Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ～50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.     

Some considerations for analyzing biodiversity using integrative metagenomics and gene networks

Background  

Improving knowledge of biodiversity will benefit conservation biology, enhance bioremediation studies, and could lead to new medical treatments. However there is no standard approach to estimate and to compare the diversity of different environments, or to study its past, and possibly, future evolution.     

MnSOD Upregulation Induces Autophagic Programmed Cell Death in Senescent Keratinocytes

Senescence is a state of growth arrest resulting mainly from telomere attrition and oxidative stress. It ultimately leads to cell death. We have previously shown that, in keratinocytes, senescence is induced by NF-kappaB activation, MnSOD upregulation and H₂O₂ overproduction. We have also shown that senescent keratinocytes do not die by apoptosis but as a result of high macroautophagic activity that targets the primary vital cell components. Here, we investigated the mechanisms that activate this autophagic cell death program. We show that corpses occurring at the senescence plateau display oxidatively-damaged mitochondria and nucleus that colocalize with autophagic vacuoles. The occurrence of such corpses was decreased by specifically reducing the H₂O₂ level with catalase, and, conversely, reproduced by overexpressing MnSOD or applying subtoxic doses of H₂O₂. This H₂O₂-induced cell death did occur through autophagy since it was accompanied by an accumulation of autophagic vesicles as evidenced by Lysotracker staining, LC3 vesiculation and transmission electron microscopy. Most importantly, it was partly abolished by 3-methyladenine, the specific inhibitor of autophagosome formation, and by anti-Atg5 siRNAs. Taken together these results suggest that autophagic cell death is activated in senescent keratinocytes because of the upregulation of MnSOD and the resulting accumulation of oxidative damages to nucleus and mitochondria.     

Prion Replication in the Hematopoietic Compartment Is Not Required for Neuroinvasion in Scrapie Mouse Model

Fatal neurodegenerative prion diseases are caused by the transmissible PrP^Sc prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B₄ receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G_i2 protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human α₅ integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB₄ binding in the presence of the purified G protein Gα_i2. The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5′-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified Gα_i2β₁γ₂ protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.     

Autoactivation of Transforming Growth Factor β-activated Kinase 1 Is a Sequential Bimolecular Process

Transforming growth factor-β-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain αG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic αG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of αG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.     

Effect of endothelin-1 on osteoblastic differentiation is modified by the level of connexin43: comparative study on calvarial osteoblastic cells isolated from Cx43+/−and Cx43+/+ mice

Bone is a dynamic tissue that undergoes a precise remodeling process involving resorptive osteoclastic cells and bone-forming osteoblastic (OB) cells. The functional imbalance of either of these cell types can lead to severe skeletal diseases. The proliferation and differentiation of OB cells play a major role in bone development and turnover. These cellular processes are coordinated by connexin43 (Cx43)-based gap-junctional intercellular communication (GJIC) and by soluble factors such as endothelin-1 (ET-1). We have used the Cx43 heterozygous (Cx43^+/−) murine model to study the possible cross-talk between Cx43 and ET-1 in cultured calvarial OB cells. On microcomputed tomographic analysis of 3-day-old pups, Cx43^+/− mice showed hypomineralized calvaria in comparison with their Cx43^+/+ littermates. Characterization of cultured OB cells clearly demonstrated the effect of the partial deletion of the Cx43 gene on its expression, on GJIC, and subsequently on OB differentiation. In this model, ET-1 (10⁻⁸ M) lost its mitogenic action in Cx43^+/− OB cells compared with Cx43^+/+ cells. Moreover, a correlation between the inhibition of cell differentiation by ET-1 and the decreased amount and function of Cx43 was found in Cx43^+/+ OB cells but not in their Cx43^+/− counterparts. Thus, as Cx43 is linked to OB differentiation, our data indicate that this mitogenic ET-1 peptide has pronounced effects on fully differentiated OB cells. With respect to roles in mechanotransduction and OB differentiation, Cx43 might modulate osteoblastic sensitivity to soluble factors.     

Decomposition dynamics and structural plant components of genetically modified <Emphasis Type="Italic">Bt</Emphasis> maize leaves do not differ from leaves of conventional hybrids

The cultivation of genetically modified Bt maize has raised environmental concerns, as large amounts of plant residues remain in the field and may negatively impact the soil ecosystem. In a field experiment, decomposition of leaf residues from three genetically modified (two expressing the Cry1Ab, one the Cry3Bb1 protein) and six non-transgenic hybrids (the three corresponding non-transformed near-isolines and three conventional hybrids) was investigated using litterbags. To elucidate the mechanisms that cause differences in plant decomposition, structural plant components (i.e., C:N ratio, lignin, cellulose, hemicellulose) were examined. Furthermore, Cry1Ab and Cry3Bb1 protein concentrations in maize leaf residues were measured from harvest to the next growing season. While leaf residue decomposition in transgenic and non-transgenic plants was similar, differences among conventional cultivars were evident. Similarly, plant components among conventional hybrids differed more than between transgenic and non-transgenic hybrids. Moreover, differences in senescent plant material collected directly from plants were larger than after exposure to soil for 5 months. While the concentration of Cry3Bb1 was higher in senescent maize leaves than that of Cry1Ab, degradation was faster, indicating that Cry3Bb1 has a shorter persistence in plant residues. As decomposition patterns of Bt-transgenic maize were shown to be well within the range of common conventional hybrids, there is no indication of ecologically relevant, adverse effects on the activity of the decomposer community.