The Yeast Prion Protein Ure2 Shows Glutathione Peroxidase Activity in Both Native and Fibrillar Forms

Abstract

Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homology to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochemical characterization of the purified protein. Here we use steady-state kinetic methods to characterize the multisubstrate peroxidase activity of Ure2p using GSH with cumene hydroperoxide, hydrogen peroxide, or tert-butyl hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concentration and showed optima at pH 8 and 40°C. Michaelis-Menten behavior with convergent straight lines in double reciprocal plots was observed. This excludes a ping-pong mechanism and implies either a rapid-equilibrium random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to wild type. Fibrillar aggregates showed the same level of activity as native protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.

The yeast prion protein Ure2: Structure, function and folding

“…Further, it was found that formation of amyloid-like fibrils had negligible effect on the level of GPx activity detected for purified protein in vitro [64]. Taken together, this indicates that Ure2 GPx activity is maintained upon structural conversion to the aggregated prion form.…”

“…Recent studies have found that deletion of the URE2 gene increases the sensitivity of S. cerevisiae cells to heavy metals and cellular oxidants, such as hydrogen peroxide [60][61][62]; and a Ure2 homologue from Aspergillus nidulans, while lacking the nitrogen metabolite repression activity of Ure2, also contributes to heavy metal and xenobiotic resistance [63]. Peroxidase activity of Ure2 towards oxidant substrates including hydrogen peroxide as well as typical organic hydroperoxides, such as cumene hydroperoxide and tert-butyl hydroperoxide (t-BH), has been demonstrated for the purified protein in vitro, confirming the function of Ure2 as a glutathione dependent peroxidase [64].…”

“…These two states of Ure2 can be distinguished by their markedly different stabilities and unfolding rates, as detected by a 'double-jump' experiment [35]. Another method for distinguishing such states is by activity assay, and so the recent development of an in vitro assay for Ure2 enzyme activity [64] provides a useful tool for further folding studies.…”

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

“…39,61,62 Ure2p displays glutathione peroxidase and glutaredoxin activities, in both the soluble and fibrillar state, and therefore contributes to cell protection against heavy metal ions and oxidant toxicity. [63][64][65] However, truncations within the N-terminal domain of Ure2p increased the glutaredoxin activity, 64 suggesting that the N-terminal domain is an important feature for the proper biological function of Ure2p. 62 Altogether, these data suggest that yeast PrDs contribute to the normal biological function of their corresponding proteins, although the extent of this contribution may not be easily detectable under normal growth conditions.…”

Role of Glutathione S-Transferases and Glutathione in Arsenic and Peroxide Resistance inSaccharomyces Cerevisiae:A Reverse Genetic Analysis Approach

“…In addition to this general defensive role, some GSTs are directly implicated in the oxidative stress response by way of their GPx function (2). Such GSTs effi ciently reduce inorganic and organic exogenous peroxides or eliminate endogenous lipid and fatty acid peroxides (3,5,8). Thus, GSTs provide general protection against electrophilic xenobiotics, toxic metals and peroxides, not only via conjugation or reduction with GSH, but also by alleviating the oxidative stress and subsequent lipid peroxidation often associated with exposure to xenobiotics.…”