Specific sequence of motifs of mitochondrial uncoupling proteins

We have searched for the exclusivity of common sequence motifs of the mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, BMCP1, and plant UCP [PUMP]) within the gene family of mitochondrial anion carrier proteins. The UCP-specific sequences, "UCP signatures", were found in the first, second, and fourth alpha-helices. First: Ala/Ser-Cys/Thr/n-n/Phe-Ala/Gly-[negatively charged residue]-n/Phe-n/Cys-Thr-Phe/n; second: Gly/Ala-Ile/Leu-Gln/X-[positively charged residue]-NH-n/Cys-Ser/nphi/X-n/Ser-OH/Gly-n-[positively charged residue]-Ile/Met-Gly/Val-n/Thr; fourth: Pro-Asn/ Thr-n-X-[positively charged residue]-Asn/Ser/Ala-n-n-Ile/Leu-n-Asn/Val-Cys/n-n/Thr-[negatively charged residue]-n-n/Thr/Pro-OH/Val (n, nonpolar; phi, aromatic; (positively charged residue/negatively charged residue, charged residue). The second and part of the third signature are also present in the yeast dicarboxylate transporter. The UCP signature excluding BMCP1 was also found in the second matrix segment: [positively charged residue]-(Pro/ del-Leu/del)-[positively charged residue]-phi-X-Gly/Ser-Thr/n-X-NH/[negatively charged residue]-Ala-phi. These UCP signatures are thought to be involved in fatty acid anion binding and translocation.     

Analysis of the mechanism by which tryptophan analogs inhibit human myeloperoxidase

Myeloperoxidase (MPO) catalyzes the formation of oxidants that have been implicated in the pathogenesis of various diseases, including cardiovascular and pulmonary diseases and cancer. Inhibition of MPO oxidant-generating activity represents an attractive target for preventing the progression of inflammatory conditions. Peroxidation and chlorination catalytic activity were utilized to screen for the most effective tryptophan analog that inhibits MPO. Rapid kinetic measurements were performed to determine the mechanisms through which these compounds inhibit the catalytic activity of MPO. Substituents on the amino and carboxyl termini of tryptophan enhance its affinity toward MPO compared to a substituent on the indole ring. Hydrogen-bond donor capabilities and a positive charge on the amino group are not required for MPO inhibition. Hydroxyl-containing indoles did not inhibit MPO H₂O₂-consumption activity. Elimination of the negative charge from the carboxyl terminus by introducing a hydrophobic character significantly enhanced tryptophan analog affinity for MPO and improved its inhibitory properties. Further mechanistic studies indicated that indole compounds inhibited MPO activity through the accumulation of compound II, an inactive MPO intermediate. Our results show that specific structural features of tryptophan analogs are involved in increasing the affinity for MPO and providing a significantly greater inhibition of MPO's catalytic activities.     

Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence

We demonstrate that addition of H₂O₂ to a mixture of myeloperoxidase (MPO), chloride and luminol immediately evokes a short intense flash of chemiluminescence (CL). This flash is diminished in the absence of MPO or chloride, and in the complete system it is suppressed by an MPO inhibitor azide, hypochlorite scavengers taurine or methionine, or an MPO peroxidase-cycle substrate guaiacol. Hence, this CL is mostly due to the MPO halogenation function; a measure of this activity is provided by the integral CL. With three independent methods (CL, taurine chlorination, and peroxidase assay) it is shown that MPO activity is suppressed by ceruloplasmin (Cp). Lactoferrin has no effect either on MPO or on the MPO-Cp complex. It is also shown that peroxidase inhibition by Cp is the stronger the larger is the MPO substrate, which suggests steric hindrances to substrate binding in the MPO-Cp complex. Importantly, the conventional chlorination and peroxidase assays detect MPO inhibition by Cp only at a large excess of the latter, whereas the CL assay reveals it at stoichiometric ratios characteristic of the naturally occurring protein complexes.     

Specific chlorination of isoquinolines by a fungal flavin-dependent halogenase

Rdc2 is the first flavin-dependent halogenase identified from fungi. Based on the reported structure of the bacterial halogenase CmlS, we have built a homology model for Rdc2. The model suggests an open substrate binding site that is capable of binding the natural substrate, monocillin II, and possibly other molecules such as 4-hydroxyisoquinoline (1) and 6-hydroxyisoquinoline (2). In vitro and in vivo halogenation experiments confirmed that 1 and 2 can be halogenated at the position ortho to the hydroxyl group, leading to the synthesis of the chlorinated isoquinolines 1a and 2a, respectively, which further expands the spectrum of identified substrates of Rdc2. This work revealed that Rdc2 is a useful biocatalyst for the synthesis of various halogenated compounds.     

Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein

Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.     

High-density lipoprotein nitration and chlorination catalyzed by myeloperoxidase impair its effect of promoting endothelial repair

High-density lipoprotein (HDL) plays a key role in protecting against atherosclerosis. In cardiovascular disease, HDL can be nitrated and chlorinated by myeloperoxidase (MPO). In this study, we discovered that MPO-oxidized HDL is dysfunctional in promoting endothelial repair compared to normal HDL. Proliferation assay, wound healing, and transwell migration experiments showed that MPO-oxidized HDL was associated with a reduced stimulation of endothelial cell (EC) proliferation and migration. In addition, we found that Akt and ERK1/2 phosphorylation in ECs was significantly lower when ECs were incubated with oxidized HDL compared with normal HDL. To further determine whether oxidized HDL diminished EC migration through the PI3K/Akt and MEK/ERK pathways, we performed experiments with inhibitors of both these pathways. The transwell experiments performed in the presence of these inhibitors showed that the migration capacity was reduced and the differences observed between normal HDL and oxidized HDL were diminished. Furthermore, to study the effects of oxidized HDL on endothelial cells in vivo, we performed a carotid artery electric injury model on nude mice injected with either normal or oxidized HDL. Oxidized HDL inhibited reendothelialization compared to normal HDL in vivo. These findings implicate a key role for MPO-oxidized HDL in the pathogenesis of cardiovascular disease.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

DNA motifs and sequence periodicities

Genomic DNA sequences contain a wealth of information about the bendability and curvature of the DNA molecule. For example, the well-known 10-11 bp periodicities within genomes can be attributed to supercoiled structures or wrapping around nucleosomes. Such periodic signals have previously been examined mainly based on mono- or dinucleotide correlations. In this study, we generalize this approach and analyze correlation functions of longer motifs such as tetramers or poly(A) sequences. Periodically placed motifs may indicate regular protein binding or curvature signals. We detected various periodic signals e.g. strong 10-11 bp oscillations of periodically placed poly(A), poly(T) or poly(W) stretches. These observations lead to a new view on the intensively studied 10-11 bp periodicities.     

Potential role of tryptophan and chloride in the inhibition of human myeloperoxidase

Myeloperoxidase (MPO) binds H2O2 in the absence and presence of chloride (Cl-) and catalyzes the formation of potent oxidants through 1e(-) and 2e(-) oxidation pathways. These potent oxidants have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Thus, inhibition of MPO and its by-products may have a wide application in biological systems. Using direct rapid kinetic measurements and H2O2-selective electrodes, we show that tryptophan (Trp), an essential amino acid, is linked kinetically to the inhibition of MPO catalysis under physiological conditions. Trp inactivated MPO in the absence and presence of plasma levels of Cl(-), to various degrees, through binding to MPO, forming the inactive complexes Trp-MPO and Trp-MPO-Cl, and accelerating formation of MPO Compound II, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO Compound II without any sign of Compound I accumulation. This behavior indicates that Trp binding modulates the formation of MPO intermediates and their decay rates. Importantly, Trp is a poor substrate for MPO Compound II and has no role in destabilizing complex formation. Thus, the overall MPO catalytic activity will be limited by: (1) the dissociation of Trp from Trp-MPO and Trp-MPO-Cl complexes, (2) the affinity of MPO Compound I toward Cl(-) versus Trp, and (3) the slow conversion of MPO Compound II to MPO-Fe(III). Importantly, Trp-dependent inhibition of MPO occurred at a wide range of concentrations that span various physiological and supplemental ranges.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1 x 10(6) M(-1)s(-1)), it reacts sluggishly with compound II (kII 7 M(-1)s(-1)). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 microM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 microM) unless superoxide dismutase was present (I50 5 microM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system

The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288-11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966-7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.     

Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride

Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8, it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. Chlorination of tyrosyl residues was not affected by bromide. With reagent hypohalous acids, bromination of tyrosyl residues was considerably more facile than chlorination. Hypochlorous acid promoted bromination to only a limited extent, which ruled out transhalogenation as a substantive route to 3-bromotyrosine. Chloramines and bromamines were also formed on albumin. Bromamines decayed much faster than chloramines and rapidly gave rise to protein carbonyls. We conclude that at physiological concentrations of chloride and bromide, hypobromous acid can be a major oxidant produced by myeloperoxidase. Its production in vivo will depend on pH and the concentration of bromide. Once produced, hypobromous acid will react with proteins to form bromamines, carbonyls, and brominated tyrosine residues. Consequently, 3-bromotyrosine should be considered as an oxidative product of myeloperoxidase and cannot be used as a specific biomarker for eosinophil peroxidase.     

Tyrosine 192 in apolipoprotein A-I is the major site of nitration and chlorination by myeloperoxidase, but only chlorination markedly impairs ABCA1-dependent cholesterol transport

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO(-)). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO(-). Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr(192) of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO(-), suggesting that solvent accessibility accounted in part for the reactivity of Tyr(192). The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) is the predominant site of nitration and chlorination when MPO or ONOO(-) oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.     

Degradation of mutagens from pyrolysates of tryptophan, glutamic acid and globulin by myeloperoxidase.

Mutagenic compounds isolated from pyrolysates of tryptophan, glutamic acid and globulin were broken down by myeloperoxidase and hydrogen peroxide with loss of their mutagenicity toward Salmonella typhimurium TA98. Lactoperoxidase and horseradish peroxidase were as effective as myeloperoxidase in degradation of the mutagens.     

Improved prediction of allergenicity by combination of multiple sequence motifs

The identification and validation of protein allergens have become more important nowadays as more and more transgenic proteins are introduced into our food chains. Current allergen prediction algorithms focus on the identification of single motif or single allergen peptide for allergen detection. However, an analysis of the 575 allergen dataset shows that most allergens contain multiple motifs. Here, we present a novel algorithm that detects allergen by making use of combinations of motifs. Sensitivity of 0.772 and specificity of 0.904 were achieved by the proposed algorithm to predict allergen. The specificity of the proposed approach is found to be significantly higher than traditional single motif approaches. The high specificity of the proposed algorithm is useful in filtering out false positives, especially when laboratory resources are limited.