Tyrosine substitution of a conserved active-site histidine residue activates Plasmodium falciparum peroxiredoxin 6.

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y revealed a preference for H2 O2 as an oxidant with second order rate constants for H2 O2 and tBuOOH around 2.5 × 107 M-1 s-1 and 3 × 106 M-1 s-1 , respectively. Differences between the oxidation kinetics of PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.

A single-cysteine mutant and chimeras of essential Leishmania Erv can complement the loss of Erv1 but not of Mia40 in yeast.

Mia40/CHCHD4 and Erv1/ALR are essential for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals. In contrast, many protists, including important apicomplexan and kinetoplastid parasites, lack Mia40. Furthermore, the Erv homolog of the model parasite Leishmania tarentolae (LtErv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 (ScMia40). Here we addressed structure-function relationships of ScErv1 and LtErv as well as their compatibility with the oxidative protein folding system in yeast using chimeric, truncated, and mutant Erv constructs. Chimeras between the N-terminal arm of ScErv1 and a variety of truncated LtErv constructs were able to rescue yeast cells that lack ScErv1. Yeast cells were also viable when only a single cysteine residue was replaced in LtErvC17S. Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of LtErv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered LtErv incompatible with ScMia40. The question whether parasite Erv homologs might also exert the function of Mia40 was addressed in another set of complementation assays. However, neither the KISS domain nor other truncated or mutant LtErv constructs were able to rescue yeast cells that lack ScMia40. The general relevance of Erv and its candidate substrate small Tim1 was analyzed for the related parasite L. infantum. Repeated unsuccessful knockout attempts suggest that both genes are essential in this human pathogen and underline the potential of mitochondrial protein import pathways for future intervention strategies.

Mothers with positive or negative depression screens evaluate a maternal resource guide.

INTRODUCTION: Social isolation is common in mothers with high depressive symptoms. This study tested the hypothesis that a maternal resource guide that provided mothers with links to community human service agencies would be deemed more helpful by mothers with positive depression screens (PDS) compared with mothers with negative depression screens (NDS). METHOD: This investigation was a cross-sectional survey study of a convenience sample from a primary care practice-based research network, the Southwestern Ohio Ambulatory Research Network (SOAR-Net). English-speaking mothers who took their child(ren) to SOAR-Net practices were eligible to participate in the study. Data were collected between May 2006 and March 2009. A total of 1048 mothers completed the survey, and 234 mothers refused to participate. RESULTS: Mothers were more likely to report that "This guide is helpful to me" if they were single (odds ratio [OR] = 4.05; 95% confidence interval [CI]: 2.77-5.94), their child had public health insurance (OR = 3.59; 95% CI: 2.39-5.40), or they had PDS (OR = 3.57; 95% CI: 2.13-5.98). After adjusting for a number of demographic variables, PDS continued to be significantly associated with "This guide is helpful to me" (adjusted OR = 2.68; 95% CI: 1.58-4.56). DISCUSSION: Mothers with PDS were more likely to report that the maternal resource guide would be personally helpful compared with mothers with NDS.