Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activitiesFAPESP, 00/11624-

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10–50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one

Genomic structure and marker-derived gene networks for growth and meat quality traits of Brazilian Nelore beef cattle

Abstract\ud \ud Background\ud Nelore is the major beef cattle breed in Brazil with more than 130 million heads. Genome-wide association studies (GWAS) are often used to associate markers and genomic regions to growth and meat quality traits that can be used to assist selection programs. An alternative methodology to traditional GWAS that involves the construction of gene network interactions, derived from results of several GWAS is the AWM (Association Weight Matrices)/PCIT (Partial Correlation and Information Theory). With the aim of evaluating the genetic architecture of Brazilian Nelore cattle, we used high-density SNP genotyping data (~770,000 SNP) from 780 Nelore animals comprising 34 half-sibling families derived from highly disseminated and unrelated sires from across Brazil. The AWM/PCIT methodology was employed to evaluate the genes that participate in a series of eight phenotypes related to growth and meat quality obtained from this Nelore sample.\ud \ud \ud Results\ud Our results indicate a lack of structuring between the individuals studied since principal component analyses were not able to differentiate families by its sires or by its ancestral lineages. The application of the AWM/PCIT methodology revealed a trio of transcription factors (comprising VDR, LHX9 and ZEB1) which in combination connected 66 genes through 359 edges and whose biological functions were inspected, some revealing to participate in biological growth processes in literature searches.\ud \ud \ud Conclusions\ud The diversity of the Nelore sample studied is not high enough to differentiate among families neither by sires nor by using the available ancestral lineage information. The gene networks constructed from the AWM/PCIT methodology were a useful alternative in characterizing genes and gene networks that were allegedly influential in growth and meat quality traits in Nelore cattle.This study was conducted with funding from EMBRAPA (Macroprograma1,\ud 01/2005) and FAPESP (process number 2012/23638-8). GBM, LLC, LCAR and\ud MMA were granted CNPq fellowships. We thank Sean McWilliam, Marina R. S.\ud Fortes, Edilson Guimaraes, Robson Rodrigues Santiago, Roselito F. da Silva,\ud Fernando F. Cardoso, Flavia Aline Bressani, Wilson Malago Jr., Avelardo U. C.\ud Ferreira, Michel E. B. Yamaguishi and Fabio D. Vieira for the help and\ud technical assistance. The authors would like to acknowledge the\ud collaborative efforts among EMBRAPA, University of Sao Paulo and CSIRO

Oligomerization of the R263E mutant EP24.15.

<p>(A) Blotting shown is representative of the anti-EP24.15 labeling of the wild and R263E mutant protein incubated at the indicated GSSG concentrations after TCEP reduction. Samples were run on 8.5% SDS-PAGE. (B) Optical density of the dimer and trimer forms of EP24.15. The OD of the 156 kDa and 234 kDa bands was normalized according to the OD values of the 78 kDa band of each sample. <i>AU</i>, arbitrary unit. Values shown are means ± SD of three independent experiments. *p≤0.0032 and ^#p≤0.024 (Student’s t-test) compared to similar WT samples.</p

EP24.15 fragments containing S-glutathiolated Cys residues generated by trypsin hydrolysis and followed by Q-ToF analysis.

<p>The wild type protein was treated with TCEP followed by 1 mM GSSG, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039408#s2" target="_blank">Materials and Methods</a>. Next, samples were digested with trypsin for Q-TOF analysis.</p><p>Sequence coverage was <b>63%</b>. <u>Underlined</u> and <i>italic</i> Cys notations were S-glutathiolated and reduced, respectively. Fragments containing the other eight EP24.15 Cys residues were not detected in this condition; those containing oxidized Cys residues to –SOH, SO₂H or –SO₃H were also investigated and not detected. Representative Q-ToF spectra detecting 305.1 mass additions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039408#pone-0039408-g001" target="_blank">Figure 1</a>.</p>1<p>The same residues were also S-glutathiolated after incubation with 50 µM GSSG.</p

Glutathione interactions to EP24.15 Cys residues.

<p>The 3D<b>-</b>EP24.15 structure focusing S-glutathiolated Cys residues is represented by ribbons and the glutathione (GSH) molecule by sticks. The Cys-sulfur, nitrogen and oxygen atoms are highlighted in yellow, blue and red, respectively. The distances (Å) of major GSH interactions to EP24.15 residues are highlighted (dashed lines).</p

Glutathione covalent docking on EP24.15 surface.

<p>The 3D<b>-</b>EP24.15 structure focusing S-glutathiolated Cys residues is surface represented and the glutathione (GSH) molecule by sticks. The Cys-sulfur, nitrogen and oxygen atoms are highlighted in yellow, blue and red, respectively. Arrows indicate protein hydrogen-acceptors. Their distance to the protein Cys-sulfur atom is shown in A (white line) and was the same in all other S-glutathiolated Cys residues (B – E).</p

Cys residues were analyzed according to their Solvent Access Surface (SAS) and distance of the Cys-sulfur atom from positively charged residues.

<p>The methodology of the analyses performed is described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039408#s2" target="_blank">Materials and Methods</a>.</p>1<p>According to MS analyses.</p>2<p>Solvent Access Surface.</p>3<p>Positively charged residues identified as close to Cys residues shown in the first column.</p>4<p>Distance between positive charge and the Cys sulfur atom.</p>5<p>The fractional values (Relative) are the surface areas of the residues relative to those of the exposed residue in the tripeptide Ala-<u>X</u>-Ala, where <u>X</u> is the residue of interest.</p

Spectra representative of Q-ToF analyses.

<p>The spectra shown were obtained after trypsin-hydrolysis of EP24.15 incubated with TCEP, followed or not by treatment with 1 mM GSSG, and refer to the fragments containing the C175 and C246 residues as follows: (A) and (C) after TCEP-treatment, respectively; (B) and (D) after incubation of the TCEP-reduced protein with 1 mM GSSG, respectively.</p