Ana Clara Guerrini Schenberg

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 l-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4′ sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.

PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae

The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle. As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1. Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS). Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays. In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2. Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E. coli SOS repair pathway.

Further characterization of the yeast pso4-1 mutant: interaction with rad51 and rad52 mutants after photoinduced psoralen lesions

Thepso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction betweenpso4-1 andrad51 and betweenpso4-1 andrad52 mutants indicated that thePSO4 gene belongs to theRAD52 epistasis group for strand-break repair. Moreover, the presence of thepso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of therad51 andrad52 mutants. Complementation tests using heterozygous diploid strains showed that thePso4 protein might interact with theRad52 protein during repair of 8-MOP photolesions. Thepso4-1 mutant, even though defective in inter- and intea-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to therad51 mutant,pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that thePSO4 gene belongs to theRAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease.

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose‐repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment. Copyright

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal‐regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram‐positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram‐negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20‐ to 53‐fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram‐negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010;107: 469–477.

Static electric fields interfere in the viability of cells exposed to ionising radiation

Purpose: The interference of electric fields (EF) with biological processes is an issue of considerable interest. No studies have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells. Materials and methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the interval 0.5–5 kGy, using a 60Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V · cm−1 static electric field and viable cells were enumerated. Cultures of Candida albicans were incubated at 36°C for 20 h, gamma-irradiated with doses from 1–4 kGy, and submitted to an electric field of 180 V · cm−1. Samples were examined under a fluorescence microscope and the number of unviable (red) and viable (apple green fluorescence) cells was determined. For crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric field of 1250 V/cm, incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a fluorescence microscope to quantify nuclei with γ-H2AX foci. Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric field on the irradiated MRC5 cells the number of nuclei with γ-H2AX foci increased 40%, approximately. Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation + EF suggests that EF likely inactivate cellular recovering processes. The result for the number of nuclei with γ-H2AX foci in MRC5 cells indicates that an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.

Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔμH(+). Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

The role played by endogenous and exogenous electric fields in DNA signaling and repair.

Comments are made and new insights are provided on the key role played by endogenous and exogenous electric fields, where the former starts and conducts the repairing chain, while the latter is able to scramble the completion of the repair process and, as a consequence, may have important potential as a radiation sensitizer for clinical application.

Expression of the 18kDa protein of Mycobacterium leprae in Saccharomyces cerevisiae

SummaryMycobacterium leprae, the etiologic agent of leprosy, until now has not been grown in vitro, resulting in exceedingly obstacles for the production of purified antigens. It is therefore of interest to clone the relevant M.leprae antigens in other easy to handle microbial hosts. Here, we describe two different systems for expressing the 18kDa antigen of M.leprae in S. cerevisiae. Each system was shown to be effective in antigen expression, but the secretion system provided easier purification. Working with different host strains under different growth conditions, large quantities of biologically active proteins were obtained.