The Interaction of Myeloperoxidase with the Industrial Contaminant 6-PPD: A Potential Pathway for Reactive Metabolites.

6-PPD (N-[1,3-dimethylbutyl]-N'-phenyl-p-phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/H2O2. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.

Neutrophil Myeloperoxidase-Mediated N-Demethylation of Quetiapine Leads to N-Desalkylquetiapine, a Pharmacologically Active Cytochrome P450 Metabolite.

The atypical antipsychotic drugs, quetiapine and clozapine, are associated with idiosyncratic drug reactions (such as agranulocytosis or neutropenia) that are thought to involve reactive metabolites. Neutrophil myeloperoxidase (MPO) metabolism of quetiapine is not well-studied, but is metabolized by cytochrome P450. Based on structural similarity to clozapine, we hypothesized that quetiapine can be metabolized by MPO and that there is overlap between cytochrome P450 and MPO metabolism of quetiapine. The interaction of quetiapine and clozapine with MPO and MPO chlorination activity was studied using UV-vis spectrophotometry. The metabolites were characterized using liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic resonance (EPR) spectroscopy was used for detecting drug-catalyzed glutathione oxidation. In the presence of quetiapine, MPO compound II accumulated for about 7.5 min, whereas in the presence of clozapine, MPO compound II was not observed as it was rapidly reduced back to the resting state. Increasing quetiapine concentrations resulted in a decrease in MPO chlorination activity, while the opposite result was found in the case of clozapine. UV-vis spectral studies showed no change when quetiapine was oxidized in the absence and presence of chloride anion (Cl-, to catalyze chlorination reactions). Significant changes, however, were observed in the same assay with clozapine, where Cl- appeared to hinder the rate of clozapine metabolism. The MPO-catalyzed hydroxylated and dealkylated metabolites of quetiapine and hydroxylated metabolites of clozapine were observed from the LC-MS analyses, particularly when Cl- was included in the reaction. In addition, hydroxylated, dealkylated, and a proposed sulfoxide metabolite of quetiapine were also observed in the reaction catalyzed by human microsomes/NADPH. Lastly, compared to quetiapine, clozapine metabolism by MPO/H2O2 and glutathione produced more glutathionyl radicals using EPR spin trapping. In conclusion, MPO/H2O2/Cl- was shown to metabolize quetiapine to S-oxidation and P450-like dealkylation products, and quetiapine metabolites were generally less reactive than clozapine.

Profiling of glycosphingolipids with SCDase digestion and HPLC-FLD-MS.

Lipid components of cells and tissues feature a large diversity of structures that present a challenging problem for molecular analysis. Glycolipids from mammalian cells contain glycosphingolipids (GSLs) as their major glycolipid component, and these structures vary in the identity of the glycan headgroup as well as the structure of the fatty acid and sphingosine (Sph) tails. Analysis of intact GSLs is challenging due to the low abundance of these species. Here, we develop a new strategy for the analysis of lyso-GSL (l-GSL), GSL that retain linkage of the glycan headgroup with the Sph base. The analysis begins with digestion of a GSL sample with sphingolipid ceramide N-deacylase (SCDase), followed by labelling with an amine-reactive fluorophore. The sample was then analyzed by HPLC-FLD-MS and quantitated by addition of an external standard. This method was compared to analysis of GSL glycans after cleavage by an Endoglycoceramidase (EGCase) enzyme and labeling with a fluorophore (2-anthranilic acid, 2AA). The two methods are complementary, with EGCase providing improved signal (due to fewer species) and SCDase providing analysis of lyso-GSL. Importantly the SCDase method provides Sph composition of GSL species. We demonstrate the method on cultured human cells (Jurkat T cells) and tissue homogenate (porcine brain).

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure.

BACKGROUND: The extension of sepsis encompassing the preterm newborn's brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins. METHODS: Male Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids. RESULTS: At both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II-III) of somatosensory cortex had a reduced surface content of polysialic acid. CONCLUSIONS: Together, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure.

Dess-Martin periodinane oxidative rearrangement for preparation of α-keto thioesters.

A Dess-Martin Periodinane (DMP) mediated oxidative rearrangement reaction was uncovered. The reaction proceeds via oxidation of a ß-hydroxy thioester to a ß-keto thioester, followed by an α-hydroxylation and then further oxidation to form a vicinal thioester tricarbonyl. This product then rearranges, extruding CO2, to form an α-keto product. The mechanism of the rearrangement was elucidated using 13C labelling and analysis of the intermediates as well as the products of the reaction. This efficient process allows for easy preparation of α-keto thioesters which are potential intermediates in the synthesis of pharmaceutically important heterocyclic scaffolds such as quinoxalinones.

Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent.

The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl-tRNA synthetase, and exhibits activity against both blood- and liver-stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non-reducing (NR) iterative type I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced-intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl-tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

A structural motif is the recognition site for a new family of bacterial protein O-glycosyltransferases.

The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a ß-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of ß-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a ß-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.

Generation of free oligosaccharides from bacterial protein N-linked glycosylation systems.

All Campylobacter species are capable of N-glycosylating their proteins and releasing the same oligosaccharides into the periplasm as free oligosaccharides (fOS). Previously, analysis of fOS production in Campylobacter required fOS derivatization or large culture volumes and several chromatography steps prior to fOS analysis. In this study, label-free fOS extraction and purification methods were developed and coupled with quantitative analysis techniques. Our method follows three simple steps: (1) fOS extraction from the periplasmic space, (2) fOS purification using silica gel chromatography followed by porous graphitized carbon purification and (3) fOS analysis and accurate quantitation using a combination of thin-layer chromatography, mass spectrometry, NMR, and high performance anion exchange chromatography with pulsed amperometric detection. We applied our techniques to analyze fOS from C. jejuni, C. lari, C. rectus, and C. fetus fetus that produce different fOS structures. We accurately quantified fOS in Campylobacter species that ranged from 7.80 (±0.84) to 49.82 (±0.46) nmoles per gram of wet cell pellet and determined that the C. jejuni fOS comprises 2.5% of the dry cell weight. In addition, a novel di-phosphorylated fOS species was identified in C. lari. This method provides a sensitive and quantitative method to investigate the genesis, biology and breakdown of fOS in the bacterial N-glycosylation systems.

Deuteration for Metabolic Stabilization of SARS-CoV-2 Inhibitors GC373 and Nirmatrelvir.

Nirmatrelvir and GC373 inhibit the SARS-CoV-2 3CL protease and hinder viral replication in COVID-19. As nirmatrelvir in Paxlovid is oxidized by cytochrome P450 3A4, ritonavir is coadministered to block this. However, ritonavir undesirably alters the metabolism of other drugs. Hydrogens can be replaced with deuterium in nirmatrelvir and GC373 to slow oxidation. Results show that deuterium slows oxidation of nirmatrelvir adjacent to nitrogen by ∼40% and that the type of warhead can switch the site of oxidative metabolism.

Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein.

Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.

Characterization of exogenous bacterial oligosaccharyltransferases in Escherichia coli reveals the potential for O-linked protein glycosylation in Vibrio cholerae and Burkholderia thailandensis.

Bacterial protein glycosylation systems from varying species have been functionally reconstituted in Escherichia coli. Both N- and O-linked glycosylation pathways, in which the glycans are first assembled onto lipid carriers and subsequently transferred to acceptor proteins by an oligosaccharyltransferase (OTase), have been documented in bacteria. The identification and characterization of novel OTases with different properties may provide new tools for engineering glycoproteins of biotechnological interest. In the case of OTases involved in O-glycosylation (O-OTases), there is very low sequence homology between those from different bacterial species. The Wzy_C signature domain common to these enzymes is also present in WaaL ligases; enzymes involved in lipopolysaccharide biosynthesis. Therefore, the identification of O-OTases using solely bioinformatic methods is problematic. The hypothetical proteins BTH_I0650 from Burkholderia thailandensis E264 and VC0393 from Vibrio cholerae N16961 contain the Wzy_C domain. In this work, we demonstrate that both proteins have O-OTase activity and renamed them PglL(Bt) and PglL(Vc), respectively, similar to the Neisseria meningitidis counterpart (PglL(Nm)). In E. coli, PglL(Bt) and PglL(Vc) display relaxed glycan and protein specificity. However, effective glycosylation depends upon a specific combination of the protein acceptor, glycan and O-OTase analyzed. This knowledge has important implications in the design of glycoconjugates and provides novel tools for use in glycoengineering applications. The codification of enzymatically active O-OTase in the genomes of members of the Vibrio and Burkholderia genera suggests the presence of still unknown O-glycoproteins in these organisms, which might have a role in bacterial physiology or pathogenesis.

Heating of consumer cannabis oils can lead to free radical initiated degradation, causing CBD and THC depletion.

Commercial cannabis oil products are widely available in Canada even though there is a significant gap in scientific information regarding them. Oils, such as vegetable oils, are known to undergo oxidative changes through free radical mechanisms when they are heated or aged, but the cannabis oils used in this study did not have expiry dates or best-before usage dates. This led to the question of how these products would be affected with time. We hypothesized that cannabis oils would produce increased concentrations of free radicals in aging-simulated conditions, which would be related to a decrease in cannabidiol (CBD) or Δ9-tetrahydrocannabinol (THC) content. Cannabis oils and their respective vehicles (oils) were heated using two protocols: One (moderate aging method) used a 2-day heating protocol at 50 °C, and the other (enhanced aging method) used a 14-day heating protocol at 70 °C. We used electron paramagnetic resonance (EPR) spectroscopy for free radical analysis using the spin trapping technique using 200 mM PBN and 0.02 mM CuCl2 (for peroxide breakdown to free radicals). For active ingredient analysis (CBD, THC), we used LC/MS. Cannabis oils that contained unsaturated oils as their vehicles, such as olive or sunflower oil, all showed varying degrees of free radical formation. In both aged and unaged oils containing CBD or THC, less free radical formation was detected compared to the vehicle controls. Cannabis oils using medium-chain triglycerides (MCT) showed little or no free radical formation. The most significant decrease in CBD or THC was observed in the products using sunflower oil, to a lesser extent in MCT oil, and THC also decreased in olive oil. These findings are important for consumers and policymakers considering using such products in hot beverages or cooking and highlighting the importance of appropriate storage conditions.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Thioimidate Bond Formation between Cardiac Troponin C and Nitrile-containing Compounds.

We have investigated the mechanism and reactivity of covalent bond formation between cysteine-84 of the regulatory domain of cardiac troponin C and compounds containing a nitrile moiety similar to the calcium sensitizer levosimendan. The results of modifications to the levosimendan framework ranged from a large increase in covalent bond formation to complete inactivity. We present the biological activity of one of the most potent compounds. Limitations, including compound solubility and degradation at acidic pH, have prevented thorough investigation of the potential of these compounds. Our studies reveal the efficacious nature of the malononitrile moiety in targeting cNTnC and its potential in future cardiotonic drug design.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I.

BACKGROUND: Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI135-209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI146-158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI1-37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury. METHODS: Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI1-77 and cTnI135-209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail. RESULTS: cTnI135-209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases. CONCLUSIONS: Both N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies. GENERAL SIGNIFICANCE: cTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury.