Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells.

Myeloperoxidase (MPO) has been reported in prostate tissue, and considering its pro-oxidant properties, this location might be linked to prostate pathology. The possibility that the glandular prostatic tissue might be the source of MPO and its potential inflammatory effects must be tested. Human prostate material was obtained from prostate biopsies and radical prostatectomies. Immunohistochemistry was performed using MPO-specific human antibody. In situ hybridization using MPO-specific probes and laser-assisted microdissection for quantitative real-time RT-PCR were performed to observe whether MPO is being produced in prostate tissue. Mass spectrometry on prostate biopsies was used to detect products of MPO activity in nucleic acids (DNA/RNA). MPO contribution to intracellular accumulation of ROS and interleukin-8 in prostatic epithelial cells was monitored in vitro. Immunohistochemistry confirmed cellular localization of MPO in epithelial cells of the prostate. The staining varied from light to high intensity. In situ hybridization did not address the presence of mRNA coding for MPO. No MPO-specific modifications on nucleic acids were detected. Mox-LDL was a major factor inducing ROS and cytokines production in prostatic epithelial cells. We did not demonstrate that MPO was synthetized by prostatic epithelial cells. However, in vitro experiments showed the ability of MPO to potentiate the ROS production and inflammation on prostate epithelial cells. Results do not allow us to demonstrate a role of MPO in prostate to date but further studies are mandatory to focus on the potential impact of MPO in the development of prostatic diseases.

Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells.

BACKGROUND AND AIMS: Oxidation of native low-density lipoproteins (LDLs-nat) plays an important role in the development of atherosclerosis. A major player in LDL-nat oxidation is myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and monocytes. MPO produces oxidized LDLs called Mox-LDLs, which cause a pro-inflammatory response in human microvascular endothelial cells (HMEC), monocyte/macrophage activation and formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of the polyunsaturated fatty acid DHA, which promotes resolution of inflammation at the ng/ml level. METHODS: In the present study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the synthesis of RvD1 and its precursors - 17(S)-hydroxy docosahexaenoic acid (17S-HDHA) and docosahexaenoic acid (DHA) - by HMEC, in the presence of several concentrations of Mox-LDLs, copper-oxidized-LDLs (Ox-LDLs), and native LDLs or in mouse plasma. The LC-MS/MS method has been validated and applied to cell supernatants and plasma to measure production of RvD1 and its precursors in several conditions. RESULTS: Mox-LDLs played a significant role in the synthesis of RvD1 and 17S-HDHA from DHA compared to Ox-LDLs. Moreover, Mox-LDLs and LDLs-nat acted in synergy to produce RvD1. In addition, different correlations were found between RvD1 and M1 macrophages, age of mice or Cl-Tyr/Tyr ratio. CONCLUSIONS: These results suggest that although Mox-LDLs are known to be pro-inflammatory and deleterious in the context of atherosclerosis, they are also able to induce a pro-resolution effect by induction of RvD1 from HMEC. Finally, our data also suggest that HMEC can produce RvD1 on their own.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

The presence of modified nucleosides in extracellular fluids leads to the specific incorporation of 5-chlorocytidine into RNA and modulates the transcription and translation.

Myeloperoxidase (MPO) is able to promote several kinds of damage and is involved in mechanisms leading to various diseases such as atherosclerosis or cancers. An example of these damages is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity. Since 5-chlorocytidine has been recently shown in healthy donor plasmas, this study aimed at discovering if these circulating modified nucleosides could be incorporated into RNA and DNA and if their presence impacts the ability of enzymes involved in the incorporation, transcription, and translation processes. Experimentations, which were carried out in vitro with endothelial and prostatic cells, showed a large penetration of all chloronucleosides but an exclusive incorporation of 5-chlorocytidine into RNA. However, no incorporation into DNA was observed. This specific incorporation is accompanied by an important reduction of translation yield. Although, in vitro, DNA polymerase processed in the presence of chloronucleosides but more slowly than in control conditions, ribonucleotide reductase could not reduce chloronucleotides prior to the replication. This reduction seems to be a limiting step, protecting DNA from chloronucleoside incorporation. This study shows the capacity of transcription enzyme to specifically incorporate 5-chlorocytidine into RNA and the loss of capacity-complete or partial-of different enzymes, involved in replication, transcription or translation, in the presence of chloronucleosides. Questions remain about the long-term impact of such specific incorporation in the RNA and such decrease of protein production on the cell viability and function.

Validation of a sensitive LC/MSMS method for chloronucleoside analysis in biological matrixes and its applications.

Myeloperoxidase promotes several kinds of damage and is involved in the development of various diseases (as atherosclerosis and cancers). An example of these damage is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity on those acids. This study aimed to develop and validate a method to analyze oxidized and MPO-specific chlorinated nucleosides in biological matrixes (cells, tissues and plasma). Although a lot of methods to quantify oxidized or chlorinated nucleosides have already been established, none of them took into account all these derivatives together. The new method used a Triple Quadrupole mass spectrometer fitted with a Jet Stream electrospray ionization source. This approach has two advantages compared with existing LC/MSMS analyses: it includes MPO-induced modifications in a unique analysis and obtains a better sensitivity. Our optimized method reached LOQs of 1.50pg and 1.42pg respectively for oxoG and oxo(d)G, being 4 times more sensitive than previous methods, and LOQs of 1.39pg, 1.30pg and 63.4 fg respectively for 5-chlorocytidine, 5-chloro-2'-deoxycytidine and 8-chloroguanosine. Developed method is also 25 times more sensitive for chloroguanosine than the best existing method. Nevertheless, this method is not specific enough for 8-chloro-(2'-deoxy)adenosine analysis. Examples of applications demonstrate the interest of this validated method. Indeed analysis of plasma from healthy donors highlighted exclusively the presence of 5-chlorocytidine (1.0±0.2nM) whereas analysis of treated endothelial cells by HOCl showed chlorination of guanosine and cytidine in cytoplasmic pools and chlorination of (deoxy)cytidine in DNA and RNA. In conclusion, this study shows that 5-chloro-2'-deoxycytidine, 5-chlorocytidine and 8-chloroguanosine are good markers allowing us to detect the MPO activity in biological fluids. The robust, specific and sensitive developed method enables future studies on MPO implications in human diseases.

Advancement in stationary phase for peptide separation helps in protein identification: application to atheroma plaque proteomics using nano-chip liquid chromatography and mass spectrometry.

In the last decades, proteomics has largely progressed. Mass spectrometry and liquid chromatography (LC) are generally used in proteomics. These techniques enable proper separation of peptides and good identification and/or quantification of them. Later, nano-scaled liquid chromatography, improvements of mass spectrometry resolution and sensitivity brought huge advancements. Enhancements in chemistry of chromatographic columns also brought interesting results. In the present work, the potency of identification of proteins by different nano-chip columns was studied and compared with classical LC column. The present study was applied to cardiovascular field where proteomics has shown to be highly helpful in research of new biomarkers. Protein extracts from atheroma plaques were used and proteomics data were compared. Results show that fewer spectra were acquired by the mass spectrometer when nano-chip columns were used instead of the classical ones. However, approximately 40% more unique peptides were identified by the recently optimized chip named Polaris-HR-chip-3C18 column, and 20% more proteins were identified. This fact leads to the identification of more low-abundance proteins. Many of them are involved in atheroma plaque development such as apolipoproteins, ceruloplasmin, etc. In conclusion, present data shows that recent developments of nanoLC column chemistry and dimensions enabled the improved detection and identification of low-abundance proteins in atheroma plaques. Several of them are of major interest in the field of cardiovascular disease.

4-Bromo-2-(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b) inhibits Na+/K(+)-ATPase and Ras oncogene activity in cancer cells.

The in vitro growth inhibitory activity of 26 thiazoles (including 4-halogeno-2,5-disubtituted-1,3-thiazoles) and 5 thienothiazoles was assessed on a panel of 6 human cancer cell lines, including glioma cell lines. (4-Chloro-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12a) and (4-bromo-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12b) displayed ~10 times greater in vitro growth inhibitory activity than perillyl alcohol (POH), which therapeutically benefits glioma patients through the inhibition of both alpha-1 Na(+)/K(+)-ATPase (NAK) and Ras oncogene activity. The in vitro cytostatic activities (as revealed by quantitative videomicroscopy) displayed by 12a and 12b were independent of the intrinsic resistance to pro-apoptotic stimuli associated with cancer cells. Compounds 12a and 12b displayed relatively similar inhibitory activities on purified guinea pig brain preparations that mainly express NAK alpha-2 and alpha-3 subunits, whereas only compound 12b was efficacious against purified guinea pig kidney preparations that mainly express the NAK alpha-1 subunit, which is also expressed in gliomas, melanomas and non-small-cell lung cancers NSCLCs.

Optimization of apolipoprotein-B-100 sequence coverage by liquid chromatography-tandem mass spectrometry for the future study of its posttranslational modifications.

Proteomic applications have been increasingly used to study posttranslational modifications of proteins (PTMs). For the purpose of identifying and localizing specific but unknown PTMs on huge proteins, improving their sequence coverage is fundamental. Using liquid chromatography coupled to mass spectrometry (LC-MS/MS), peptide mapping of the native apolipoprotein-B-100 was performed to further document the effects of oxidation. Apolipoprotein-B-100 is the main protein of low-density lipoprotein particles and its oxidation could play a role in atherogenesis. Because it is one of the largest human proteins, the sequence recovery rate of apolipoprotein-B-100 only reached 1% when conventional analysis parameters were used. The different steps of the peptide mapping process-from protein treatment to data analysis-were therefore reappraised and optimized. These optimizations allowed a protein sequence recovery rate of 79%, a rate which has never been achieved previously for such a large human protein. The key points for improving peptide mapping were optimization of the data analysis software; peptide separation by LC; sample preparation; and MS acquisition. The new protocol has allowed us to increase by a factor of 4 the detection of modified peptides in apolipoprotein-B-100. This approach could easily be transferred to any study of PTMs using LC-MS/MS.