Schematic model of the citrus multiprotein complex comprising the PthA4-interacting partners.

<p>Protein-protein and protein-RNA contacts involving the PthA4 interactors based on the yeast two-hybrid, GST-pulldown and gel-shift assays described here, and literature data. The citrus multiprotein complex is reminiscent of that of mammalian miRISC involved in miRNA-mediated deadenylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Fabian1" target="_blank">[51]</a>. Importin-α, which interacts with all PthA variants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Domingues1" target="_blank">[16]</a> is also a component of the cap-binding complex (CBC) which inhibits mRNA deadenylation when in the presence of a poly(A)-specific ribonucleases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Balatsos1" target="_blank">[55]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Sato1" target="_blank">[58]</a>. It is suggested that by interacting with such proteins and with poly(U) RNA (not necessarily simultaneously), PthA proteins may displace some of the components of this complex thought to promote deadenylation and mRNA decay and thus increase mRNA stabilization and translation initiation. U-rich sequences found in both 5′ and 3′ ends of mRNAs could represent binding sites of CsHMG and PthA4.</p

Protein-protein interactions among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.

<p>Yeast cells double-transformed with the indicated prey-bait constructs were grown in SC -Trp -Leu -His -Ade in the presence of 5 mM 3AT. (A) Positive interactions observed between CsTRAX and CsSMC, CsPABP1, CsTRAX, CsRRMP1 and CsVIP2. (B) Protein-protein interactions observed between CsSMC and CsPABP2, CsSMC, CsPABP1, CsVIP2 and CsTRAX, but not between CsSMC and CsKH. (C) Interactions of CsVIP2 with CsKH, reciprocal interactions between CsPABP2 and CsVIP2, and self interactions of CsVIP2 and CsPABP2. (D) Weak interactions between CsHMG and the poly(A)-binding proteins CsPABP2 and CsPABP1. (E) A diagram illustrating the network of interactions observed among the citrus PthA targets. (F) Silver-stained SDS polyacrylamide gels of citrus proteins trapped in cobalt beads carrying the recombinant 6xHis-tagged CsSMC or CsTRAX as baits (bands 9 and 10, respectively). Protein bands excised from the gels, indicated by the numbers, were identified by mass spectrometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone-0032305-t002" target="_blank">Table 2</a> for details). The molecular markers (MM) are shown on the left.</p

Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

<p>Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA</p

PthA binds to CsHMG <i>in vivo</i> through its invariable LRR region.

<p>(A) PthA2-GST, PthA4-GST or GST alone bound to glutathione resins were incubated with a citrus cell lisate. Bound proteins were separated by gel electrophoresis and CsHMG was detected by the anti-CsHMG serum in the PthA samples only. (B) Western blot of GST-pulldown assay of immobilized GST or GST-CsHMG as baits and purified 6xHis-PthA5.5rep+CT2 as prey. The eluted 6xHis-PthA5.5rep+CT2 (∼63 kDa) was detected by the anti-PthA serum only when GST-CsHMG was used as bait. The purified 6xHis-PthA5.5rep+CT2 was added in the first lane of the gel as reference. (C) Western blot analysis of eluted fractions of GST-pulldown assay of immobilized GST or GST-PthALRR as baits and purified 6xHis-CsHMG as prey. The eluted 6xHis-CsHMG (∼22 kDa) was detected only when GST-PthALRR was used as bait. (D) Yeast two-hybrid assay showing the interaction between CsHMG and the PthA LRR domain. Yeast double-tranformants, including controls (GAL4AD+GAL4BD-PthALRR and GAL4BD+GAL4AD-CsHMG), were grown in SC -Trp -Leu -His -Ade in the presence of 5 mM 3AT.</p

<i>Citrus sinensis</i> proteins identified as targets of PthA4 are homologous to nuclear factors involved in chromatin remodeling and repair, transcription regulation and mRNA stabilization/modification.

<p><i>Citrus sinensis</i> proteins identified as targets of PthA4 are homologous to nuclear factors involved in chromatin remodeling and repair, transcription regulation and mRNA stabilization/modification.</p

CsHMG shows identity to plant HMGBs of group B.

<p>(A) Schematic representation of the CsHMG primary structure showing its central HMG-box domain flanked by the basic K-rich N-terminal and the acidic DE-rich C-terminal. (B) Phylogenetic analysis of plant HMGB proteins showing that CsHMG belongs to group B HMGBs. (C) Western-blot detection of the recombinant 6xHis-CsHMG (∼22 kDa) made in bacteria compared to bands detected in citrus cell extracts with the expected molecular size for the endogenous CsHMG (∼16 kDa). The anti-CsHMG serum also cross-reacted with a band of similar size in the cell extracts of <i>A. thaliana</i> wild-type and heterozygous <i>hmg-b1</i> mutant. This band, which has the expected molecular weight for AtHMGB1 (∼18 kDa), is less pronounced in the heterozygous <i>hmgb-1</i> mutant, thus indicating that CsHMG is structurally related to AtHMGB1.</p

<i>Citrus sinensis</i> proteins identified as binding partners of CsSMC and CsTRAX by mass spectrometry.

<p>Protein bands are numbered and they correspond to those depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone-0032305-g002" target="_blank">Fig. 2F</a>.</p

Protein-protein interactions between PthAs and citrus nuclear proteins.

<p>(A) Citrus preys fused to yeast GAL4-AD (GAL4AD-prey) or control plasmid (GAL4AD) were moved into yeast cells carrying one of the four PthA variants fused to GAL4-BD domain as shown in the diagram (1 to 4, respectively). Yeast double-transformants were grown on SC -Trp -Leu -His -Ade in the presence of 5 mM of 3AT. None of prey fusions transactivated the reporter genes when co-transformed with empty bait vector (5). The PthA baits also did not transactivate the reporter genes when co-transformed with the empty prey vector in the same growth conditions (GAL4AD). (B) Western blot detection of eluted fractions from GST pulldown assays using the purified 6xHis-PthAs 1–4 as prey and immobilized GST or GST-fusion proteins as baits. Arrows indicate bands corresponding to the expected size for the GST-fusion proteins CsHMG (∼45 kDa), CsTRAX (∼55 kDa), CsSMC (∼45 kDa), CsRRPMP1 (∼50 kDa), CsRRMP2 (∼46 kDa), CsPABP1 (∼53 kDa) and CsVIP2 (∼85 kDa) detected by the GST anti-serum. PthA proteins (∼116–122 kDa) were detected using the anti-PthA serum. Recombinant PthAs 3 and 4 were added as references in the first lanes of the gels shown in the middle and right panels, respectively.</p

P-I Snake Venom Metalloproteinase Is Able to Activate the Complement System by Direct Cleavage of Central Components of the Cascade

<div><p>Background</p><p>Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the <i>Bothrops</i> genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from <i>Bothrops pirajai</i> venom.</p><p>Results</p><p>Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from <i>Bothrops pirajai</i> venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. <i>In vivo</i>, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.</p><p>Conclusion</p><p>We show here that a P-I metalloproteinase from <i>Bothrops pirajai</i> snake venom activated the Complement system by direct cleavage of the central C-components, <i>i.e.</i>, C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.</p></div

Action of C-SVMP on complement pathways.

<p>Samples (50 µl) of normal human serum (NHS), as the complement source, were incubated for 30 min at 37°C with 50 µl of C-SVMP (0.5 µg) and with 50 µl of <i>Bothrops</i> venom (1 µg) or PBS as positive and negative controls, respectively. The residual complement activity was determined by ELISA for the Classical [A], Alternative [B] and Lectin [C] pathways. The results were expressed as the percentage reduction of component deposition relative to the negative control sample (NHS+PBS). Generation of anaphylatoxins in the serum samples was measured using ELISA kits for C3a [D], C4a [E] and C5a [F]. The results were expressed as the concentration of each anaphylatoxin <i>per</i> mL of human serum. Data are representative of three separate experiments and are expressed as the mean of the duplicates +/− SD. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p