Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.     

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), while Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.Key words: Drosophila, tumor suppressor gene, cell polarity, Hippo pathway, Crb, Lgl, aPKC     

Neutrophils are a key component of the antitumor efficacy of topical chemotherapy with ingenol-3-angelate

Harnessing neutrophils for the eradication of cancer cells remains an attractive but still controversial notion. In this study, we provide evidence that neutrophils are required to prevent relapse of skin tumors following topical treatment with a new anticancer agent, ingenol-3-angelate (PEP005). Topical PEP005 treatment induces primary necrosis of tumor cells, potently activates protein kinase C, and was associated with an acute T cell-independent inflammatory response characterized by a pronounced neutrophil infiltrate. In Foxn1(nu) mice depleted of neutrophils and in CD18-deficient mice (in which neutrophil extravasation is severely impaired) PEP005 treatment was associated with a >70% increase in tumor relapse rates. NK cell or monocyte/macrophage deficiency had no effect on relapse rates. Both in vitro and in mice, PEP005 induced MIP-2/IL-8, TNF-alpha, and IL-1beta, all mediators of neutrophil recruitment and activation. In vitro, PEP005 activated human endothelial cells resulting in neutrophil adhesion and also induced human neutrophils to generate tumoricidal-reactive oxygen intermediates. Treatment of tumors with PEP005 significantly elevated the level of anticancer Abs, which were able to promote neutrophil-mediated Ab-dependent cellular cytotoxicity (ADCC) in vitro. PEP005 treatment of tumors grown in SCID mice was also associated with >70% increase in tumor relapse rates. Taken together, these data suggest a central role for neutrophil-mediated ADCC in preventing relapse. PEP005-mediated cure of tumors therefore appears to involve initial chemoablation followed by a neutrophil-dependent ADCC-mediated eradication of residual disease, illustrating that neutrophils can be induced to mediate important anticancer activity with specific chemotherapeutic agents.     

Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.     

Differences in microbial activity and microbial populations of peat associated with suppression of damping-off disease caused by Pythium sylvaticum

The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.