Human Peroxiredoxin 1 and 2 Are Not Duplicate Proteins

Weonsup Lee,Jong-Hoon Park,Jonah Riddell,Young-Mee Park,Debashis Ghosh,Clement Ip,Kyoung-Soo Choi

doi:10.1074/jbc.m610330200

Abstract

Human peroxiredoxins 1 and 2, also known as Prx1 and Prx2, are more than 90% homologous in their amino acid sequences. Prx1 and Prx2 are elevated in various cancers and are shown to influence diverse cellular processes. Although their growth regulatory role has traditionally been attributed to the peroxidase activity, the physiological significance of this function is unclear because the proteins are highly susceptible to inactivation by H(2)O(2). A chaperone activity appears to emerge when their peroxidase activity is lost. Structural studies suggest that they may form a homodimer or doughnut-shaped homodecamer. However, little information is available whether human Prx1 and Prx2 are duplicative in structure and function. We noted that Prx1 contains a cysteine (Cys(83)) at the putative dimer-dimer interface, which is absent in Prx2. We studied the role of Cys(83) in regulating the peroxidase and chaperone activities of Prx1, because the redox status of Cys(83) might influence the oligomeric structure and consequently the functions of Prx1. We show that Prx1 is more efficient as a molecular chaperone, whereas Prx2 is better suited as a peroxidase enzyme. Substituting Cys(83) with Ser(83) (Prx1C83S) results in dramatic changes in the structural and functional characteristics of Prx1 in a direction similar to those of Prx2. Here we also report the first crystal structure of human Prx1 and the presence of the Cys(83)-Cys(83) bond at the dimer-dimer interface of decameric Prx1. These findings are consistent with the hypothesis that human Prx1 and Prx2 possess unique functions and regulatory mechanisms and that Cys(83) bestows a distinctive identity to Prx1.

Highlights

Human peroxiredoxins 1 and 2, known as Prx1 and Prx2, are more than 90% homologous in their amino acid sequences
Prx1 and Prx2 are elevated in various cancers and are shown to influence diverse cellular processes. Their growth regulatory role has traditionally been attributed to the peroxidase activity, the physiological significance of this function is unclear because the proteins are highly susceptible to inactivation by H2O2
The Cys173 residue of Prx1 (Cys172 in Prx2) has traditionally been referred to as a “resolving” Cys. This is based on the fact that the transient inter-molecular disulfide bond formation between the Cys173/172-SH of one Prx molecule and the Cys52/51-SOH of another Prx molecule engaged in peroxide catalysis is a necessary step for reduction of the Cys52/51-SOH back to Cys52/51-SH

Summary

EXPERIMENTAL PROCEDURES

Materials—Dithiothreitol (DTT), iodoacetamide (IAA), NADPH, hydrogen peroxide, citrate synthase, and insulin were obtained from Sigma. 1 ␮M of MDH, citrate synthase, or insulin was mixed with various concentrations of Prx, Prx, or Prx1C83S, in a degassed 50 mM Hepes (pH 7.0) solution containing 0.1 M NaCl. The reaction mixture was incubated at 45 °C for 30 min, and the increase of light scattering as a result of thermal aggregation of substrate proteins was monitored at 360 nm. Consistent with the result obtained from the gel structure of human Prx has not been determined, the filtration study, the Trp fluorescence spectra indicated that the atomic structure of the well characterized bacterial Prx, tertiary packing property of Prx is significantly different from AhpC (Protein Data Bank code 1YEP), was used as a basis for that of Prx1C83S or Prx. The theoretical and experimental masses as well as the sequences of TABLE 1 Theoretical and experimental molecular masses of Prx, Prx1C83S, and Prx protein ions

Theoretical Experimental

Findings

Peptide sequence