Serial Review: Peroxiredoxins Serial Review Editor: H. Forman
Peroxiredoxins: A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling

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Abstract

The observation that purified yeast glutamine synthetase is rapidly inactivated in a thiol-containing buffer yet retains activity in crude extracts containing the same thiol led to our discovery of an enzyme that protects against oxidation in a thiol-containing system. This novel antioxidant enzyme was shown to reduce hydroperoxides and, more recently, peroxynitrite with the use of electrons provided by a physiological thiol like thioredoxin. It defined a family of proteins, present in organisms from all kingdoms, that was named peroxiredoxin (Prx). All Prx enzymes contain a conserved Cys residue that undergoes a cycle of peroxide-dependent oxidation and thiol-dependent reduction during catalysis. Mammalian cells express six isoforms of Prx (Prx I to VI), which are classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys) based on the number and position of Cys residues that participate in catalysis. The relative abundance of Prx enzymes in mammalian cells appears to protect cellular components by removing the low levels of peroxides produced as a result of normal cellular metabolism. During catalysis, the active site cysteine is occasionally overoxidized to cysteine sulfinic acid. Contrary to the general belief that oxidation to the sulfinic state is an irreversible process in cells, studies on the fate of the overoxidized Prx species revealed a mechanism by which the catalytically active thiol form is recovered. This sulfinic reduction is a slow, ATP-dependent process that is specific to 2-Cys Prx isoforms. This reversible overoxidation may represent an adaptation unique to eukaryotic cells that accommodates the intracellular messenger function of H2O2, but experimental validation of such speculation is yet to come.

Section snippets

Discovery of a 25-kDa antioxidant protein which inhibits enzyme inactivation by a thiol/Fe(III)/oxygen mixed-function oxidation system

While studying the regulation of glutamine synthetase in Saccharomyces cerevisiae in 1995, we noticed that yeast glutamine synthetase, which had been purified to an apparent homogeneity, lost activity and degraded gradually when stored in a buffer containing dithiothreitol (DTT) or 2-mercapteothanol [1]. Neither the addition of various protease inhibitors nor further purification on several HPLC columns had an effect on the rate of inactivation and degradation. Therefore, it seemed unlikely

The essential role of a conserved cysteine as the primary site of oxidation

The yeast, rat, and human genes that encode the 25-kDa TSA proteins were cloned and sequenced [4], [5], [6]. Alignment revealed 65% amino acid identity between mammalian and yeast TSA. The deduced amino acid sequence shows no significant homology to any known catalase, SOD, or peroxidase enzymes. This lack of homology is consistent with the observation that the 25-kDa protein does not possess catalytic activity characteristic of the conventional antioxidant enzymes that protect against reactive

Identification of peroxides as the substrates and thioredoxin as the immediate electron donor for the peroxidase function

Subsequently, the bacterial AhpC was also shown to be as effective as TSA in protecting glutamine synthetase from inactivation by the thiol mixed-function oxidation system [4]. This result, together with the sequence similarity between AhpC and TSA, suggested that TSA may also act on peroxides and the reduction of the resulting disulfide may be achieved by an enzyme with a function similar to that of AhpF. Thus, TSA proteins may possibly function as an antioxidant against both the ascorbate and

Three distinct types of mammalian peroxiredoxins and their reaction mechanisms

Although peroxiredoxin (formerly TSA and TPx, now Prx) was identified initially in yeast, the presence of multiple isoforms and their characterization were achieved first in mammalian cells. Mammalian cells express six Prx isoforms (Prx I–VI), which can be divided into three subgroups as follow: 2-Cys Prx proteins, which contain both the N- and C-terminal-conserved Cys residues and require both of them for catalytic function; atypical 2-Cys proteins, which contain only the N-terminal Cys but

Peroxynitrite reductase activity of peroxiredoxins

Nitric oxide (NOradical dot) and superoxide (O2radical dot), which are often produced simultaneously in cells, rapidly form peroxynitrite (ONOO). Peroxynitrite is extremely toxic to cells because it is readily converted to two radical species HOradical dot and NO2radical dot via hemolytic decomposition [46] or to CO3radical dot through a rapid reaction with CO2 . However, defense mechanisms against this toxic nitro oxidant had not been known. Like H2O2, peroxynitrite oxidizes thiols in a two-electron process that leads to the formation of

Intracellular messenger function of H2O2 and its regulation by peroxiredoxins

Peroxides like H2O2 and peroxynitrite are produced as the result of normal cellular processes that involve oxygen. The long-term presence of even a small amount of those peroxides is a risk to cells because they can be converted to the toxic radicals and thereby damage cellular components. Cells are therefore equipped with peroxide-eliminating enzymes like catalase, glutathione peroxidase, and Prx.

Besides the peroxides produced at basal levels, cells produce H2O2 transiently in response to the

Inactivation of the peroxidase activity of Prx I and Prx II via Cdc2-dependent phosphorylation

Like many enzymes involved in the metabolism of intracellular messengers, the peroxidase activity of Prx I and Prx II is regulated by phosphorylation, which in this instance is mediated by cyclin-dependent kinases (Cdks)[65]. Phosphorylation at Thr90 by several Cdks, including Cdc2, in vitro, results in inhibition of its peroxidase activity. Experiments with HeLa cells arrested at various stages of the cell cycle showed that phosphorylation of Prx I on Thr90 occurs in parallel with the

Reversible inactivation of 2-Cys Prx enzymes via hyperoxidation of the catalytic cysteine to cysteine sulfinic acid

Another regulatory mechanism involving a novel type of posttranslational modification was revealed as a result of studies on the inactivation of Prx enzymes during catalysis [66]. Spectrophotometric monitoring of Prx–dependent oxidation of NADPH revealed that Prx activity decreased gradually with time [67]. The decay in activity was coincident with the conversion of Prx to a more acidic species as assessed by two-dimensional gel electrophoresis [67], [68]. Mass spectrometric analysis and

Conclusions

We initially identified the Prx family of peroxidase in yeast and subsequently showed that Prx enzymes are present in organisms from all kingdoms. All Prx enzymes contain a conserved cysteine residue in the N-terminal region that is the primary site of oxidation by H2O2. Mammalian cells express at least six Prx isoforms, which can be divided into three subgroups designated 2-Cys, atypical 2-Cys, and 1-Cys. Members of the 2-Cys subgroup include Prx I through Prx IV and contain an additional

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    This article is part of a series of reviews on “Peroxiredoxins”. The full list of papers may be found on the home page of the journal.

    1

    Present address: Department of Food and Nutrition, College of Home Economics, Chonnam National University, Kwanju 500-757, Korea.

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