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

Functional effect of indole-3 carbinol in the viability and invasive properties of cultured cancer cells

Cancer treatment typically involves multiple strategies, such as surgery, radiotherapy, and chemotherapy, to remove tumors. However, chemotherapy often causes side effects, and there is a constant search for new drugs to alleviate them. Natural compounds are a promising alternative to this problem. Indole-3-carbinol (I3C) is a natural antioxidant agent that has been studied as a potential cancer treatment. I3C is an agonist of the aryl hydrocarbon receptor (AhR), a transcription factor that plays a role in the expression of genes related to development, immunity, circadian rhythm, and cancer.In this study, we investigated the effect of I3C on cell viability, migration, invasion properties, as well as mitochondrial integrity in hepatoma, breast, and cervical cancer cell lines. We found that all tested cell lines showed impaired carcinogenic properties and alterations in mitochondrial membrane potential after treatment with I3C. These results support the potential use of I3C as a supplementary treatment for various types of cancer

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.

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

Decoding Aging: Understanding the Complex Relationship among Aging, Free Radicals, and GSH

N-aryl maleimides can undergo a 1,4-Michael-type addition reaction with reduced glutathione (GSH), leading to a decreased concentration of GSH and an increased concentration of free radicals (FRs) in cells. GSH is a critical scavenging molecule responsible for protecting cells from oxidation and for maintaining redox homeostasis. N-aryl maleimides disturb redox homeostasis in cells because they scavenge thiol-containing molecules, especially GSH. This study aimed at measuring the concentrations of GSH and FRs by electronic paramagnetic resonance (EPR), in the brain and liver tissue of male Wistar rats (ex vivo) at different ages and after treatment with 3,5-dimaleimylbenzoic acid (3,5-DMB). Our results showed a relationship between age and the concentrations of GSH and FRs in cells. In young rats, the concentration of GSH was higher than in old rats, while the concentration of FRs was higher in adult rats than in young rats, suggesting an inverse relationship between GSH and FRs. On the other hand, the reaction of 3,5-DMB (an electrophilic maleimide) with cellular GSH increased the FR content. The results of this study contribute to the awareness that the process of aging implies not only a loss of tissue function but also essential changes in the molecular contents of cells, especially the concentrations of FRs and GSH

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

Putrescine effect on TvCP39 transcript and protein.

<p>A) Semi-quantitative RT-PCR analysis performed with total RNA from untreated parasites grown in normal medium (N)(lane 1); DAB-treated parasites (D)(lane 2,); DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP)(lane 3); DAB-treated trichomonads transferred to normal medium (DN)(lane 4), and trichomonads grown in normal medium and transferred into 40 mM exogenous putrescine medium (NP)(lane 5) to amplify 238 bp from the <i>tvcp39</i>. A 112 pb amplicon from <i>β-tubulin</i> was amplify as a loading control. B) qRT-PCR of samples described in A. The Ct levels of <i>tvcp39</i> mRNA in trichomonads after DAB treatment (bar D) decreased at 20% but the <i>tvcp39</i> mRNA were restored (70%) by adding 40 mM exogenous putrescine to DAB-treated parasites (bar DP). C) Total protein extract from <i>T. vaginalis</i> grown in normal media (N)(lane 1); DAB-treated parasites (D)(lane 2); DAB-treated trichomonads transferred into exogenous putrescine media (DP)(lane 3); DAB-treated parasites transferred into normal medium (DN)(lane 4) and trichomonads grown in normal medium transferred to medium with 40 mM exogenous putrescine (NP)(lane 5) were blotted onto nitrocellulose membranes and incubated with anti-TvCP39 and anti-α-tubulin (loading control) antibodies. Arrowheads indicate the immunodetected protein for each antibody employed.</p

The <i>tvcp39</i> mRNA and protein stabilities are regulated by putrescine.

<p>A) RNAm levels of <i>tvcp39</i> by semi-quantitative RT-PCR analysis using total RNA from parasites treated with actinomycin D and grown in normal culture media (N); or DAB-treated parasites (D); or DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP); or DAB-treated trichomonads transferred to normal medium (DN); or trichomonads grown in normal medium and transferred into 40 mM exogenous putrescine medium (NP). Samples were taken at 0, 1, 3, 6, 8, 12 and 24 h for amplification of 110 pb of <i>tvcp39</i> mRNA and 112 bp of <i>β-tubulin</i> mRNA (<i>β-tub)</i>(loading control). Arrowheads indicate the amplification products obtained. B) Transcriptional blockade using actinomycin D. Trichomonads grown in normal medium (N), DAB-treated trichomonads (D), DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP), DAB-treated trichomonads transferred into normal medium (DN), and trichomonads grown in normal medium and transferred into an exogenous putrescine medium (NP) were treated with actinomycin D. Samples taken at several times (0, 1, 2, 6, 8, 12, and 24 h) were use to amplified the <i>tvcp39</i> mRNA which was quantified by densitometric analysis and normalized. Bars represent the mean of each sample and the standard errors were included. C) Blockage of protein synthesis by cycloheximide. Trichomonads were treated with 10 µg of cycloheximide and grown in normal culture media (N); or DAB-treated parasites (D); or DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP). Samples were taken at several times for Western blot analysis using anti-TvCP39 (dilution 1: 1000) and α-tubulin (dilution 1∶100) antibodies. Arrowheads indicate the TvCP39 and α-tubulin proteins. D) Densitometric analysis of the samples described in C. The bands corresponded to TvCP39 were quantified and normalized to α tubulin. Bars represent the mean of three biological triplicates.</p