Homocysteine Thiolactone Research Articles

Are heme proteins major targets for homocysteine‐induced modifications?

The prime cause of homocysteine (Hcy) toxicity towards proteins has been attributed to the modification of protein by homocysteine thiolactone (HTL). But the effects of Hcy (the parent molecule) on protein structure and function are still scarce. Hcy also has the ability to modify proteins, which could in turn affect the functionality and proper folding of the modified protein. Hcy is a highly pro‐oxidizing agent, thus Hcy‐induced oxidative stress has been commonly used to explain the toxicity of Hcy on endothelial cells and other tissues. Moreover, Hcy is known to modify cysteine residues in proteins in a process termed as S‐homocysteinylation. However, the structural and functional consequences of such modification are not yet clearly understood. The present study aims at investigating possible effects of Hcy on proteins structure and function. Using RNase‐A, Lyz and horseradish peroxidise (HRP) as models, we studied the effects of Hcy on the enzyme function and conformational status of these proteins. Enzyme activity measurements after overnight incubation with Hcy led to gradual decrease in enzyme function of HRP in a concentration dependent manner but not in Lyz and RNase‐A. Conformational analyses revealed no gross structural alterations in all proteins studied. However, certain alteration in the heme‐Trp distance was observed in case of HRP as evident from Trp fluorescence measurements. The loss of function in HRP was due to disruption of heme redox state in the presence of Hcy. Similar findings were obtained for three other common heme metalloprotein (Cytochrome c, Cyt c; Hemoglobin, Hb; and Myoglobin, Myo), wherein Hcy induced certain alterations in the heme environment rendering it reduced with further disruption of the heme‐Trp distance. In addition, Hcy was found to alter the heme redox states without direct interaction with the proteins (in case of Cyt c and Myo). The findings suggest that heme and heme containing proteins could be major targets for Hcy‐induced modifications and involved in Hcy‐induced cytotoxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N-Acetylcysteine supplementation decreased brain lipid and protein oxidations produced by experimental homocysteine thiolactone exposure: Relevance to neurodegeneration

High levels of homocysteine (Hcy) have neurotoxic effects. Homocysteine thiolactone (HcyT), a thioester of Hcy, plays a role in Hcy-induced toxicity. In this study, effects of HcyT treatment (500 mg/kg body weight/day in drinking water) for 6 weeks on serum Hcy levels and brain prooxidant-antioxidant balance were investigated in rats. The effect of N-acetylcysteine (NAC) treatment (1 g/kg body weight/day in chow) for 6 weeks on HcyT-induced neurotoxicity was evaluated. Reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), diene conjugate (DC), protein carbonyl (PC) and advanced oxidation protein products (AOPP) were determined in the brain tissue. Ferric reducing antioxidant power (FRAP) and non-protein sulfydryl groups (NPSH) levels as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were also measured to evaluate the antioxidant potential of brain the tissue. HcyT elevated serum Hcy levels and brain ROS, TBARS, DC, PC and AOPP levels. However, HcyT did not affect FRAP levels and SOD, and GSH-Px activities. NAC treatment decreased serum Hcy and brain ROS, TBARS, DC, PC and AOPP levels in HCyT-treated rats. Our results indicate that NAC supplementation may be effective in decreasing serum Hcy levels and HcyT-induced oxidative stress in brain of rats.

Paraoxonases: metabolic role and pharmacological projection

Atherosclerosis is one of the leading causes of death in Western countries, with high-density lipoproteins (HDL) playing an important protective role due to their ability to inhibit oxidation of low-density lipoproteins (LDL), thus relieving vascular subendothelial damage. One of the proteins constituting HDL particles is paraoxonase-1 (PON1), an enzyme able to hydrolyze aryl esters, lactones, and organophosphates. Other closely related paraoxonases are designated as PON2, which is a protein localized inside many different kinds of cells, and PON3, not only present in HDL but also in mitochondria and endoplasmic reticulum, as well. Given that the amount and the activity of PON1 in human serum are significantly lower in people suffering from cardiovascular diseases, enhancing both parameters might contribute to their treatment and prevention. One of the physiologically interesting substrates for the abovementioned hydrolytic cleavage is homocysteine thiolactone (HTL), an atherothrombotic active form of homocysteine. Although it was therefore postulated that PON1 would participate in preventing the HTL-mediated lipid peroxidation, some attention is recently paid to other enzymes, like biphenyl hydrolase-like protein, that seem to more selectively involved in lowering this risk factor. The aim of this paper is to elucidate the role of paraoxonases, especially PON1, by reviewing the latest studies in order to understand both its physiological role and modulation by drugs, nutrients, and plant extracts.

Chemical proteomic profiling of protein N-homocysteinylation with a thioester probe.

Hyperhomocysteinemia (HHcy) refers to a medical condition of abnormally high level of homocysteine (Hcy) in blood (>15 μmol L-1) and has been clinically implicated with cardiovascular diseases and neurodegenerative disorders. Excessive Hcy can be converted to a reactive thioester intermediate, Hcy thiolactone (HTL), which selectively reacts with protein lysine residues ("N-homocysteinylation") and this non-enzymatic modification largely contributes to manifestations of HHcy. However, the proteome-wide detection of protein N-homocysteinylation remains a challenge to date. In this work, we report a chemoselective reaction to label and enrich N-homocysteinylation from complex proteome samples as inspired by native chemical ligation for protein synthesis. Alkynyl thioester probes are synthesized and the reaction is validated with small molecule and purified protein models successfully. We performed quantitative chemical proteomics to identify more than 800 N-homocysteinylated proteins as well as 304 N-homocysteinylated sites directly from HTL-treated HeLa cells. The chemical proteomics strategies will facilitate functional study of protein N-homocysteinylations in the HHcy-implicated diseases.

Spontaneous Release of Human Serum Albumin S-Bound Homocysteine in a Thiol-Free Physiological Medium

Elevated plasma homocysteine (Hcy) concentrations independently predict cardiovascular disease. However, the transport of Hcy into pathological tissues such as atherosclerotic plaques, characterized by relatively low local thiol concentrations, is largely unknown. We sought to address if albumin-bound Hcy can be released in a thiol-free medium with or without previous incubation with Hcy and homocysteine thiolactone (HTL). We found that Hcy release was dependent on the baseline amount of albumin-bound Hcy. After 48 h incubation in a thiol- free medium the quantity of albumin-bound Hcy released from commercial human serum albumin (HSA) was 1.15 mmol/mol prot. If HSA was pre-incubated with 50 µmol/L reduced Hcy and then transferred into a free-thiol medium (HSA-S-Hcy), the amount of Hcy released after 48 h increased to 33.5 mmol/mol prot. If HSA was pre-incubated with 5 mmol/L HTL and then with 50 µmol/L of reduced Hcy (HSA-HTL-S-Hcy), the amount of Hcy released increased to 92.8 mmol/mol prot. Hcy release from HSA-HTL-S-Hcy further increased in presence of cysteine (Cys), glutathione (GSH), or Cys + GSH in the medium. Therefore, a significant amount of albumin-bound Hcy is released into thiol-free environments, similar to atherosclerotic plaques, with potential deleterious effects on vascular homeostasis, atherosclerosis and thrombosis.

EFFECT OF A COMBINED HIGH-FATTY DIET AND THIOLACTONE HYPERHOMOCYSTEINEMIA ON MASS, GROWTH AND ON BIOCHEMICAL MARKERS OF THE LIVER IN RATS

The impact of high fat diet (HFD) and thiolactone hyperhomocysteinemia combination on mass-growth parameters and biochemical markers of liver state in rats has been studied. It was proved that using HFD (54 % of kilocalories from fat) combined with the administration of homocysteine thiolactone (100 mg/kg intragastrically) for 60 days in rats lead to a significant increase of homocysteine concentration in the serum (by 55,0 %), BMI increase (by 8,9 %), total fat weight (by 39,8 %) and obesity index (by 18,5 %). In addition, the functional state of the liver was disturbed significantly and the processes of hepatic steatogenesis and fibrogenesis were accelerated, in comparison with single HFD use.

Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction.

Homocysteine and its cyclic ester homocysteine thiolactone (HTL) have been involved in the detrimental consequences associated to hyperhomocysteinemia, an independent risk factor for vascular diseases. HTL reacts with protein lysine residues in a process named N-homocysteinylation. The aim of our study was to evaluate the in vitro effects of HTL on the fibrinogen through electrophoretic methods. Fibrinogen was incubated with HTL at different molar ratios and structural changes of the protein were assessed by polyacrylamide gel electrophoresis (PAGE), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF). Ellman´s reaction, CZE and proton nuclear magnetic resonance (1H NMR) were used to evaluate HTL hydrolyisis. On denaturing PAGE numerous bands were observed, being the three lower bands identical to those obtained by treatment with 2-mercaptoethanol. This effect was also detected by CZE. The results show a reducing action of HTL on the fibrinogen molecule, probably attributed to the sulfhydryl groups generated by N-homocysteinylation and/or by the ones present in the homocysteine molecule yielded by HTL hydrolysis. In order to distinguish between these two options, HTL stability was evaluated at different pH and incubation times. The results showed minimum HTL hydrolysis in our experimental conditions. We postulate that the reducing effect observed would be mainly associated to the new sulfhydryl groups generated by the N-homocysteinylation process. Moreover, a displacement of the HTL-treated fibrinogen isoforms towards more acidic pH values was detected. The structural changes of N-homocysteinylated fibrinogen could be involved in the pathological consequences of hyperhomocysteinemia.

Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2′-deoxyuridine 5′-monophosphate (pTFT). The conjugate allows for direct optical and 19F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.

Lowered PON1 activities are strongly associated with depression and bipolar disorder, recurrence of (hypo)mania and depression, increased disability and lowered quality of life

Objectives: Mood disorders (MDs) frequently co-exist with cardiovascular disease (CVD) and immune-inflammatory and oxidative stress are important shared pathophysiological pathways. Even though there has been an extensive investigation of the enzyme paraoxonase 1 (PON1) as a biomarker of susceptibility for CVD, there are few reports studying PON1 in MDs. The aim of this study was to determine the association between PON1 activities as well as functional genotypes and MD diagnosis, clinical characteristics and outcomes.Methods: PON1 activities and functional genotypes were assayed in 58 bipolar disorder (BD) and 32 major depressed patients (MDD) and compared with 59 controls.Results: Our findings show significantly lower PON1 total and CMPAase activities in MDs, which are partly related to the number of previous depressive and manic episodes. Lowered CMPAase activity is associated with a worse outcome of MDs as indicated by lowered quality of life (WHOQoL-BREF scale) and increased disability in the Sheeham scale.Conclusions: We hypothesise that lowered PON1 total and CMPAase activities may play a role in the pathophysiology of MDs by lowering antioxidant defences thereby increasing the risk of lipid peroxidation and inflammation; lowered inhibition of quorum-sensing lactones thereby increasing bacterial proliferation; and attenuated homocysteine thiolactone catabolism which may trigger immune-inflammatory response and/or induce neurotoxicity.

Influence of hypolipidemic agents on the level of steatogenic and fibrogenic modulators in rats with non-alcoholic fatty liver disease associated with hyperhomocysteinemia

The aim was to determine the influence of Simvastatin and PUFA ω-3 remedy on the level of pro-fibrogenic neurotransmitters (Homocysteine, TNFα) and the level of neurotransmitters that determine anti-fibrous and liver regeneration potential (H 2 S and IGF-1) under experimental NAFLD associated with HHC. Materials and methods. The study was carried out using 100 white laboratory male rats divided into 7 experimental and 3 control groups. NAFLD associated with HHC was modeled in 7 groups of rats by applying high fat diet with simultaneous administration of Homocysteine thiolactone (100 mg / kg / intragastrically) for 60 days. From the 61st day and till the end of the experiment, 6 groups of rats with NAFLD + HHC had a standard diet. On this background, the animals of 4 groups were treated by lipid-lowering drugs - Simvastatin or ω-3 PUFAs remedy. The levels of Homocysteine, TNF-α and IGF-1 in bloodserum, as well as the content of triglycerides, cholesterol, phospholipids, hydroxyproline and H 2 S in rat liver were determined. Results. The use of Simvastatin did not cause statistically significant changes in the levels of Homocysteine, TNFα in blood serum and H 2 S content in the liver, but it led to IGF-1 worsening deficits in blood serum. At the same time, the use of PUFA ω-3 resulted in a significant reduction in the levels of Homocysteine, TNFα and increase in the levels of IGF-1 in serum and H 2 S in rat liver. Anti-steatosis and anti-fibrous effects of ω-3 PUFA remedy were significantly higher comparing to Simvastatin. Conclusion. Therefore, PUFA ω-3 drug significantly exceeds Simvastatin by hypohomocystinemic effect, more effectively reduces TNFα in blood serum and increases the H 2 S content in liver, and stimulates an IGF-1 level increase in blood serum, while Simvastatin enhances its deficit

Рівень інсуліноподібного фактору росту-1 та гідроген сульфіду у щурів з неалкогольною жировою хворобою печінки, асоційованою з гіпергомоцистеїнемією

Introduction. Evolution of nonalcoholic fatty liver disease (NAFLD) is often accompanied by increased levels of homocysteine, on the other hand hyperhomocysteinemia (HHC) is considered as an independent factor for steatosis and fibrosis of the liver. An important role in regulating of liver functional state has IGF-1 and gas mediator – hydrogen sulfide (H2S), but their role in the pathogenesis NAFLD associated with HHC, is not certain.The aim of the study – to investigate level of insulin-like growth factor-1 (IGF-1) in blood and concentration of H2S in the liver of rats with NAFLD induced by high fat diet (HFD) and its combination with HHC.Methods of the research. The experiment was performed on 56 white male rats. Animals were divided into 4 groups. Group 1 and group 2 received standard diet, group 3 and group 4 received HFD for 60 days. Rats from groups 2 and 4 were injected іntragastrically daily by thiolakton homocysteine in the dose of 100 mg/kg. After experiment the levels of homocysteine and IGF-1 in serum and H2S content in the liver of rats were determined.Results and Discussion. Homocysteine thiolactone or HFD and especially their combination causes a reduction of IGF-1 in serum and H2S content in the liver of rats. Rats with NAFLD associated with HHC had levels of H2S and IGF-1 significantly lower (respectively 22.6 % and 32.2 %) than that of rats with purely NAFLD. The level of homocysteine is inversely correlated with the level of IGF-1 in blood and the level of H2S in the liver (r= -0.68, -0.73, р<0.01). Formation of deficit аntifibrotic mediators (IGF-1 and H2S) may be a factor in the progression NAFLD associated with HHC.Conclusions. 60-day use of HFD or homocysteine thiolactone causes reduction of IGF-1 in serum and H2S content in the liver, but the most significant shortage of mediators is as result of the combination HFD with HHC.

Insights Into The Mechanism Of Protein Functional Loss Upon Covalent Modification By Homocysteine Thiolactone

Increased level of homocysteine (Hcy), associated with homocystinuria (or hyperhomocysteinemia), and is known to result in various clinical manifestations including cardiovascular complications and neurodegeneration. One likely mechanism underlying harmful effects of Hcy is the covalent post‐translational modification of protein by homocysteine thiolactone (HTL), a highly reactive cyclic thioester of Hcy formed by methionyl‐tRNA synthetase in an error editing reaction. Modification of proteins by HTL (a process termed N‐homocysteinylation) is known to result in protein functional loss. In the present study, the effects of HTL on structural and functional activity of three proteins RNase‐A, Lysozyme and Carbonic anhydrase were studied. We observed that RNase‐A and Lysozyme did not exhibit any functional loss upon overnight incubation with HTL. However, these enzymes required several days incubation to come to complete loss of function. On the other hand, CA was rendered non‐functional upon overnight incubation. The results indicate that certain protein requires exceptional longer incubation to bring about functional loss upon modification by HTL. Analyses of the mechanisms for the loss of enzyme function revealed that CA loses its enzyme function due to certain conformational alterations leading to aggregates formation. On the other hand, loss of activity in case of RNase‐A and Lysozyme was due to generation of exceptionally higher oligomeric spherical ensembles with varying sizes. The study revealed two independent mechanisms occurring for protein functional loss upon post‐translational modification by HTL.

Precisely Alternating Functionalized Polyampholytes Prepared in a Single Pot from Sustainable Thiolactone Building Blocks.

Polyampholytes with precisely alternating cationic and anionic functional groups were prepared using sustainable thiolactone building blocks in a simple one-pot procedure at room temperature and in water. Ring opening of the N-maleamic acid-functionalized homocysteine thiolactone monomer enabled the introduction of different functional groups into the polymer chain, which contributed to both ionic and hydrogen bonding interactions. The resulting polyampholytes exhibited various isoelectric points while maintaining high solubility in water under different pH and ionic strengths, which expands their potential applications. Finally, it is shown that the upper critical solution temperature (UCST) of these alternating polyampholytes in water/ethanol (30/70% vol) solutions can be tuned as a function of the content of ionic and hydroxyl groups.

The effect of N-acetylcysteine supplementation on serum homocysteine levels and hepatic and renal oxidative stress in homocysteine thiolactone-treated rats

The effect of N-acetylcysteine (NAC) (1 g/kg body weight/day) on serum homocysteine (Hcy) levels, insulin resistance (IR), and hepatic and renal prooxidant-antioxidant balance was evaluated in rats treated with homocysteine thiolactone (HcyT) (500 mg/kg body weight/day for 6 weeks). Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione, ferric reducing antioxidant power, and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined in the liver and kidney. HcyT elevated serum Hcy levels and caused IR, but liver and kidney function tests remained unchanged. HcyT increased ROS and MDA without any change in hepatic antioxidants, but it elevated renal SOD and GSH-Px activities. NAC decreased serum Hcy, hepatic and renal ROS and MDA levels, and renal SOD and GSH-Px activities in rats with high Hcy levels. However, it did not ameliorate IR. Our results indicate that NAC supplementation may be effective in decreasing Hcy levels and Hcy-induced hepatic and renal oxidative stress.

Lovastatin upregulates microRNA-29b to reduce oxidative stress in rats with multiple cardiovascular risk factors.

AimsProteasome-linked oxidative stress is believed to cause endothelial dysfunction, an early event in cardiovascular diseases (CVD). Statin, as HMG-CoA reductase inhibitor, prevents endothelial dysfunction in CVD. However, the molecular mechanism of statin-mediated normalization of endothelial function is not completely elucidated.Methods and ResultsLovastatin time/dose-dependently increased miR-29b expression and decreased proteasome activity in cultured human umbilical vein endothelial cells (HUVECs). Anti-miR-29b or overexpression of PA200 abolished lovastatin-induced inhibition of proteasome activity in HUVECs. In contrast, pre-miR-29b or PA200 siRNA mimics these effects of lovastatin on proteasome activity. Lovastatin inhibited oxidative stress induced by multiple oxidants including ox-LDL, H2O2, TNFa, homocysteine thiolactone (HTL), and high glucose (HG), which were reversed by inhibition of miR-29b in HUVECs. Ex vivo analysis indicated that lovastatin normalized the acetylcholine-induced endothelium-dependent relaxation and the redox status in isolated rat aortic arteries exposure to multiple cardiovascular risk factors. In vivo analysis revealed that administration of lovastatin remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats with hyperglycemia, dyslipidemia, and hyperhomocysteinemia, as well as increased miR-29b expressions, reduced PA200 protein levels, and suppression of proteasome activity in aortic tissues.ConclusionUpregulation of miR-29b expression is a common mechanism contributing to endothelial dysfunction induced by multiple cardiovascular risk factors through PA200-dependent proteasome-mediated oxidative stress, which is prevented by lovastatin.

4-BENZOYLPYRIDINE OXIME DERIVATIVE (GIZH-298) VERSUS VALPROIC ACID: THE ANTICONVULSANT POTENTIAL EFFECT IN A MODEL OF EPILEPSY IN RATS WITH COBALT-INDUCED LESIONS

Objective of the research is to evaluate the anticonvulsant effect of the new original compound GIZH-298 (4-benzoylpyridine oxime derivative) versus valproic acid (VPA) in a model of epilepsy in rats with cobalt-induced lesions. Materials and methods. Modeling of epileptic status was performed using the technique of creating a chronic epileptogenic focus caused by the application of cobalt to the sensorimotor zone of the rat cortex, followed by intraperitoneal administration of homolecysteine thiolactone. Compounds GIZH-298 and VPA were introduced against the background of development of electrographic status with behavioral convulsive seizure manifestations. Results. The study revealed that GIZH-298 at a dose of 60 mg/kg (i. p.) in 50 minutes after injection reduces the number of high-amplitude generalized discharges caused by homocysteine thiolactone in the ipsilateral and contralateral cortex (46-fold decrease), in the hippocampus and hypothalamus (28-fold decrease); eliminates (in 100% of the animals) the generalized tonic-clonic seizures that arise in the advanced stage of status epilepticus. VPA at a dose of 100 mg/kg (i. p.) in 3 hours after injection significantly suppresses the EpA in all evaluated structures with the maximum value in the hypothalamus (28-fold decrease), and after 5 hours in the ipsilateral and contralateral (33-fold decrease). At the same time, VPA eliminates generalized motility of status epilepticus only in 71% of the animals and protects from death 86% of the rats. Conclusion. The compound GIZH-298 significantly earlier (for 2 hours) than the VPA (100 mg/kg) and at a lower dose (60 mg/kg) fully eliminates electrographic (in all evaluated brain structures with the greatest efficiency in the contralateral cortex and the hypothalamus) and behavioral manifestations of unfolded status epilepticus and prevents deaths in 100%.

EFFECTS OF ISOLATED VITAMIN B6 SUPPLEMENTATION ON OXIDATIVE STRESS AND HEART FUNCTION PARAMETERS IN EXPERIMENTAL HYPERHOMOCYSTEINEMIA

Introduction : The purpose of this study was to investigate the effects of isolated vitamin B 6 (VB 6 ) supplementation on experimental hyperhomocysteinemia (Hhe) induced by homocysteine thiolactone (HcyT). Methods: Fifteen male Wistar rats were divided into three groups according to their treatment. Animals received water and food ad libitum and an intragastric probe was used to administer water for 60 days (groups: CB 6, HcyT, and HB 6 ). On the 30th day of treatment, two groups were supplemented with VB 6 in the drinking water (groups: CB 6 and HB 6 ). After 60 days of treatment, homocysteine (Hcy), cysteine, and hydrogen peroxide concentration, nuclear factor (erythroid-derived 2)-like 2 (NRF2) and glutathione S-transferase (GST) immunocontent, and superoxide dismutase (SOD), catalase (CAT), and GST activities were measured. Results: The HcyT group showed an increase in Hcy concentration (62%) in relation to the CB 6 group. Additionally, GST immunocontent was enhanced (51%) in the HB 6 group compared to the HcyT group. Also, SOD activity was lower (17%) in the HB 6 group compared to the CB 6 group, and CAT activity was higher in the HcyT group (53%) compared to the CB 6 group. Ejection fraction (EF) was improved in the HB 6 group compared to the HcyT group. E/A ratio was enhanced in the HB 6 group compared to the CB 6 group. Correlations were found between CAT activity with myocardial performance index (MPI) (r = 0.71; P = 0.06) and E/A ratio (r = 0.6; P = 0.01), and between EF and GST activity (r = 0.62; P = 0.02). Conclusions: These findings indicate that isolated VB 6 supplementation may lead to the reduction of Hcy concentration and promotes additional benefits to oxidative stress and heart function parameters. Keywords: Homocysteine; oxidative stress; vitamin B 6.

Conformational status of cytochrome c upon N-homocysteinylation: Implications to cytochrome c release

One of the proposed mechanisms of homocysteine (Hcy) toxicity is the post-translational modification of proteins by its metabolite, homocysteine thiolactone (HTL). Incubation of proteins with HTL has been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation) which ultimately results in structural and functional alterations of the modified proteins. In the present study, the effects of HTL on the conformational and heme status of cytochrome c (cyt c) were investigated. Spectroscopic analyses revealed that HTL-modified cyt c undergoes certain conformational alterations leading to disturbed heme-Trp distance and packing of the apolar groups. These alterations were accompanied with the reduction of the heme moiety and activation of peroxidase-like function of cyt c, which is known to be a crucial event for initiation of the intrinsic apoptotic pathway. Further structural characterization revealed that disruption of the heme-Met80 interaction, thereby converting the hexa-coordinate cyt c to a penta-coordinate species (with a free heme ligand), was responsible for the activation of the peroxidase activity. The study provides insights for the possible role of cyt c N-homocysteinylation in eliciting its toxicity and cell death.

Low efficacy of simvastatin in treatment of nonalcoholic fatty liver disease in rats

We determined the possibility of simvastatin in non-alcoholic fatty liver disease in rats. The animals were kept for 2 months at high-fatty diet with additional input homocysteine thiolactone. Then, the animals were divided into groups according to the doses (20 or 40 mg/kg body weight) and duration of treatment (14 or 28 days). At the end of experiment there was determined the concentration of homocysteine, serum biochemical parameters, intensity of oxidative stress and antioxidant defense, and the degree of hepatic fibrogenesis and steatohenesis. It was shown that simvastatin did not improve the health of rats.

Processes of plasma protein N-homocysteinylation in multiple sclerosis

Background: Homocysteine thiolactone (HTL) is a cyclic thioester of homocysteine (Hcy) contributing to the toxicity of this amino acid. HTL spontaneously reacts with protein lysine residues leading to altered properties of target proteins and induction of immune response. HTL is hydrolyzed to Hcy by plasma enzyme, paraoxonase 1 (PON1). Although both Hcy and PON1 may be involved in the pathogenesis of multiple sclerosis (MS), protein modification by HTL in this disease has not been studied so far. Purpose/Aim: The aim of this study was to assess the level of Hcy, HTL and autoantibodies against N-homocysteinylated proteins as well as PON1 activity in patients with MS. Methods: The studies were performed in 61 MS patients with relapsing-remitting (RR group, n = 25) and secondary-progressive type of MS (SP group, n = 36), and in healthy people (C – control group, n = 44). Results: Homocysteine level was significantly higher in MS patients comparing to control group (C vs. RR p < 0.01; C vs. SP p < 0.05). The level of HTL tended to be higher in RR-MS in comparison to control group, but it did not reach the level of significance. The level of antibodies against N-homocysteinylated proteins did not differ significantly between studied groups. PON1 activity was significantly lower in SP type of MS (SP vs. C p < 0.05; SP vs. RR p < 0.05). Conclusions: Although plasma Hcy concentration is higher in MS patients and PON1 activity is reduced in the SP form, MS is associated with minor or no changes in protein-attached HTL and anti-homocysteinylated protein immune response.