Quantitative proteomic analysis of the tizoxanide effect in vero cells.

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.

Hydroxyl radical-induced hydrogen/deuterium exchange in amino acid carbon-hydrogen bonds.

Hydroxyl radicals produced by radiolysis under anaerobic conditions in the presence of dithiothreitol and D2O have been shown to be capable of inducing hydrogen/deuterium (1H/2H) exchange in carbon-hydrogen bonds of amino acids. When the solution is saturated with N2O, a 1H/2H exchange efficiency of 38% (based on the G value of 5.6 x 10(-7) mol J(-1) for hydroxyl radical) was determined by measuring the amino acid isotope ratio [M+H+1]+/[M+H]+ using electrospray ionization-mass spectrometry. The incorporation of 2H was proportional to the amount of hydroxyl radical generated and required the presence of dithiothreitol. Using standard anaerobic reaction conditions with dithiothreitol and N2O, incorporations of 2H of 3% and 8% into L-valine (100 microM, 35 microM DTT) and L-leucine (100 microM, 31 microM DTT), respectively, were achieved after a dose of 89 Gy and, using d8-DL-valine (100 microM, 35 microM DTT) with H2O as the solvent, approximately 2% incorporation of protium was detected. Additionally, 1H/2H exchange into the peptide (Ala2)-leucine enkephalin produced 6% incorporation of 2H. These results directly demonstrate the ability of sulfhydryl groups to mediate the chemical repair of proteins through hydrogen-atom donation to an amino acid carbon-centered radical, thus providing a means of isotopically labeling solvent-accessible amino acid residues of peptides and proteins.

Determination of amino acid isotope ratios by electrospray ionization-mass spectrometry.

Electrospray ionization-mass spectrometry has been shown capable of measuring the isotope ratios in the amino acids, proline, leucine, and arginine with standard deviations of around 0.1%, obviating the need for derivatization and GC separation. The efficiency of electrospray ionization coupled with the sensitivity of the quadrupole mass filter and ion multiplier allows the isotope ratio to be measured with less than 50 nmol of amino acid. The resolution of a standard commercial quadrupole mass filter is capable of providing sufficient resolution such that there is minimal contribution of the primary molecular mass ion, [M+H]+, to either the isotopically important [M+ H + 1)+ or [M+ H - 1]+ peak. The isotope dilution curve between 0 and 25% is linear, with a correlation coefficient of 0.993. It is shown that the precision is great enough that the addition of 0.85 mol% of a single 13C-labeled isotopomer was easily detected and quantified.

Sites of hydroxyl radical reaction with amino acids identified by (2)H NMR detection of induced (1)H/(2)H exchange.

Hydroxyl radical reacts with the aliphatic C-H bonds of amino acids by H atom abstraction. Under anaerobic conditions inclusion of a (2)H atom donor results in (1)H/(2)H exchange into these C-H bonds [Goshe et al. Biochemistry 2000, 39, 1761--1770]. The site of (1)H/(2)H exchange can be detected and quantified by (2)H NMR. Integration of the (2)H NMR resonances within a single spectrum permits the relative rate of H atom abstraction from each position to be determined. Analysis of the aliphatic amino acid spectra indicates that the methine and methylene positions were more reactive than the methyl positions. The (2)H NMR spectra of isoleucine and leucine show that H-atom abstraction distal to the alpha-carbon occurs preferentially. Significant (1)H/(2)H exchange was observed into the delta positions of proline and arginine and into the epsilon-methylene of lysine, indicating that a positive charge on a geminal N does not inhibit the (1)H/(2)H exchange. Comparisons of (2)H NMR integrations between amino acid spectra indicated that (1)H/(2)H exchange occurred in the following descending order: L > I > V > R > K > Y > P > H > F >M> T > A > [C, S, D, N, E, Q, G, W]. The extent of (1)H/(2)H exchange into methionine, N-glycyl-methionine, and methionine sulfoxide suggests that a prominent solvent exchange pathway involving hydroxyl radical mediated oxidation of methionine exists to account for the large (2)H incorporation into the gamma-methylene of methionine sulfoxide that is absent for N-glycyl-methionine. Analysis of the (1)H NMR spectra of the reactions with phenylalanine and tyrosine indicated that hydroxyl radical addition to the phenyl ring under the anaerobic reductive reaction conditions did not result in either exchange or hydroxylation.

Identification of the sites of hydroxyl radical reaction with peptides by hydrogen/deuterium exchange: prevalence of reactions with the side chains.

Hydroxyl radical-effected protium/deuterium ((1)H/(2)H) exchange into the C-H bonds present in peptides has been used to identify the site of hydrogen atom abstraction by hydroxyl radical. Radiolysis of anaerobic, N(2)O-saturated D(2)O solutions containing peptide and dithiothreitol generates a hydroxyl radical that mediates (1)H/(2)H exchange into the side chains of peptides of up to 66 atom % excess (2)H. The (1)H/(2)H exchange is determined by measuring the isotope ratio, [M + H + 1](+)/[M + H](+), of the peptide using electrospray ionization-mass spectrometry. The (1)H/(2)H exchange within each residue of the peptide was determined by measuring the isotope ratio of each isolated dansyl amino acid following hydrolysis and derivatization. Generation of 0.40 mM hydroxyl radical effected (1)H/(2)H exchange into each of the five different residues of (Ala(2))-leucine enkephalin (YAGFL). The propensity of the residues to undergo exchange was L > Y > A congruent with F > G, independent of whether they were radiolyzed separately or as the peptide. The minimal exchange into glycine suggests that reaction of hydroxyl radical with the side chain hydrogens predominates over reaction with the polypeptide alpha-hydrogens. The ability of radiolysis to effect (1)H/(2)H exchange into a larger peptide, SNEQKACKVLGI, was also demonstrated.

Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.

A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein. After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry. PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.