S-homocysteinylation of transthyretin is detected in plasma and serum of humans with different types of hyperhomocysteinemia

While the association of homocystinuria with disease is known for more than four decades, mild hyperhomocysteinemia has been detected more recently as a risk factor for a number of diseases. However, the mechanism which apparently renders (even mild) hyperhomocystenemia harmful is not known. Following reports on N-homocysteinylation of proteins by the homocysteine derivative homocysteine thiolactone, it has been suggested that homocysteinylation of proteins may contribute to the induction of biological effects by homocysteine. This has prompted us to study by electrospray ionization mass spectrometry homocysteinylation of transthyretin (TTR) in plasma and serum of humans with different types of hyperhomocysteinemia. We did not detect any N-homocysteinylation, but found pronounced S-homocysteinylation of TTR, if the concentration of total homocysteine was high. Our findings support a possible role of S-homocysteinylation of proteins in the mediation of detrimental effects of hyperhomocysteinemia. Careful study of posttranslational modifications of individual proteins may contribute to a better understanding of diseases associated with hyperhomocysteinemia.     

Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the ?-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.     

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.     

Mechanisms of homocysteine toxicity in humans

Summary. Homocysteine, a non-protein amino acid, is an important risk factor for ischemic heart disease and stroke in humans. This review provides an overview of homocysteine influence on endothelium function as well as on protein metabolism with a special respect to posttranslational modification of protein with homocysteine thiolactone. Homocysteine is a pro-thrombotic factor, vasodilation impairing agent, pro-inflammatory factor and endoplasmatic reticulum-stress inducer. Incorporation of Hcy into protein via disulfide or amide linkages (S-homocysteinylation or N-homocysteinylation) affects protein structure and function. Protein N-homocysteinylation causes cellular toxicity and elicits autoimmune response, which may contribute to atherogenesis. Present address: Department of Biochemistry and Biotechnology, Agricultural University, 60637 Poznań, Poland     

Effect of N-homocysteinylation on physicochemical and cytotoxic properties of amyloid β-peptide

Abstract Hyperhomocysteinemia has recently been identified as an important risk factor for Alzheimer's disease (AD). One of the potential mechanisms underlying harmful effects of homocysteine (Hcy) is site-specific acylation of proteins at lysine residues by homocysteine thiolactone (HCTL). The accumulation of amyloid β-peptide (Aβ) in the brain is a neuropathological hallmark of AD. In the present study we were interested to investigate the effects of N-homocysteinylation on the aggregation propensity and neurotoxicity of Aβ(1-42). By coupling several techniques, we demonstrated that the homocysteinylation of lysine residues increase the neurotoxicity of the Aβ peptide by stabilizing soluble oligomeric intermediates.     

The disturbance of hemostasis induced by hyperhomocysteinemia; the role of antioxidants

Elevated concentration of homocysteine (Hcy) in human tissues, definied as hyperhomocysteinemia has been correlated with some diseases, such as cardiovascular, neurodegenerative, and kidney disorders. Homocysteine occurs in human blood plasma in several forms, including the most reactive one, the homocysteine thiolactone (HTL) - a cyclic thioester, which represents up to 0.29% of total plasma Hcy. In the article, the effects of hyperhomocysteinemia on the complex process of hemostasis, which regulates the flowing properties of blood, are described. Possible interactions of homocysteine and its different derivatives, including homocysteine thiolactone, with the major components of hemostasis such as endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen, are also discussed. Modifications of hemostatic proteins (N-homocysteinylation or S-homocysteinylation) induced by Hcy or its thiolactone seem to be the main cause of homocysteine biotoxicity in hemostatic abnormalities. It is suggested that Hcy and HTL may also act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. We also discuss the role of phenolic antioxidants in hyperhomocysteinemia -induced changes in hemostasis.     

Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation

Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys(525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys(525), is affected by the status of Cys(34). The disulfide forms of circulating albumin, albumin-Cys(34)-S-S-Cys and albumin-Cys(34)-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys(34)-SH. Although N-homocysteinylations of albumin-Cys(34)-SH and albumin-Cys(34)-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys(34) of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys(34)-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.     

Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.     

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.     

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.     

N-Homocysteinylation Induces Different Structural and Functional Consequences on Acidic and Basic Proteins

One of the proposed mechanisms of homocysteine toxicity in human is the modification of proteins by the metabolite of Hcy, homocysteine thilolactone (HTL). Incubation of proteins with HTL has earlier been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation). It has been believed that protein N-homocysteinylation is the pathological hallmark of cardiovascular and neurodegenerative disorders as homocysteinylation induces structural and functional alterations in proteins. In the present study, reactivity of HTL towards proteins with different physico-chemical properties and hence their structural and functional alterations were studied using different spectroscopic approaches. We found that N-homocysteinylation has opposite consequences on acidic and basic proteins suggesting that pI of the protein determines the extent of homocysteinylation, and the structural and functional consequences due to homocysteinylation. Mechanistically, pI of protein determines the extent of N-homocysteinylation and the associated structural and functional alterations. The study suggests the role of HTL primarily targeting acidic proteins in eliciting its toxicity that could yield mechanistic insights for the associated neurodegeneration.     

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.     

Modification by homocysteine thiolactone affects redox status of cytochrome C

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.     

N-homocysteinylation of α-synuclein promotes its aggregation and neurotoxicity

The aggregation of α-synuclein plays a pivotal role in the pathogenesis of Parkinson's disease (PD). Epidemiological evidence indicates that high level of homocysteine (Hcy) is associated with an increased risk of PD. However, the molecular mechanisms remain elusive. Here, we report that homocysteine thiolactone (HTL), a reactive thioester of Hcy, covalently modifies α-synuclein on the K80 residue. The levels of α-synuclein K80Hcy in the brain are increased in an age-dependent manner in the TgA53T mice, correlating with elevated levels of Hcy and HTL in the brain during aging. The N-homocysteinylation of α-synuclein stimulates its aggregation and forms fibrils with enhanced seeding activity and neurotoxicity. Intrastriatal injection of homocysteinylated α-synuclein fibrils induces more severe α-synuclein pathology and motor deficits when compared with unmodified α-synuclein fibrils. Increasing the levels of Hcy aggravates α-synuclein neuropathology in a mouse model of PD. In contrast, blocking the N-homocysteinylation of α-synuclein ameliorates α-synuclein pathology and degeneration of dopaminergic neurons. These findings suggest that the covalent modification of α-synuclein by HTL promotes its aggregation. Targeting the N-homocysteinylation of α-synuclein could be a novel therapeutic strategy against PD.     

Characterization of N-homocysteinylated Albumin Adducts

Increased plasma homocysteine levels are considered an important risk factor for vascular disease. Homocysteine, an intermediate compound in methionine metabolism, is an amino acid that includes a thiol group, and circulates as different species. One of them, Homocysteine thiolactone (HTL) forms adducts through irreversible reactions with epsilon-NH₂ groups of lysine residues. These processes can alter the structure and biological function of diverse proteins that may be involved in the detrimental effects of homocysteine. Particularly, in this work we evaluated HTL-mediated molecular changes in human serum albumin (HSA) through electrophoretic techniques. Albumin and HTL were incubated (37 °C, 6 h) at HSA:HTL molar ratios of 1:25, 1:50 and 1:100. Polyacrylamid gel electrophoresis showed that electrophoretic mobility was increased in the treated HSA respect to control, in an HTL-concentration-dependent manner. That anodic shift of the treated samples was also observed in crossed immunoelectrophoresis profiles. As expected, a decrease in the isoelectric point of the homocysteinylated albumin (pI 4.7) in comparison to that of control (pI 4.8) was shown by the isoelectric focusing technique. Moreover, the electropherogram acquired by capillary zone electrophoresis indicated that migration times and full width at half height were enhanced with the rise of HTL concentration. We propose that the in vitro structural changes of albumin described in the present work would be involved in the harmful effects of the N-homocysteinylation process.     

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.     

Relative roles of albumin and ceruloplasmin in the formation of homocystine, homocysteine-cysteine-mixed disulfide, and cystine in circulation

Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Büdy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.     

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine–cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein–protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.     

Novel proteins for homocysteine biosynthesis in anaerobic microorganisms

The metabolic network for sulfide assimilation and trafficking in methanogens is largely unknown. To discover novel proteins required for these processes, we used bioinformatics to identify genes co‐occurring with the protein biosynthesis enzyme SepCysS, which converts phosphoseryl‐tRNA^Cys to cysteinyl‐tRNA^Cys in nearly all methanogens. Exhaustive analysis revealed three conserved protein families, each containing molecular signatures predicting function in sulfur metabolism. One of these families, classified within clusters of orthologous groups (COG) 1900, possesses two conserved cysteine residues and is often found in genomic contexts together with known sulfur metabolic genes. A second protein family is predicted to bind two 4Fe‐4S clusters. All three genes were also identified in more than 50 strictly anaerobic bacterial genera from nine distinct phyla. Gene‐deletion and growth experiments in Methanosarcina acetivorans, using sulfide as the sole sulfur source, demonstrate that two of the proteins (MA1821 and MA1822) are essential to homocysteine biosynthesis in a background lacking an additional gene for sulfur insertion into homocysteine. Mutational analysis confirms the importance of several structural elements, including a conserved cysteine residue and the predicted 4Fe‐4S cluster‐binding domain.     

Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-¹³C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys²⁸² and Cys²⁶⁵ are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys²⁶⁵ and Cys²⁸² varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys²⁶⁵ appears to be in a relatively hydrophilic, oxidizable environment compared with Cys¹²⁷ and Cys²⁸². Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.