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.     

Glial innate immunity generated by non-aggregated alpha-synuclein in mouse: differences between wild-type and Parkinson's disease-linked mutants

Background

Parkinson''s disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown.

Methods and Findings

In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T).

Conclusions

These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.     

Repair of DNA damage induced by the mycotoxin alternariol involves tyrosyl-DNA phosphodiesterase 1

Alternariol (AOH) was reported recently to act as a topoisomerase poison. To underline the relevance of topoisomerase targeting for the genotoxic properties of AOH, we addressed the question whether human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital to the repair of covalent DNA-topoisomerase adducts, affects AOH-mediated genotoxicity. The relevance of TDP1 activity on AOH-induced genotoxicity was investigated by the comet assay in human cells overexpressing GFP chimera of TDP1 or the inactive mutant TDP1^H263A as well as in cells subjected to siRNA-mediated knock-down of endogenous TDP1. Cells overexpressing TDP1 exhibited significantly less DNA damage after treatment with AOH in comparison to cells expressing the inactive mutant TDP1^H263A. In accordance with these results, levels of AOH inducing DNA strand breaks were increased in TDP1-suppressed cells in comparison to cells transfected with control siRNA. The specific topoisomerase poisons camptothecin and etoposide caused comparable effects, underlining that TDP1 plays an important role in the repair of topoisomerase-mediated DNA damage. In summary, the repair enzyme TDP1 was identified as a factor for the modulation of AOH-mediated DNA damage in human cells.     

Unique Impact of RB Loss on Hepatic Proliferation: TUMORIGENIC STRESSES UNCOVER DISTINCT PATHWAYS OF CELL CYCLE CONTROL

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.     

Discovery of N-methyl-4-(4-methoxyanilino)quinazolines as potent apoptosis inducers. Structure–activity relationship of the quinazoline ring

As a continuation of our efforts to discover and develop apoptosis inducing N-methyl-4-(4-methoxyanilino)quinazolines as novel anticancer agents, we explored substitution at the 5-, 6-, 7-positions of the quinazoline and replacement of the quinazoline by other nitrogen-containing heterocycles. A small group at the 5-position was found to be well tolerated. At the 6-position a small group like an amino was preferred. Substitution at the 7-position was tolerated much less than at the 6-position. Replacing the carbon at the 8-position or both the 5- and 8-positions with nitrogen led to about 10-fold reductions in potency. Replacement of the quinazoline ring with a quinoline, a benzo[d][1,2,3]triazine, or an isoquinoline ring showed that the nitrogen at the 1-position is important for activity, while the carbon at the 2-position can be replaced by a nitrogen and the nitrogen at the 3-position can be replaced by a carbon. Through the SAR study, several 5- or 6-substituted analogs, such as 2a and 2c, were found to have potencies approaching that of lead compound N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (1g, EP128495, MPC-6827, Azixa®).     

Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.     

A fungal metallothionein is required for pathogenicity of Magnaporthe grisea

The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.     

Role of the chaperonin CCT/TRiC complex in G protein betagamma-dimer assembly

Gbetagamma dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, (35)S-labeled Ggamma subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, (35)S-labeled Gbeta subunits traveled at a high apparent molecular mass (approximately 700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with Ggamma. When translated Ggamma was added to translated Gbeta, a new band of low apparent molecular mass (approximately 50 kDa) was observed, which was labeled by either (35)S-labeled Gbeta or Ggamma, indicating that it is a dimer. Formation of the Gbetagamma dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg(2+). This inhibition led to increased association of Gbeta with CCT/TRiC. Although Ggamma did not bind CCT/TRiC, addition of Ggamma to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbetagamma dimer formation by an ATP-dependent reaction.     

Relationship between the ability of oral streptococci to interact with platelet glycoprotein Ibalpha and with the salivary low-molecular-weight mucin, MG2

The oral streptococci Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis are common aetiological agents of infective endocarditis, and their ability to adhere to and induce the aggregation of platelets is thought to be a virulence trait. The platelet glycoprotein GPIbalpha has been implicated as the adhesion receptor for S. sanguinis and S. gordonii, but it is not known if this is the case for S. oralis and other species. The aim of this study was to determine the GPIbalpha-interactive capability of a range of oral streptococci and to determine the relationship between this capability and their ability to interact with the salivary constituents that they would encounter in their normal habitat. All platelet-adhesive S. sanguinis strains and most S. gordonii strains adhered in a GPIbalpha-dependent manner, but strains of S. oralis, Streptococcus cristatus, Streptococcus parasanguinis and Streptococcus mitis had no direct affinity for platelets. Those strains that were able to bind GPIbalpha also bound to the low-molecular-weight submandibular salivary mucin, MG2, and this interaction was sialic acid-dependent. The data suggest that S. sanguinis and S. gordonii may be efficient colonizers of platelet vegetations because of their adaptation to recognize sialylated salivary mucins. In contrast, S. oralis does not interact with platelets and so is likely to colonize vegetations through an as yet unidentified mechanism.