Identification of the Phosphorylated Residues in TveIF5A by Mass Spectrometry

AbstractThe initiation factor eIF5A in Trichomonas vaginalis (TveIF5A) is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TveIF5A have been reported. The most acidic isoform (pI 5.2) corresponds to the precursor TveIF5A, whereas the mature TveIF5A appears to be the most basic isoform (pI 5.5). In addition, the intermediary isoform (pI 5.3) is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TveIF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TveIF5A contains four phosphorylated residues (S3, T55, T78 and T82). Phosphorylation at S3 and T82 is also identified in the intermediary TveIF5A, while no phosphorylated residues are found in the precursor TveIF5A. It has been demonstrated that eIF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 (CK2). Interestingly, a gene encoding a protein highly similar to CK2 (TvCK2) is found in T. vaginalis, which might be involved in the phosphorylation of TveIF5A in T. vaginalis

Lipidomics as a Tool in the Diagnosis and Clinical Therapy

The lipids are essential compounds of cells, with biochemical and structural properties. Lipids are classified according to their chain length or saturation levels and biogenesis. Lipidomics is a spectroscopic and spectrometric technique, like Mass Spectrometry and Nuclear Magnetic Resonance, as well as bioinformatics to quantify and characterize the lipid profile. Lipidomics enables the fundamental understanding of lipid biology, the identification of drug targets for therapy, and the discovery of lipid biomarkers of disease cohorts. Therefore, lipidomics allows knowing the diagnosis and clinical follow-up in medical therapy towards any disease. In this way, the lipid profile allows us to monitor the administration of a clinical treatment and assertively diagnose human diseases

Lipoproteomics: Methodologies and Analysis of Lipoprotein-Associated Proteins along with the Drug Intervention

Lipoproteins are specialized particles involved in the transport and distribution of hydrophobic lipids, as cholesterol and triglycerides, throughout the body. The lipoproteins exhibit a basic spherical shape as complexes of lipids and proteins, and these latter are known as apolipoproteins. Initially, the proteins associated with lipoproteins were recognized as integral or peripheral proteins that only maintain the dynamics and metabolism of lipoproteins. However, there exist many studies on different lipoproteins evidencing that the quantity and type of apolipoproteins and lipoprotein-associated proteins are diverse and could be associated with different lipoprotein function outcomes. Here, we summarized recent processes in the determination of apolipoproteins and lipoprotein-associated proteins profiles through a proteomic approach, analyzing the major methods available and are used to achieve this. We also discuss the relevance of these lipoproteomic analyses on the human disease outcomes

The Role of miR-107 in Prostate Cancer: A Review and Experimental Evidence

Over the past two decades, several research groups have focused on the functioning of microRNAs (miRNAs), because many of them function as positive or negative endogenous regulators of processes that alter during the development of cancer. Prostate cancer is the second most commonly occurring cancer in men. New biomarkers are needed to support the diagnosis of prostate cancer. Although it is necessary to deepen the research on this molecule to explore its potential utility in the diagnosis, follow-up, and prognosis of cancer, our results support a role of miR-107 in the signaling cascades that allow cancer progression, and as shown here, in the progression of Prostate Cancer (PCa). These findings strongly suggest that miR-107 may be a potential circulating biomarker for the diagnosis and prognosis of prostate cancer

Proteins of <em>Streptococcus pneumoniae</em> Involved in Iron Acquisition

Streptococcus pneumoniae is a human pathogen bacterium capable of using hemoglobin (Hb) and haem as a single iron source but not in presence of lactoferrin. This bacterium has developed a mechanism through the expression of several membrane proteins that bind to iron sources, between them a lipoprotein of 37 kDa called Spbhp-37 (Streptococcus pneumoniae haem-binding protein) involved in iron acquisition. The Spbhp-37 role is to maintain the viability of S. pneumoniae in presence of Hb or haem. This mechanism is relevant during the invasion of S. pneumoniae to human tissue for the acquisition of iron from hemoglobin or haem as an iron source

The effect of Zn2+ on prostatic cell cytotoxicity caused by Trichomonas vaginalis

Our investigation focused on the study of the proteome, morphology, and cytotoxicity of T. vaginalis during interactions with prostatic DU-145 cells. The results suggest that approximately 37 different proteins are expressed in the presence of Zn2+, which also down-regulates the protein and transcriptional levels of TvCP65. The result is a negative effect on trichomonal cytotoxicity. The differentially expressed proteins were identified by mass spectrometry analysis.

Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation

Abstract Background Bovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future. Results To isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro. Conclusion Babesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite

Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization

Putrescine effect on the TvCP39 activity from <i>T. vaginalis</i>.

<p>A) Putrescine effect on the proteolytic activity of <i>T. vaginalis</i>. Zimograms using total proteinases from parasites grown in normal media (N)(lane 1), DAB-treated parasites (D)(lane 2), DAB-treated parasites transferred into exogenous putrescine (DP)(lane 3), DAB-treated trichomonads transferred into a normal medium (DN)(lane 4) and parasites grown in normal medium transferred into an exogenous putrescine media (NP)(lane 5). B) Polyamine effect on the proteinases activity bound to HeLa cells. Ligand-proteinases assays using untreated parasites grown in normal medium (N)(lane 1); DAB-treated parasites (D)(lane 2); DAB-treated parasites transferred into exogenous putrescine media (DP)(lane 3), DAB-treated parasites transferred into normal media (DN)(lane 4) and parasites grown in normal media and transferred into an exogenous putrescine media (NP)(lane 5). Arrowhead shows the TvCP30 proteolytic activity. C) Densitometry analyses of TvCP39 proteolytic activity bands from panel B. Bars indicate the average of the intensity of TvCP39 activity bands from three independent ligand-proteinases assays and error bars represent the standard deviations.</p

TvCP39 re-localization after DAB treatment and putrescine restoration.

<p>A) Cytoplasmic (Cyt) and nuclear (Nuc) protein extract from DAB-treated parasites transferred into exogenous putrescine media (DP) (lanes 1 and 2) and from untreated parasites grown in normal media (N)(lanes 3 and 4) were blotted into a nitrocellulose membrane and incubated with anti-TvCP39, anti-TveIF-5A (control of cytoplasmic protein), anti-nucleoporin (control of nuclear protein) and anti-PCNA (control of nuclear protein) antibodies. Arrowheads show TvCP39 (39 kDa), the TveIF-5A (20 kDa), the nucleoporin (53 kDa), and the PCNA (28 kDa) protein bands. B) Zymograms from Cytoplasmic (Cyt) and nuclear (Nuc) protein extract from DAB-treated parasites transferred into exogenous putrescine media (DP) (lanes 1 and 2) and from untreated parasites grown in normal media (N)(lanes 3 and 4). Arrowhead indicates the TvCP39 proteolytic activity.</p