Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

Existence of Molten Globule State in Homocysteine-Induced Protein Covalent Modifications

Homocysteine thiolactone is a toxic metabolite produced from homocysteine by amino-acyl t-RNA synthetase in error editing reaction. The basic cause of toxicity of homocysteine thiolactone is believed to be due to the adduct formation with lysine residues (known as protein N-homocysteinylation) leading to protein aggregation and loss of enzyme function. There was no data available until now that showed the effect of homocysteine thiolactone on the native state structural changes that led to aggregate formation. In the present study we have investigated the time dependent structural changes due to homocysteine thiolactone induced modifications on three different proteins having different physico-chemical properties (cytochrome-c, lysozyme and alpha lactalbumin). We discovered that N-homocysteinylation leads to the formation of molten globule state—an important protein folding intermediate in the protein folding pathway. We also found that the formation of the molten globule state might be responsible for the appearance of aggregate formation. The study indicates the importance of protein folding intermediate state in eliciting the homocysteine thiolactone toxicity.     

Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in the yeast Saccharomyces cerevisiae.

Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coli methionyl-tRNA synthetase, which prevents incorporation of homocysteine into tRNA and protein, both in vitro and in vivo. Here, the thiolactone is also shown to occur in cultures of the yeast Saccharomyces cerevisiae. In yeast, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 500 molecules of methionine incorporated into protein. Homocysteine, added exogenously to the medium or overproduced by some yeast mutants, is detrimental to cell growth. The cost of homocysteine editing in yeast is minimized by the presence of a pathway leading from homocysteine to cysteine, which keeps intracellular homocysteine at low levels. These results not only directly demonstrate that editing of errors in amino acid selection by methionyl-tRNA synthetase operates in vivo in yeast but also establish the importance of proofreading mechanisms in a eukaryotic organism.     

Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection

A sensitive and simple method utilising fluorometric detection for the simultaneous routine monitoring of homocysteine thiolactone (HTL) and homocysteine (Hcy) in biological samples has been developed. Separation relies on isocratic ion-pairing and reversed-phase chromatography while the principle of the detection is that the lactone ring in HTL molecule is cleaved with an alkali to produce Hcy, which reacts with ortho-phthalaldehyde (OPA) in the absence of an added thiol reagent to form a stable fluorescent derivative. The method has a sensitivity of 200 fmol of HTL and 100 fmol for Hcy in the sample. The present method was applied to the determination of HTL and Hcy in Hep G2 cell.     

Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels.

Homocysteine thiolactone, a cyclic thioester, is synthesized by certain aminoacyl-tRNA synthetases in editing or proofreading reactions that prevent translational incorporation of homocysteine into proteins. Although homocysteine thiolactone is expected to acylate amino groups in proteins, virtually nothing is known regarding reactivity of the thiolactone. Here it is shown that reactions of the thiolactone with protein lysine residues were robust under physiological conditions. In human serum incubated with homocysteine thiolactone, protein homocysteinylation was a major reaction that could be observed with as little as 10 nM thiolactone. Individual proteins were homocysteinylated at rates proportional to their lysine contents. Homocysteinylation led to protein damage, manifested as multimerization and precipitation of extensively modified proteins. Model enzymes, such as methionyl-tRNA synthetase and trypsin, were inactivated by homocysteinylation. Metabolic conversion of homocysteine to the thiolactone, protein homocysteinylation, and resulting protein damage may underlie involvement of Hcy in the pathology of vascular disease.-Jakubowski, H. Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels.     

Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N-homocysteinylation

Homocysteine thiolactone is formed in all cell types studied thus far as a result of editing reactions of some aminoacyl-tRNA synthetases. Because inadvertent reactions of thiolactone with proteins are potentially harmful, the ability to detoxify homocysteine thiolactone is essential for biological integrity. This work shows that a single specific enzyme, present in mammalian but not in avian sera, hydrolyzes thiolactone to homocysteine. Human serum thiolactonase, a 45-kDa protein component of high density lipoprotein, requires calcium for activity and stability and is inhibited by isoleucine and penicillamine. Substrate specificity studies suggest that homocysteine thiolactone is a likely natural substrate of this enzyme. However, thiolactonase also hydrolyzes non-natural substrates, such as phenyl acetate, p-nitrophenyl acetate, and the organophospate paraoxon. N-terminal amino acid sequence of pure thiolactonase is identical with that of human paraoxonase. These and other data indicate that paraoxonase, an organophosphate-detoxifying enzyme whose natural substrate and function remained unknown up to now, is in fact homocysteine thiolactonase. By detoxifying homocysteine thiolactone, the thiolactonase/paraoxonase would protect proteins against homocysteinylation, a potential contributing factor to atherosclerosis.     

An in vitro evaluation of the effects of homocysteine thiolactone on key steps of angiogenesis and tumor invasion

Homocysteine thiolactone is a highly reactive homocysteine derivative that can react easily with proteins. Protein homocysteinylation has been suggested as a possible mechanism underlying the pathological consequences of impaired homocysteine metabolism. Homocysteine inhibits key steps of angiogenesis and tumor invasion. It can be hypothesized that homocysteine thiolactone could mimic the described anti-angiogenic and anti-invasive effects of homocysteine. Therefore, we studied the effects of homocysteine thiolactone on different key steps of angiogenesis and tumor invasion, using model endothelial and tumor cell lines. This study demonstrates that homocysteine thiolactone, in high contrast to homocysteine, is not an anti-angiogenic compound. Furthermore, our results suggest that homocysteine thiolactone could behave as a pro-angiogenic compound.     

Proofreading in trans by an aminoacyl-tRNA synthetase: a model for single site editing by isoleucyl-tRNA synthetase.

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code. Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine). That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme. Although IleRS.Val-AMP undergoes thiolysis as efficiently as do IleRS.Ile-AMP and IleRS.Ile-tRNAIle, IleRS.Val-tRNAIle does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS.Val-tRNAIle is, most likely, positioned off the enzyme.     

Selection and characterization of a murine lymphoid cell line partially deficient in S-adenosylhomocysteine hydrolase

The exact role of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) in mediating the toxic effects of adenosine toward mammalian cells has not been ascertained. The selection and characterization of S-adenosylhomocysteine hydrolase-deficient cell lines offers a biochemical genetic approach to this problem. In the present experiments, a mutant clone (Sahn 12) with 11-13% of wild-type S-adenosylhomocysteine hydrolase activity was selected from the murine T lymphoma cell line R 1.1 after mutagenesis and culture in adenosine, deoxycoformycin, uridine and homocysteine thiolactone-supplemented medium. In the presence of 0.5 mM homocysteine thiolactone and 10-200 microM adenosine, wild-type and mutant cells synthesized S-adenosylhomocysteine intracellularly at markedly different rates, and excreted the compound extracellularly. Thus, at time points up to 10 h, the S-adenosylhomocysteine hydrolase-deficient lymphoblasts required 5-10-fold higher concentrations of adenosine in the medium to achieve the same intracellular S-adenosylhomocysteine levels as wild-type cells. Similarly, the Sahn 12 lymphoblasts were 5-10-fold more resistant than R 1.1 cells to the toxic effects of adenosine plus homocysteine thiolactone. These results establish that (i) 11-13% of wild-type S-adenosylhomocysteine hydrolase activity is compatible with normal growth, (ii) in medium supplemented with both adenosine and homocysteine thiolactone, intracellular S-adenosylhomocysteine is synthesized by S-adenosylhomocysteine hydrolase, (iii) the net intracellular level of S-adenosylhomocysteine is determined by both the rate of S-adenosylhomocysteine synthesis and its rate of excretion, (iv) under such conditions the accumulation of S-adenosylhomocysteine is related to cytotoxicity, (v) in the absence of an exogenous homocysteine source, S-adenosylhomocysteine derives from endogenous sources, and the accumulation of S-adenosylhomocysteine is not the primary cause of adenosine induced cytotoxicity.     

Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes

We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experimental conditions (8 SH/LDL), the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.     

Antithrombin activity is inhibited by acrolein and homocysteine thiolactone: Protection by cysteine

Conditions in which serum or tissue acrolein levels are high (e.g.: renal failure, heavy smoking, oxidative stress) are also associated with increased thrombogenicity. Another emerging cardiovascular risk factor is homocysteine, and its derivative, homocysteine thiolactone. Antithrombin is one of the most important inhibitors of blood coagulation Since its activation by heparin binding requires critical interactions involving 3 Lys residues; we hypothesized that acrolein or homocysteine thiolactone impair antithrombin activity. When we incubated human antithrombin with increasing concentrations of acrolein (0-2 mmol/L) over a short period of time (0-4 h), a time and concentration dependent loss of activity was apparent (IC(50)=0.25 mmol/L). At 2 mmol/L, maximum inhibition (60%) is achieved at 1 h. This loss of activity was mirrored by changes in the electrophoretic pattern (homogeneity of the native antithrombin band as well as polymerization). In the same conditions, homocysteine thiolactone produces a significant, yet far less pronounced effect; acrolein being 3 times more potent than homocysteine thiolactone. When antithrombin was co-incubated with acrolein and cysteine, only less than 10% of antithrombin activity was lost. Aminoguanidine or carnosine displayed a significant yet, minor protective effect. The results suggest that in conditions where circulating or local acrolein concentrations are increased (atheroma plaque, thrombosis, sites of lipoperoxidation, smokers), acrolein-mediated loss of antithrombin activity could be a plausible phenomenon. This could contribute to explain increased thrombogenicity in smokers and in other conditions, as well as pointing at dietary intervention or the use of thiol-conserving reducing compounds as putative coadjuvant therapeutic measures.     

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Molecular mechanisms of biotoxicity of homocysteine--facts and hypotheses

In the article, the main pathways of homocysteine metabolism are described, i.e. transsulfuration to cysteine and glutathione, as well as remethylation to methionine. Furthermore, formation of homocysteine thiolactone through editing mechanism with methionyl t-RNA syntethase and unusual reactivity of thiolactone against lysine epsilonNH2 groups of proteins as well as calcium dependent enzymatic hydrolysis of thiolactone are discussed. The effects of oxidative stress related to homocysteine are also reviewed. Finally, possible links of homocysteine to NO and arginine metabolism are discussed, including ADMA (N(G),N(G)-dimethylarginine). The links between metabolism of homocysteine, adenosine and other nucleosides are emphasized. In conclusion, the N-homocysteilation of proteins with thiolactone changing enormously their properties seems to be the main reason of biotoxicity of homocysteine during atherosclerosis and other diseases.     

Homocysteine and other structurally-diverse amino thiols can alter pancreatic beta cell function without evoking cellular damage

Homocysteine and related amino thiols, homocysteic acid, cysteic acid, homocysteine sulphinic acid and cysteine sulphinic acid have been labelled as neurotoxins. Homocysteine thiolactone, a metabolic derivative of homocysteine, is cytotoxic to endothelial cells and other cell lineages. Since pancreatic beta cells share many phenotypic similarities with neuronal cells, the present study uses clonal pancreatic BRIN-BD11 cells to investigate possible detrimental effects of these amino thiols on insulin secretion and pancreatic beta cell function. Insulin secretion was concentration-dependently inhibited at both basal (1.1 mM) and stimulatory (16.7 mM) glucose by homocysteine, homocysteine thiolactone and homocysteine sulphinic acid. Cysteic acid concentration-dependently inhibited insulin secretion at 16.7 mM glucose. Cell viability was not compromised by any of the amino thiols. Insulin secretory responses to alanine were inhibited by homocysteine, homocysteine thiolactone, homocysteic acid and cysteic acid. Insulin secretion in the presence of elevated Ca(2+) and forskolin were lowered by all amino thiols, except homocysteic acid. The secretory responsiveness to PMA, GLP-1 and KCl were only impaired in the presence of homocysteine and homocysteine thiolactone. These findings indicate that homocysteine, homocysteine thiolactone and, to a lesser extent, other amino thiols cause dysfunctional insulin secretion from pancreatic beta cells.     

Altered tubulin assembly dynamics with N-homocysteinylated human 4R/1N tau in vitro

Tau isoforms promote neuronal integrity through binding and stabilization of microtubule proteins (MTP). It has been shown that hyperphosphorylation of tau contributes to Alzheimer’s disease (AD) pathology and related tauopathies. However, other pathogenic modifications of tau have not been well characterized. It is well accepted that elevated level of homocysteine (Hcy) is associated with neurodegenerative diseases such as AD. As a result of N-homocysteinylation of lysine residues, Hcy becomes a component of proteins, as a protein–homocystamide adduct, which affects protein structure and function. Here we demonstrate that N-homocysteinylation of human tau (4R/1N isoform) inhibits its function via impaired tau–tubulin specific binding and MTP assembly dynamics in vitro.     

Effect of homocysteinylation of low density lipoproteins on lipid peroxidation of human endothelial cells

Homocysteine-thiolactone (HcyT) is a toxic product whose synthesis is directly proportional to plasma homocysteine (Hcy) levels. Previous studies demonstrated that the interaction between HcyT and low density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL). Structural and functional alterations of Hcy-LDL have been described and it has been suggested that homocysteinylation could increase atherogenicity of LDL. Oxidative damage of endothelial cells (EC) is considered to be a critical aspect of the atherosclerotic process. To further investigate the molecular mechanisms involved in the atherogenicity of homocysteinylated LDL, we studied the effect of interaction between Hcy-LDL and EC on cell oxidative damage, using human aortic endothelial cells (HAEC) as experimental model. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy normolipemic subjects, with HcyT (10-100 microM). In our experimental conditions, homocysteinylation treatment was not accompanied by oxidative damage of LDL. No modifications of apoprotein structure and physico-chemical properties were observed in Hcy-LDL with respect to control LDL (c-LDL), as evaluated using the intrinsic fluorescence of tryptophan and the probe Laurdan incorporated in lipoproteins. Our results demonstrated that Hcy-LDL incubated at 37 degrees C for 3 h with HAEC, induced an oxidative damage on human EC with a significant increase of lipid hydroperoxides in cells incubated with Hcy-LDL with respect to cell incubated with c-LDL. The compositional changes were associated with a significant decrease viability in cells treated with Hcy-LDL. The relationship between the levels of -SH groups of LDL and the oxidative damage of HAEC has been demonstrated. These results suggest that Hcy-LDL exert a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of EC.     

Major metabolic homocysteine-derivative, homocysteine thiolactone, exerts changes in pancreatic beta-cell glucose-sensing, cellular signal transduction and integrity

Homocysteine can be converted to its reactive thioester, homocysteine thiolactone. Cytotoxic properties of these amino thiols have been attributed to protein homocysteinylation, increased oxidative stress, DNA damage and apoptosis. This study used pancreatic BRIN-BD11 beta-cells to examine functional defects caused by acute and long-term exposure to homocysteine thiolactone in comparison with homocysteine. Acute and long-term exposure to both agents caused concentration-dependent inhibitions of glucose-induced insulin secretion while impairing the insulin-secretory responses to alanine, KCl, elevated Ca(2+), forskolin and PMA. Acute exposures also caused significant reduction in the amplitude of KCl-induced membrane depolarisation but no effects on changes of intracellular Ca(2+) induced by alanine or KCl. Cellular insulin content and DNA damage were not altered following culture, however, there were early signs of apoptosis consistent with impaired cellular integrity. In conclusion, exposure to homocysteine thiolactone, like homocysteine, induced beta-cell dysfunction and demise by mechanisms independent of changes in membrane potential and [Ca(2+)](i).     

Predicting protein homocysteinylation targets based on dihedral strain energy and pKa of cysteines

A multitude of complex diseases have been linked to elevated homocysteine levels; however, till date there is no plausible explanation for a single amino acid's involvement in so many diseases. Since homocysteine is a reactive thiol amino acid and the majority of plasma homocysteine is protein thiol bound, we hypothesized that homocysteine might bind to accessible cysteine residues in target proteins, thereby modulating its structure or function or both. The parameters that dictate homocysteine-protein interaction are not well understood, and the few known homocysteine binding proteins were identified by a candidate protein approach. In this study, we identified potential homocysteine interacting proteins based on cysteine content, solvent accessibility of cysteine residues, and dihedral strain energies and pKa of these cysteines. Pathway mapping of the cysteine-rich proteins revealed that proteins in the coagulation cascade, notch receptor-mediated signaling, LDL endocytosis, programmed cell death, and extracellular matrix proteins were significantly over-represented with cysteine-rich proteins, and we believe that homocysteine has a high probability to bind to proteins in these pathways. In fact, several clinical studies have implicated high homocysteine levels to be associated with diseases like thrombosis, neural tube defects, and so forth, which result from dysfunction of one or more of the proteins identified in our study. Further, we successfully validated our prediction parameters on the proteins that have already been experimentally shown to bind homocysteine, and our structural analysis argues a plausible explanation for these prior reported protein interactions with homocysteine that could not be previously explained.     

Homocysteine metabolism and its role in pathology

Results of the study of homocysteine metabolism and its role in physiological and pathological processes are given in this review. The participation of homocysteine in the process of methionine synthesis, transsulfuration, formation of homocysteine thiolacton and their regulation, polymorphism of homocysteine metabolism enzymes, the ways of homocysteine and thiolactone incorporation into protein molecule, sources and forms of homocysteine in the blood plasma, the role of hyperhomocysteinemia in pathogenesis of cardiovascular and other diseases have been considered. Principles of homocysteine determination in the blood plasma are described here.     

N-methyl-D-aspartate receptor agonists modulate homocysteine-induced developmental abnormalities.

We showed previously that the induction of neural crest (NC) and neural tube (NT) defects is a general property of N-methyl-D-aspartate receptor (NMDAR) antagonists. Since homocysteine induces NC and NT defects and can also act as an NMDAR antagonist, we hypothesized that the mechanism of homocysteine-induced developmental defects is mediated by competitive inhibition of the NMDAR by homocysteine. If this hypothesis is correct, homocysteine-induced defects will be reduced by NMDAR agonists. To test the hypothesis, we treated chicken embryos during the process of neural tube closure with sufficient homocysteine thiolactone to induce NC and NT defects in approximately 40% of survivors or with homocysteine thiolactone in combination with each of a selected set of NMDAR agonists in 0. 05-5000 nmol doses. Glutamate site agonists selected were L-glutamate and N-methyl-D-aspartate. Glycine site agonists were glycine, D-cycloserine, and aminocyclopropane-carboxylic acid. Glycine was the most effective overall, reducing defects significantly at two different doses (each P>0.001). These results support the hypothesis that homocysteine may affect NC and NT development by its ability to inhibit the NMDAR. One potentially important consequence of this putative mechanism is that homocysteine may interact synergistically with other NMDAR antagonists to enhance its effect on development.