Emerging roles of low-molecular-weight thiols at the host–microbe interface

Low-molecular-weight (LMW) thiols are an abundant class of cysteine-derived small molecules found in all forms of life that maintain reducing conditions within cells. While their contributions to cellular redox homeostasis are well established, LMW thiols can also mediate other aspects of cellular physiology, including intercellular interactions between microbial and host cells. Here we discuss emerging roles for these redox-active metabolites at the host–microbe interface. We begin by providing an overview of chemical and computational approaches to LMW-thiol discovery. Next, we highlight mechanisms of virulence regulation by LMW thiols in infected cells. Finally, we describe how microbial metabolism of these compounds may influence host physiology.     

Female mice carrying a defective Alox15 gene are protected from experimental colitis via sustained maintenance of the intestinal epithelial barrier function

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.     

Redox modulation of homomeric rho1 GABA receptors

The activity of many receptors and ion channels in the nervous system can be regulated by redox-dependent mechanisms. Native and recombinant GABA_A receptors are modulated by endogenous and pharmacological redox agents. However, the sensitivity of GABA_C receptors to redox modulation has not been demonstrated. We studied the actions of different reducing and oxidizing agents on human homomeric GABAρ₁ receptors expressed in Xenopus laevis oocytes. The reducing agents dithiothreitol (2 mM) and N -acetyl- l -cysteine (1 mM) potentiated GABA-evoked Cl⁻ currents recorded by two-electrode voltage-clamp, while the oxidants 5-5'-dithiobis-2-nitrobenzoic acid (500 μM) and oxidized dithiothreitol (2 mM) caused inhibition. The endogenous antioxidant glutathione (5 mM) also enhanced GABAρ₁ receptor-mediated currents while its oxidized form GSSG (3 mM) had inhibitory effects. All the effects were rapid and easily reversible. Redox modulation of GABAρ₁ receptors was strongly dependent on the GABA concentration; dose–response curves for GABA were shifted to the left in the presence of reducing agents, whereas oxidizing agents produced the opposite effect, without changes in the maximal response to GABA and in the Hill coefficient. Our results demonstrate that, similarly to GABA_A receptors and other members of the cys-loop receptor superfamily, GABA_C receptors are subjected to redox modulation.     

Glutathione peroxidase 8 is transcriptionally regulated by HIFα and modulates growth factor signaling in HeLa cells

GPx8 is a mammalian Cys-glutathione peroxidase of the endoplasmic reticulum membrane, involved in protein folding. Its regulation is mostly unknown. We addressed both the functionality of two hypoxia-response elements (HREs) within the promoter, GPx8 HRE1 and GPx8 HRE2, and the GPx8 physiological role. In HeLa cells, treatment with HIFα stabilizers, such as diethyl succinate (DES) or 2-2′-bipyridyl (BP), induces GPx8 expression at both mRNA and protein level. Luciferase activity of pGL3^GPx8wt, containing a fragment of the GPx8 promoter including the two HREs, is also induced by DES/BP or by overexpressing either individual HIFα subunit. Mutating GPx8 HRE1 within pGL3^GPx8wt resulted in a significantly higher inhibition of luciferase activity than mutating GPx8 HRE2. Electrophoretic mobility-shift assay showed that both HREs exhibit enhanced binding to a nuclear extract from DES/BP-treated cells, with stronger binding by GPx8 HRE1. In DES-treated cells transfected with pGL3^GPx8wt or mutants thereof, silencing of HIF2α, but not HIF1α, abolishes luciferase activity. Thus GPx8 is a novel HIF target preferentially responding to HIF2α binding at its two novel functional GPx8 HREs, with GPx8 HRE1 playing the major role. Fibroblast growth factor (FGF) treatment increases GPx8 mRNA expression, and reporter gene experiments indicate that induction occurs via HIF. Comparing the effects of depleting GPx8 on the downstream effectors of FGF or insulin signaling revealed that absence of GPx8 results in a 16- or 12-fold increase in phosphorylated ERK1/2 by FGF or insulin treatment, respectively. Furthermore, in GPx8-depleted cells, phosphorylation of AKT by insulin treatment increases 2.5-fold. We suggest that induction of GPx8 expression by HIF slows down proliferative signaling during hypoxia and/or growth stimulation through receptor tyrosine kinases.     

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

Effects of diet quality on phenotypic flexibility of organ size and digestive function in Mongolian gerbils (Meriones unguiculatus)

In the context of evolution and ecology, there is a trade-off between the benefits of processing food through a digestive system with specific phenotypic attributes and the cost of maintaining and carrying the digestive system. In this study, we tested the hypothesis that digestive modulations at several levels can match each other to meet the energy and nutrient demands of Mongolian gerbils, a small granivorous rodent species, by acclimating them to a high-quality diet diluted with alfalfa powder. Mongolian gerbils on the diluted diet maintained metabolizable energy intake by an integrated processing response (IPR), which included increases in dry matter intake, gut capacity and rate of digesta passage after 2-weeks of acclimation. Down-regulation of hydrolytic enzyme activity in the intestinal brush-border membrane supported the adaptive modulation hypothesis. The absence of up-modulation of summed enzyme hydrolytic capacity on the diluted diet indicated that greater mass of small intestine on a high-fibre diet is not a direct indicator of digestive or absorptive capacity. Changes in mass of vital organs and carcass suggested that the amount of energy allocated to various organs and hence physiological functions was regulated in response to diet shift.     

ESEEM and Mössbauer studies of the ferriheme model compound bis(3-aminopyrazole)tetraphenylporphyrinatoiron(III) chloride, [TPPFe(NH2PzH)2]Cl

A model heme complex, bis(3-aminopyrazole)tetraphenylporphinatoiron(III) chloride, [TPPFe (NH₂PzH)₂]Cl, for which the EPR g-values lead to a rhombicity V/Δ=1.2 if g _zz is the largest g-value, have been investigated by electron spin echo envelope modulation (ESEEM) and Mössbauer spectroscopies. The ESEEM studies focus on the proton sum frequency peaks at near twice the proton Larmor frequency. Analysis of the distant proton peak (mainly due to the pyrrole-H) at exactly twice the proton Larmor frequency shows conclusively that g _zz is aligned along the normal to the porphyrin plane, and thus the electron configuration is (d _xy )²(d _xz ,d _yz )³, with g _zz >g _yy >g _xx . This system is thus another violation to Taylor's "proper axis system" rule. The near proton (the α-H and N-H of the axial ligands) peaks provide distance information for those protons from the metal. Magnetic Mössbauer studies of the same complex confirm the (d _xy )²(d _xz ,d _yz )³ ground state and indicate that, as is the case for cytochrome P450_cam, A _xx is the largest magnitude A-value, and is negative in sign. Other low-spin iron(III) porphyrinates also have A _xx of negative sign, but usually the magnitude is only about half that of A _zz , which is always positive in sign.