A Disordered Region in the EvpP Protein from the Type VI Secretion System of <i>Edwardsiella tarda</i> is Essential for EvpC Binding

<div><p>The type VI secretion system (T6SS) of pathogenic bacteria plays important roles in both virulence and inter-bacterial competitions. The effectors of T6SS are presumed to be transported either by attaching to the tip protein or by interacting with HcpI (haemolysin corregulated protein 1). In <i>Edwardsiella tarda</i> PPD130/91, the T6SS secreted protein EvpP (<i><u>E</u>. tarda</i><u>v</u>irulent <u>p</u>rotein P) is found to be essential for virulence and directly interacts with EvpC (Hcp-like), suggesting that it could be a potential effector. Using limited protease digestion, nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa) contains a substantial proportion (40%) of disordered regions but still maintains an ordered and folded core domain. We show that an N-terminal, 10-kDa, protease-resistant fragment in EvpP connects to a shorter, 4-kDa protease-resistant fragment through a highly flexible region, which is followed by another disordered region at the C-terminus. Within this C-terminal disordered region, residues Pro143 to Ile168 are essential for its interaction with EvpC. Unlike the highly unfolded T3SS effector, which has a lower molecular weight and is maintained in an unfolded conformation with a dedicated chaperone, the T6SS effector seems to be relatively larger, folded but partially disordered and uses HcpI as a chaperone.</p></div

GST-EvpC pull-down assay for truncation mutants of EvpP.

<p>SDS-PAGE showing pull-down results using GST-EvpC and truncation mutants of EvpP. Lane 1: M.W. marker; lane2: GST-EvpC+EvpP; lane 3: GST+EvpP; lane 4: EvpP; lane 5: GST-EvpC+EvpP_1–168; lane 6: GST+EvpP_1–168; lane 7: EvpP_1–168; lane 8: GST-EvpC+EvpP_1–142; lane 9: GST+EvpP_1–142; and lane 10: EvpP_1–142. The region from residues Pro143 to Ile168 is essential for the interaction between EvpC and EvpP.</p

Heteronuclear NOE experiment on P143T EvpP.

<p>(A) Overlay spectra from heteronuclear NOE experiments showing cross-peaks from an experiment without proton saturation (red) and an experiment with proton saturation (black). The label next to each peak corresponds to sequential assignment of the residue. (B) A plot showing the relative ratio of peak intensities with proton saturation against those without proton saturation for all residues that can be assigned. Residue with a low value for the ratio is located at a disordered region. Boundaries of the three identified ordered regions are boxed by dashed lines and labeled Box 1, 2 and 3.</p

EvpP is a dimeric protein in solution.

<p>(A) The recombinant wild type EvpP purified as a dimeric protein with an elution volume of 72 ml from the Superdex 75 gel filtration column (HiLoad 16/60, GE Healthcare). (B) Dynamic light scattering data showing that the hydrodynamic radius of EvpP ranged from 2.92 nm to 3.09 nm, with a mean value of 3.01 nm. The estimated molecular weight ranged from 41.2 kDa to 47.3 kDa, with mean value of 44.4 kDa (data not shown).</p

Limited trypsin digestion of EvpP and EvpP P143T.

<p>(A) Limited trypsin digestion of both wild type and P143T EvpP resulted in two protease-resistant fragments of 10 kDa and 4 kDa consistently within a digestion period of 20–60 min. (B) Gel filtration elution profile showed that the 10-kDa and 4-kDa fragments remain associated with each other after protease digestion.</p

¹H-¹⁵N HSQC spectra of wild type, P143T and trypsin digested EvpP.

<p>(A) Overlay of the ¹H-¹⁵N HSQC spectra of wild type EvpP (black) and P143T EvpP (red). (B) ¹H-¹⁵N HSQC spectrum of trypsin digested EvpP. (C) Sequence of P143T EvpP from <i>E. tarda</i> PPD130/91, with the boundaries of the 10-kDa (residues Met1 to Arg85) and 4-kDa (residues Ile106 to Gly141) protease-resistant fragments boxed. Residues that can be assigned by NMR experiments are shaded in grey. Residues that are predicted to be disordered by the software RONN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110810#pone.0110810-Yang1" target="_blank">[33]</a> and PrDOS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110810#pone.0110810-Ishida1" target="_blank">[34]</a> are marked by black and red asterisks, respectively, above the sequence.</p

Low-Temperature Diffusion of Oxygen through Ordered Carbon Vacancies in Zr₂C_<i>x</i>: The Formation of Ordered Zr₂C_<i>x</i>O_<i>y</i>

Investigations are performed on low-temperature oxygen diffusion in the carbon vacancy ordered ZrC_0.6 and thus induced formation of the oxygen atom ordered ZrC_0.6O_0.4. Theoretically, a superstructure of Zr₂CO can be constructed via the complete substitution of carbon vacancies with O atoms in the Zr₂C model. In the ordered ZrC_0.6, the consecutive arrangement of vacancies forms the vacancy channels along some zone axes in the C sublattice. Through these vacancy channels, the thermally activated oxygen diffusion is significantly facilitated. The oxygen atoms diffuse directly into and occupy the vacancies, producing the ordered ZrC_0.6O_0.4. Relative to the ordered ZrC_0.6, the Zr positions are finely tuned in the ordered ZrC_0.6O_0.4 because of the ionic Zr–O bonds. Because of this fine adjustment of Zr positions and the presence of oxygen atoms, the superstructural reflections are always observable in a selected area electron diffraction (SAED) pattern, despite the invisibility of superstructural reflections in ZrC_0.6 along some special zone axes. Similar to the vacancies in ordered ZrC_0.6, the ordering arrangement of O atoms in the ordered ZrC_0.6O_0.4 is in nanoscale length, thus forming the nano superstructural domains with irregular shapes

MiR-663 inhibits radiation-induced bystander effects by targeting <i>TGFB1</i> in a feedback mode

<div><p>The mechanisms of radiation-induced bystander effects (RIBE) have been investigated intensively over the past two decades. Although quite a few reports demonstrated that cytokines such as TGF-β1 are induced within the directly irradiated cells and play critical roles in mediating the bystander effects, little is known about the signaling pathways that occur in bystander cells. The crucial question as to why RIBE signals cannot be infinitely transmitted, therefore, remains unclear. In the present study, we showed that miR-663, a radiosensitive microRNA, participates in the regulation of biological effects in both directly irradiated and bystander cells via its targeting of TGF-β1. MiR-663 was downregulated, while <i>TGFB1</i> was upregulated in directly irradiated cells. The regulation profile of miR-663 and <i>TGFB1</i>, on the other hand, was reversed in bystander cells, in which an elevated miR-663 expression was exhibited and led to downregulation of TGF-β1. Further studies revealed that miR-663 interacts with <i>TGFB1</i> directly and that through its binding to the core regulation sequence, miR-663 suppresses the expression of <i>TGFB1</i>. Based on the results, we propose that miR-663 inhibits the propagation of RIBE in a feedback mode, in which the induction of TGF-β1 by reduced miR-663 in directly irradiated cells leads to increased level of miR-663 in bystander cells. The upregulation of miR-663 in turn suppresses the expression of TGF-β1 and limits further transmission of the bystander signals.</p></div