichia coli glutaredoxin 2

INTRODUCTION

Intracellular sulfhydryl homeostasis is maintained in Escherichia coli (E. coli) through the glutaredoxin (Grx) and thioredoxin systems. In the Grx system, reducing equivalents are transferred from NADPH to glutathione reductase (GR), glutathione (GSH) and finally to the three different Grxs (Grx1, 2, 3). Grx1 can reduce ribonucleotide reductase 1a (RR1a), and PAPS reductase. Grx3 has 5 % of the catalytic activity of Grx1 in reducing RR and no catalytic activity to PAPS reductase. Grx2 does not reduce PAPS reductase or RR1a but is the most potent antioxidant redoxin. In E. coli crude extracts, Grx2 comprises more than 80 % of total GSH-disulfide oxidoreductase activity and prevents oxidative damage (carbonylation) of E. coli proteins by hydrogen peroxide. The antioxidant function of Grx2 has been demonstrated also in rat neurons, where the protein affects signal transduction pathways leading to the activation of NF-kappaB via Ref-1, preventing thus dopamine-induced apoptosis. In relation to its role as an antioxidant, Grx2 is upregulated at the stress conditions of stationary phase to up to 1 % of total protein by ppGpp and RpoS. However, the physiological electron acceptor(s) of Grx2 remains so far unknown.

AIMS/EXPERIMENTAL APPROACH

The aim of this proposal is to identify potential electron acceptors for Grx2.
To address this problem we will use two biochemical approaches:

Approach 1: Formation of a stable complex between glutaredoxin-substrate
Using a monothiol Grx2, 3 fused to a His tag, we will make a stable complex of monothiol Grx2 with its substrates. The complex will be purified using an affinity matrix, then reduced to release the Grx2 partner which will finally be identified with electron spray mass spectrometry.

2. Identification of dithiol substrates by alkylation of their thiols with fluorescent tag
We will employ a modification of the method used previously for the identification of substrates for plant thioredoxin h. Briefly bacterial extracts will be treated with reduced Grx, alkylate the thiols with the ALEXA meleimide fluorophore and then subject the lysates to electrophoresis. A control lysate without glutaredoxin would serve as a negative control. With this approach we should be able to differentiate specific disulfide substrates reduced by glutaredoxin. Any wild type strain would be suitable for this work.

Candidates from combined results from the two approaches will be overexpressed and purified and their interaction with Grx2 confirmed with in vitro assays measuring consumption of NADPH in the presence of GSH, and GR. Future work will include the construction of appropriate null mutants.

IMPORTANCE

1. Many glutaredoxins and glutaredoxin like molecules exist in human cells. The function of these proteins is greatly unknown. Elucidation of their function in E. coli will give good leads for pathways in mammalian cells.

2. Studies on glutaredoxins unravel the cellular defences against oxidative stress considered to be related with Altzeimer¿s, Parkinson¿s diseases, atherosclerosis, and ageing. For example, we have recently shown that glutaredoxins can rescue neurons from dopamine induced oxidative stress by activating NF-kB through Ref-1.

For further information check Medline:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Limits&DB=PubMed
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