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A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase
Karolinska Institutet, Stockholm.
Karolinska Institutet, Stockholm.
Karolinska Institutet, Stockholm.
Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 26, 24544-24552 p.Article in journal (Refereed) Published
Abstract [en]

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750–2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3′,5′-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

Place, publisher, year, edition, pages
2005. Vol. 280, no 26, 24544-24552 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-41980DOI: 10.1074/jbc.M500678200Local ID: 59458OAI: oai:DiVA.org:liu-41980DiVA: diva2:262835
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Protein Structure and Interaction in Health and Disease
Open this publication in new window or tab >>Protein Structure and Interaction in Health and Disease
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on protein structure, dynamics and interaction and their relation to human disease. In particular, the biophysical and structural properties of both well-ordered and partially disordered proteins are studied using a range of biophysical techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. Pseudomonas aeruginosa is a human pathogen due to its multidrug resistance (MDR) caused by overexpression of efflux pump systems. This thesis describes how MDR mutations within the MexR repressor of the MexAB-OprM system reduce the DNA affinity by altering its stability with maintained structure. The oncogenic protein c-Myc is involved in many essential biological functions such as cell proliferation, differentiation and apoptosis and is also highly associated with several forms of human cancers, and where the N-terminal domain is regulated by a plethora of protein interactions. In this thesis the intrinsically disordered N-terminal part of c-Myc and its interactions with the proteins Bin1 and TBP are described. Myc binds Bin1 with maintained disorder in a multivalent manner, which may explain why the onco-protein can interact with such a wide range of binding partners. A similarly dynamic interaction is observed for Myc with the TATA-binding protein (TBP). The essential human multidomain glutaredoxin Grx3 is associated with several biological functions such as redox signaling, proliferation and signal transduction. We have solved the structure and analyzed the dynamic properties in the ps-ns and ms time scale for the two N-terminal domains, providing a platform for further analysis of the Grx3 protein and its interactions. Taken together, this thesis emphasizes the importance of joint structural, biophysical and dynamic studies to better understand protein function in health and disease.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. 67 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1394
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-70837 (URN)978-91-7393-077-2 (ISBN)
Public defence
2011-10-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 14:00 (Swedish)
Opponent
Supervisors
Available from: 2011-09-20 Created: 2011-09-20 Last updated: 2011-09-20Bibliographically approved

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Andrésen, CeciliaSunnerhagen, Maria

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