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.     

“Missing perikymata”—fact or fiction? A study on chimpanzee (Pan troglodytes verus) canines

Recently, a lower than expected number of perikymata between repetitive furrow‐type hypoplastic defects has been reported in chimpanzee canines from the Fongoli site, Senegal (Skinner and Pruetz: Am J Phys Anthropol 149 (2012) 468–482). Based on an observation in a localized enamel fracture surface of a canine of a chimpanzee from the Taï Forest (Ivory Coast), these authors inferred that a nonemergence of striae of Retzius could be the cause for the “missing perikymata” phenomenon in the Fongoli chimpanzees. To check this inference, we analyzed the structure of outer enamel in three chimpanzee canines. The teeth were studied using light‐microscopic and scanning‐electron microscopic techniques. Our analysis of the specimen upon which Skinner and Pruetz (Am J Phys Anthropol 149 (2012) 468–482) had made their original observation does not support their hypothesis. We demonstrate that the enamel morphology described by them is not caused by a nonemergence of striae of Retzius but can be attributed to structural variations in outer enamel that result in a differential fracture behavior. Although rejecting the presumed existence of nonemergent striae of Retzius, our study provided evidence that, in furrow‐type hypoplastic defects, a pronounced tapering of Retzius increments can occur, with the striae of Retzius forming acute angles with the outer enamel surface. We suggest that in such cases the outcrop of some striae of Retzius is essentially unobservable at the enamel surface, causing too low perikymata counts. The pronounced tapering of Retzius increments in outer enamel presumably reflects a mild to moderate disturbance of the function of late secretory ameloblasts. Am J Phys Anthropol 157:276–283, 2015. © 2015 Wiley Periodicals, Inc.     

Identification and characterization of Escherichia coli thioesterase III that functions in fatty acid beta-oxidation

When Escherichia coli is grown on oleic acid as the sole carbon source, most of this fatty acid is completely degraded by beta-oxidation. However, approximately 10% of the oleic acid is only partially degraded to 3,5- cis-tetradecadienoyl-CoA, which is hydrolyzed to 3,5- cis-tetradecadienoic acid and released into the growth medium. An investigation of thioesterases involved in this novel pathway of beta-oxidation led to the identification of a new thioesterase (thioesterase III) that is induced by growth of E. coli on oleic acid. This enzyme was partially purified and identified as the ybaW gene product by mass spectrometric analysis of tryptic peptides. The ybaW gene, which has a putative consensus sequence for binding the fatty acid degradation repressor, was cloned and expressed in E. coli. Thioesterase III was shown to be a long-chain acyl-CoA thioesterase that is most active with 3,5-tetradecadienoyl-CoA, a minor metabolite of oleate beta-oxidation. Its substrate specificity and induction by fatty acids agree with its proposed function in the thioesterase-dependent pathway of beta-oxidation. Thioesterase III is proposed to hydrolyze metabolites of beta-oxidation that are resistant to further degradation and that would inhibit the flux through the pathway if they were allowed to accumulate.     

Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion

Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1β (IL-1β) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.     

Segmental duplication associated with the human-specific inversion of chromosome 18: a further example of the impact of segmental duplications on karyotype and genome evolution in primates

The human-specific pericentric inversion of chromosome 18 was analysed using breakpoint-spanning BACs from the chimpanzee and human genome. Sequence and FISH analyses disclosed that the breakpoints map to an inverted segmental duplication of 19-kb, which most likely mediated the inversion by intrachromosomal homologous recombination. The 19-kb duplication encompasses the 3 end of the ROCK1 gene and occurred in the human lineage. Only one copy of this segment is found in the chimpanzee. Due to the inversion, the genomic context of the ROCK1 and USP14 genes is altered. ROCK1 flanks USP14 in the long arm of the chimpanzee chromosome 17, which is homologous to human chromosome 18. This order is interrupted by the inversion in humans. ROCK1 is localized close to the pericentromeric region in 18q11 and USP14 is inverted to distal 18p11.3 in direct neighbourhood to LSAU-satellites, -satellites and telomere-associated repeats. Our findings essentially confirm the analysis of Dennehey et al. (2004). Intriguingly, USP14 is differentially expressed in human and chimpanzee cortex as well as fibroblast cell lines determined previously by the analysis of oligonucleotide arrays. Either position effects mediated by the proximity to the telomeric region or nucleotide divergence in regulatory regions might account for the differential expression of USP14. The assignment of the breakpoint region to a segmental duplication underlines the significance of the genomic architecture in the context of genome and karyotype evolution in hominoids.     

Thiol Peroxidase Protects Salmonella enterica from Hydrogen Peroxide Stress In Vitro and Facilitates Intracellular Growth

At present, Salmonella is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in Salmonella enterica and show that a single tpx mutant is susceptible to exogenous hydrogen peroxide (H₂O₂), that it has a reduced capacity to degrade H₂O₂ compared to the ahpCF and tsaA mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.Salmonella is an important human pathogen which causes a variety of diseases, including gastroenteritis, septicemia, and typhoid fever. In the host, salmonellae reside inside phagocytic cells and are exposed to various host defense mechanisms, including oxidative stress (13). The production of superoxide anion (O₂⁻) is crucial, as individuals with chronic granulomatous disease, which is due to a defective phagocyte NADPH oxidase, are more susceptible to infections with Salmonella (10). Likewise, diminished NADPH oxidase activity leads to increased susceptibility to Salmonella in murine macrophages (20-22, 25). Superoxide anion (O₂⁻) is weakly reactive and fails to pass through the bacterial cell wall. After conversion to H₂O₂ by either spontaneous or enzymatic dismutation by superoxide dismutases, it readily diffuses into the bacterial cell and forms reactive hydroxyl radicals (OH) that damage macromolecules such as DNA, proteins, and lipids (12, 17).In principle, Salmonella possesses two classes of enzymes to degrade H₂O₂. Catalases degrade H₂O₂ to water and molecular oxygen independent of an additional reductant. Peroxiredoxin-type peroxidases (peroxiredoxins) reduce organic hydroperoxides to alcohols and hydrogen peroxide to water at the expense of NADH or NADPH. In a recent study by Hébrard et al., three members of the catalase family, KatG, KatE, and KatN, and two members of the peroxiredoxin family, AhpC and TsaA, were characterized in Salmonella (14). Previously it had been shown that single katE, katG, and katN Salmonella mutants did not show increased susceptibility to exogenous H₂O₂ (3, 24). In macrophages a katG katE katN triple mutant had no growth defect, whereas an ahpCF tsaA double mutant showed a reduced growth rate in macrophages (14). These observations point out the multiple routes that have evolved in Salmonella to protect the pathogen against oxidative stress and suggest that peroxiredoxins play a dominant role in the antioxidant defense during infection. In this study we characterized a third peroxiredoxin-type peroxidase, Tpx. Surprisingly, a simple tpx mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) was more susceptible to exogenous H₂O₂ than the wild type (WT). The mutant grew less well in activated macrophages and showed a reduced peroxidase activity toward H₂O₂.     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.