Iodination Of Tyrosine Research Articles

Metabolomic screening of radioiodine refractory thyroid cancer patients and the underlying chemical mechanism of iodine resistance

Radioiodine refractory (RAIR) patients do not benefit from iodine-131 therapy. Thus, timely identification of RAIR patients is critical for avoiding ineffective radioactive iodine therapy. In addition, determining the causes of iodine resistance will facilitate the development of novel treatment strategies. This study was comprised of 20 RAIR and 14 non-radioiodine refractory (non-RAIR) thyroid cancer patients. Liquid chromatography-mass spectrometry was used to identify differences in the serum metabolites of RAIR and non-RAIR patients. In addition, chemical assays were performed to determine the effects of the differential metabolites on iodine uptake. Metabolic pathway enrichment analysis of the differential metabolites revealed significant differences in the phenylalanine and tyrosine metabolic pathways. Notably, quinate and shikimic acid, metabolites of the tyrosine pathway, were significantly increased in the RAIR group. In contrast, the phenylalanine pathway metabolites, hippuric acid and 2-phenylacetamide, were markedly decreased in the RAIR group. Thyroid peroxidase plays an important role in catalyzing the iodination of tyrosine residues, while the ionic state of iodine promotes the iodination reaction. Quinate, shikimic acid, hippuric acid, and 2-phenylacetamide were found to be involved in the iodination of tyrosine, which is a key step in thyroid hormone synthesis. Specifically, quinate and shikimic acid were found to inhibit iodination, while hippuric acid and 2-phenylacetamide promoted iodination. Abnormalities in phenylalanine and tyrosine metabolic pathways are closely associated with iodine resistance. Tyrosine is required for thyroid hormone synthesis and could be a potential cause of iodine resistance.

The Impact of Tyrosine Iodination on the Aggregation and Cleavage Kinetics of MMP-9-Responsive Peptide Sequences.

Matrix metalloproteinase (MMP) enzymes are over-expressed by some metastatic cancers, in which they are responsible for the degradation and remodeling of the extracellular matrix. In recent years, MMPs have emerged as promising targets for enzyme-responsive diagnostic probes because oligopeptides can be designed to be selectively hydrolyzed by exposure to these enzymes. With the ultimate goal of developing radio-iodinated peptides as supramolecular building blocks for MMP-sensitive tools for nuclear imaging and therapy, we designed three MMP-9-responsive peptides containing either tyrosine or iodotyrosine to assess the impact of iodotyrosine introduction to the peptide structure and cleavage kinetics. We found that the peptides containing iodotyrosine underwent more rapid and more complete hydrolysis by MMP-9. While the peptides under investigation were predominantly disordered, it was found that iodination increased the degree of aromatic residue-driven aggregation of the peptides. We determined that these iodination-related trends stem from the improved overall intramolecular order through H- and halogen bonding, in addition to intermolecular organization of the self-assembled peptides due to steric and electrostatic effects introduced by the halogenated tyrosine. These fundamental observations provide insights for the development of enzyme-triggered peptide aggregation tools for localized radioactive iodine-based tumor imaging.

Free‐Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication

AbstractA free‐radical footprinting approach is described for integral membrane protein (IMP) that extends, significantly, the “fast photochemical oxidation of proteins” (FPOP) platform. This new approach exploits highly hydrophobic perfluoroisopropyl iodide (PFIPI) together with tip sonication to ensure efficient transport into the micelle interior, allowing laser dissociation and footprinting of the transmembrane domains. In contrast to water soluble footprinters, PFIPI footprints both the hydrophobic intramembrane and the hydrophilic extramembrane domains of the IMP vitamin K epoxide reductase (VKOR). The footprinting is fast, giving high coverage for Tyr (100 %) and Trp. The incorporation of the reagent with sonication does not significantly affect VKOR's enzymatic function, and tyrosine iodination does not compromise protease digestion and the subsequent analysis. The locations for the modifications are largely consistent with the corresponding solvent accessibilities, recommending this approach for future membrane protein footprinting.

Free-Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication.

A free-radical footprinting approach is described for integral membrane protein (IMP) that extends, significantly, the "fast photochemical oxidation of proteins" (FPOP) platform. This new approach exploits highly hydrophobic perfluoroisopropyl iodide (PFIPI) together with tip sonication to ensure efficient transport into the micelle interior, allowing laser dissociation and footprinting of the transmembrane domains. In contrast to water soluble footprinters, PFIPI footprints both the hydrophobic intramembrane and the hydrophilic extramembrane domains of the IMP vitamin K epoxide reductase (VKOR). The footprinting is fast, giving high coverage for Tyr (100 %) and Trp. The incorporation of the reagent with sonication does not significantly affect VKOR's enzymatic function, and tyrosine iodination does not compromise protease digestion and the subsequent analysis. The locations for the modifications are largely consistent with the corresponding solvent accessibilities, recommending this approach for future membrane protein footprinting.

Lithium from breast-milk inhibits thyroid iodine uptake and hormone production, which are remedied by maternal iodine supplementation.

Lithium is especially taken as a maintenance medication for Bipolar Disorder. In women with bipolar disorder, lithium is often effective during postpartum period, but breast-feeding for medicated mothers is controversial because of harmful effects for her child. At present, the biological mechanisms of lithium are not well-understood, affecting its usage and overall health implications. We developed a rat lithium and breast-feeding model at human therapeutic levels to study the effects of lithium exposure through breast-milk on pups' thyroid function. Novel laser analytical spectroscopy, along with traditional blood and immunohistochemical tests, were applied to further investigate the mechanisms behind the thyroid dysfunction. Maternal iodine supplementation was evaluated as a therapeutic method to address the pups' thyroid dysfunction. Pups exposed to lithium via breastmilk, even with the dam on a sub-therapeutic level, experienced weight gain, reduced blood thyroxine (T4 ), and elevated blood urea nitrogen, indicating effects on thyroid and kidney function. We show that lithium inhibited iodine uptake by thyroid follicles, initiating a mechanism that reduced iodination of tyrosine, thyroglobulin cleavage, and thyroid hormone production. Importantly, infant thyroid function can be significantly improved by administering supplementary iodine to the medicated dam's diet during breast-feeding. These results elucidate the mechanisms of lithium in thyroid function, provide valuable information on use postpartum, and suggest a clinically applicable remedy to side-effects. The results are particularly important for patients (and their infants) who respond well to lithium and need, or choose, to breast-feed.

Effect of Different Chemical Species on the Behavior of Tyrosine in a Typical BR Oscillatory Chemical Reaction

AbstractEffect on the behavior of Tyrosine through a malonic acid (MA) based Briggs‐Rauscher (BR) oscillatory system was studied in order to understand the mechanistic details of thyroid metabolism in vitro, specifically the iodination of tyrosine. It was observed that the present BR system employing tyrosine as a main additive and malonic acid as substrate provides an example of entrainment oscillations, in which malonic acid is responsible for oscillations and in presence of tyrosine, the system showed different oscillatory behavior. The effects of various other chemical species such as SeO2, KSCN, KNO3, KBr and Fe(III) on the tyrosine in this malonic acid based BR system are studied. The study was supported by UV‐Visible spectrophotometry to study the interactions of these chemical species with Dushmann reaction, which is one of the important reaction steps in BR reaction mechanism, in order to investigate the key factors responsible for tyrosine iodination responsible for thyroxin formation. Needless to mention that, all the aforesaid chemical species have due role in normal thyroid metabolism in vivo and as such, the present BR system containing tyrosine as main (first) additive can help in understanding of thyroid metabolism.

Effects of Halogenation on Tyrosine Phosphorylation and Peptide Binding to the Src Homology 2 Domain of Lymphocyte-Specific Protein Tyrosine Kinase

Phosphorylation of tyrosine residues by protein tyrosine kinases (PTK) and phosphotyrosine/Src homology 2 (SH2) domain interactions are crucial not only for signal transduction but also for regulation of PTK activity. Tyrosine residues also receive nitration and halogenation under oxidative conditions. It has been reported that nitration of tyrosine residue caused peptides to be a poor substrate for PTK and that nitrotyrosine residues could bind to SH2 domains as a phosphotyrosine mimic to activate Src family kinase. However, the effect of halogenation on tyrosine phosphorylation or SH2 domain binding is not well understood. We examined the phosphorylation of model peptides containing 3-halotyrosine or 3-nitrotyrosine using typical receptor tyrosine kinase, epidermal growth factor receptor (EGFR), and nonreceptor tyrosine kinase, lymphocyte-specific protein tyrosine kinase (Lck). The EGFR- and Lck-mediated phosphorylation was markedly inhibited by tyrosine halogenation. Iodination showed the strongest inhibition of the phosphorylation among four types of halogenation, and its inhibitory effect was stronger than that of nitration. We also examined the effect of iodination and nitration of tyrosine residues on binding to the SH2 domain of Lck, using a model peptide containing the phosphoTyr-Glu-Glu-Ile motif, which has a high affinity for the SH2 domain. The relative affinities of the modified peptides whose phosphotyrosine was substituted with unphosphorylated tyrosine, 3-nitrotyrosine, and 3-iodotyrosine, and of the model peptide were 0.024, 0.26, 1, and 16, respectively. These results suggest that tyrosine iodination may have an effect on the phosphorylation or binding to the SH2 domain similar to nitration. Tyrosine iodination possibly modulates signal transduction, with the potential impairment of cell function.

Capillary HPLC−ICPMS and Tyrosine Iodination for the Absolute Quantification of Peptides Using Generic Standards

The validity of using tyrosine iodination chemistry for the absolute and generic quantification of peptides by capillary high-performance liquid chromatography (capHPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is investigated in detail. In this approach, two iodine atoms are specifically bioconjugated to the meta positions of the aromatic ring of every tyrosine residue. Characterization studies by capHPLC with parallel ICPMS and electrospray ionization tandem mass spectrometry (ESIMS/MS) detection clearly showed that such labeling iodination reaction affords one to obtain most accurate peptide determinations (after translation of the picomoles of iodine, quantified by ICPMS in each chromatographic peak, into picomoles of the corresponding labeled peptide). It is demonstrated that only, but every, tyrosine residue present in the peptide is completely diiodinated. The excellent detection limits for iodine using ICPMS allowed robust and highly sensitive tyrosine-containing peptide quantification (480 pM, 480 amol absolute). Derivatization is easily accomplished in a water/acetonitrile solution in only 2 min. Moreover, since the signal in ICPMS is completely independent from the chemical species containing the detected element, any iodine-containing standard (e.g., iodobenzoic acid) could be used as internal standard for the absolute quantification of every iodine-labeled tyrosine-containing peptide separated and detected along the gradient. The approach was optimized for tyrosine labeling and then validated by application to the absolute quantification of the three standard peptides present in the only reference material for peptide quantity (NIST 8327) commercially available. Identification of the species quantified by ICPMS was carried out by parallel capHPLC-ESI quadrupole time-of-flight (Q/TOF) analysis and corresponded, as expected, to the diiodinated peptides. The collision-induced dissociation (CID) spectra obtained demonstrated unequivocally the specific and complete derivatization of the tyrosine residues. The obtained quantitative results closely matched the reference values reported by the National Institute of Standards and Technology (NIST). In terms of precision, the relative standard deviation was as low as 3% RSD. Finally the approach was tested for the absolute quantification of proteins using a model standard protein (beta-casein). Results agreed again with the value specified showing that this labeling reaction is compatible with tryptic digestion.

Combined Extracellular Matrix Cross-linking Activity of the Peroxidase MLT-7 and the Dual Oxidase BLI-3 Is Critical for Post-embryonic Viability in Caenorhabditis elegans

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

Contact area of bovine somatotropin dimer: involvement of tyrosine 142

The presence of tyrosine residues in the contact area between protomers of bovine somatotropin dimers (Fernandez & Delfino, Biochem. J. 209, 107-115, 1983) was investigated taking advantage of the impaired self-associating ability of molecules iodinated at such residues. Reaction of bovine somatotropin dissolved in 8 M urea with the NaI-Chloramine T couple (2.1 x 10(-4) M) rendered a preparation with 3.1 iodine atoms per molecule which, by stepwise elimination of the denaturant and gel filtration through Sephadex G-100, originated two distinguishable populations: one able (iododerivatives I), the other unable (iododerivatives II) to self-associate. After frontal analysis, iododerivatives II were found to be unable to interact even with native molecules. Identification of the reacting tyrosine residues indicated that iodination of tyrosine 142 was responsible for the loss of the ability to form dimers in iododerivatives II. Iodohormones retained the ability to bind to somatogenic mouse hepatocyte receptors--the relative potency for iododerivatives I and II being 0.60 (0.34-1.03) and 0.71 (0.41-1.22) respectively.

Deciphering the Peptide Iodination Code: Influence on Subsequent Gas-Phase Radical Generation with Photodissociation ESI-MS

Iodination of tyrosine was recently discovered as a useful method for generating radical peptides via photodissociation of carbon-iodine bonds by an ultraviolet photon in the gas phase. The subsequent fragmentation behavior of the resulting odd-electron peptides is largely controlled by the radical. Although previous experiments have focused on mono-iodination of tyrosine, peptides and proteins can also be multiply iodinated. Tyrosine and, to a lesser extent, histidine can both be iodinated or doubly iodinated. The behavior of doubly iodinated residues is explored under conditions where the sites of iodination are carefully controlled. It is found that radical peptides generated by the loss of a single iodine from doubly iodinated tyrosine behave effectively identically to singly iodinated peptides. This suggests that the remaining iodine does not interfere with radical directed dissociation pathways. In contrast, the concerted loss of two iodines from doubly iodinated peptides yields substantially different results that suggest that radical recombination can occur. However, sequential activation can be used to generate multiple usable radicals in different steps of an MS(n) experiment. Furthermore, it is demonstrated that in actual peptides, the rate of iodination for tyrosine versus mono-iodotyrosine cannot be predicted easily a priori. In other words, previous assumptions that mono-iodination of tyrosine is the rate-limiting step to the formation of doubly iodinated tyrosine are incorrect.

Mechanism of enzymatic and non-enzymatic tyrosine iodination. Inhibition by excess hydrogen peroxide and/or iodide.

Non-enzymatic (I2-mediated) and lactoperoxidase-catalyzed iodination of tyrosine are inhibited by excess iodide (I-) and/or hydrogen peroxide (H2O2). This phenomenon is a consequence of the concentration-dependent dual role of I- and H2O2 in the iodinating system. I- and H2O2, in addition to their function as primary substrates of peroxidase, may act as alternative 'iodine acceptors' and therefore compete with tyrosine for the active iodinating agent, irrespective of whether this compound is an enzyme-associated iodinium cation (E X I delta +) or an equivalent oxidized iodine species (IOH, IC1, I2). The competitive reaction pathways resulting from excess I- and/or H2O2 in the iodination system are I2/I-3 generation and/or pseudo-catalatic degradation of H2O2, respectively. Our results also demonstrate that I2 (and alternative medium-dependent oxidized iodine species such as IOH and IC1) generated in the iodination system may play an important role as iodinating agent(s). They serve as a substitute for the enzyme-bound iodinium species (E X I delta +), if the prevailing I- concentration favours this pathway. The proposed mechanism of the various antagonistic and interactive reaction pathways is summarized in a scheme.

The insulin-like action of beta-cell-tropin on glucose and lipid metabolism in adipocytes.

The insulin-like activity of the pituitary pars-intermedia insulin secretagogue beta-cell-tropin, ACTH22-39, has been studied on rat adipocytes. The peptide was prepared by tryptic digestion of synthetic human CLIP, ACTH18-39. beta-Cell-tropin stimulated the incorporation of 3H2O into total lipids. The 50% maximal activity concentration was 5 X 10(-2) ng/ml-1 about 2.5 X 10(-11) M. Iodination of tyrosine, the penultimate amino-acid of the N-terminal, eliminated lipogenic activity, and acetylation of the N-terminal valine reduced activity. ACTH and CLIP (ACTH18-39) had no lipogenic action on the adipocyte system studied. beta-Cell-tropin stimulated the oxidation of glucose and the conversion of glucose into saponified fatty acids and glyceride glycerol. The influence of beta-cell-tropin and insulin on the incorporation of glucose into total lipids, saponified fatty acids and glyceride glycerol was not additive. The results suggest that beta-cell-tropin is either a potent lipogenic hormone, stimulating the conversion of glucose into lipids or that it activates endogenous insulin. The biological activity is associated with the N-terminal amino-acids of the peptide. The possible significance of beta-cell-tropin in obesity is discussed.

Targeted tyrosine iodination in a multi‐tyrosine vasopressin analog

Iodination of the conserved 2-tyrosine (Tyr(2)) residue in the pressin and tocin rings of arginine- or lysine-vasopressin (AVP or LVP), and oxytocin, respectively, impairs binding to their respective receptors. Synthetic antagonists that have their Tyr(2) either replaced by another amino acid or irreversibly blocked by an O-methyl or O-ethyl ether, but have, instead, an iodinatable phenol moiety outside the pressin/tocin ring, are used for radiolabeling. We explored another approach to avoid iodinating Tyr(2) by capping this residue with a reversible O-acetyl group, incorporated during peptide synthesis. The O-acetyl-Tyr(2) LVP peptide, with a free iodinatable tyrosine attached to the epsilon-amine of 8-lysine, is iodinated at a neutral pH and purified by reverse-phase high-pressure liquid chromatography (HPLC) at an acidic pH, conditions under which the O-acetyl groups are stable. Deacetylation with hydroxylamine is selective, and leaves intact the disulfide bridge. The marked shortening of the HPLC retention time after deblocking produces a chemically homogeneous label, iodinated exclusively on the free tyrosine residue attached to the epsilon-amine of LVP. Hitherto, this (125)I labeled vasopressin agonist could be obtained only in low yield, via conjugation labeling with iodinated N-t-Boc-tyrosine succinimidyl ester. This fully reversible tyrosine protection strategy does not require special equipment, and retains the conserved Tyr(2), typical of vasopressin and oxytocin agonists.

Interaction of anti-thyroid drugs with iodine: the isolation of two unusual ionic compounds derived from Se-methimazole

The inhibition of lactoperoxidase (LPO)-catalyzed iodination of l-tyrosine by the anti-thyroid drug methimazole (MMI) and its selenium analogue (MSeI) is described. MSeI inhibits LPO with an IC(50) value of 12.4 microM, and this inhibition could be completely reversed by increasing the peroxide concentration. In addition to the inhibition, MSeI reacts with molecular iodine to produce novel ionic diselenides, and the nature of the species formed in this reaction appear to be solvent-dependent. The formation of ionic species in the reaction is confirmed by single-crystal X-ray studies, FT-IR and FT-Raman spectroscopic investigations. This study provides the first experimental evidence that MSeI not only effectively inhibits the LPO-catalyzed iodination of tyrosine, but also reacts with I(2) to produce novel ionic diselenides. These results also suggest that MSeI reacts with iodine, even in its oxidized form, to form ionic diselenides containing iodide or polyiodide anions, which might be effective intermediates in the inhibition of thyroid hormones.

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.

Coupling tandem affinity purification and quantitative tyrosine iodination to determine subunit stoichiometry of protein complexes

Rapid protein purification methodologies, such as strategies involving the tandem affinity purification module, have resulted in the identification of a tremendous number of multisubunit protein complexes. Furthermore, in this modern genomic age, mass spectrometry methods are often coupled with affinity purification to identify the genes that encode each protein subunit. However, simple methodologies to determine the stoichiometry of individual subunits within a multisubunit complex have not received much attention. In this article we describe a procedure to rapidly and efficiently determine the stoichiometry of subunits within multisubunit complexes using a combination of tandem affinity purification and quantitative 125 I labeling of subunit tyrosines.

Elevated temperature and tyrosine iodination aid in the crystallization and structure determination of an antifreeze protein.

Production and refolding of recombinant Choristoneura fumiferana antifreeze protein (CfAFP) leads to a disulfide-bonded product containing dynamic conformational microheterogeneity. Difficulties in the crystallization of this protein arising from its microheterogeneity were overcome by screening of crystallization conditions at various temperatures and finally using a temperature of 318 K to obtain diffraction-quality crystals. In addition, heavy-atom derivatization of this protein required the iodination of a specific tyrosine residue, leading to the successful single anomalous scattering (SAS) structure determination. The techniques of higher temperature screening, to reduce dynamic conformational microheterogeneity, and defined tyrosine iodination, for specific heavy-atom incorporation, are methods which can be employed with other proteins to aid in structure determination.

Purification and partial characterization of haloperoxidase from fresh water algae Cladophora glomerata

Many haloperoxidases have been purified from diverse organisms, including lichen, fungi, bacteria, and marine algae. In this study a haloperoxidase was purified from the fresh water algae, Cladophora glomerata, by homogenization and centrifugation, ammonium sulfate fractionation, ion-exchange and gel filtration chromatography. Molecular weight was determined by SDS-PAGE and by size exclusion HPLC and found to be approximately 43 kDa. The isoelectric point was determined to be approximately 8.1 by isoelectric focusing. The UV spectrum of the peroxidase showed a strong absorbance in the Soret band indicating a heme protein, unlike vanadium-dependent haloperoxidases from marine algae. Fresh water algal haloperoxidase catalyzed the iodination of tyrosine at a pH of 3.1. This haloperoxidase also catalyzes the oxidation of guaiacol and oxidation of iodide as well as catalyzing a peroxide-dependent reaction in both the presence and absence of chloride and bromide ions.

Presence of a soluble inhibitor of thyroid iodination in primary cultures of thyroid cells.

Monolayer cultures of thyroid cells lose their iodide organification capacity a few days before the disappearance of thyroid peroxidase (TPO) activity. The present studies were performed in order to clarify this point. The above mentioned difference was due to the presence of an inhibitor in the monolayer thyroid cells culture, given that total homogenate prepared from confluent cells caused a significant inhibition of activity of TPO from fresh tissue. The inhibitor was localized in the 105000g supernatant of the homogenate of the cell culture, but not in a similar preparation obtained from fresh thyroid. It is thermostable, dialyzable and has a molecular weight of less than 2 kDa. Addition of the inhibitor at the end of the reaction of tyrosine iodination failed to alter the results. This fact suggests that the compound does not destroy the iodinated product. The presence of the cytosolic inhibitor was observed in monolayer thyroid cell cultures of different species (bovine, porcine, rat and human) but not in free follicles cultures.