COVID-19 and One-Carbon Metabolism.

Dysregulation of one-carbon metabolism affects a wide range of biological processes and is associated with a number of diseases, including cardiovascular disease, dementia, neural tube defects, and cancer. Accumulating evidence suggests that one-carbon metabolism plays an important role in COVID-19. The symptoms of long COVID-19 are similar to those presented by subjects suffering from vitamin B12 deficiency (pernicious anemia). The metabolism of a cell infected by the SARS-CoV-2 virus is reshaped to fulfill the need for massive viral RNA synthesis, which requires de novo purine biosynthesis involving folate and one-carbon metabolism. Many aspects of host sulfur amino acid metabolism, particularly glutathione metabolism underlying antioxidant defenses, are also taken over by the SARS-CoV-2 virus. The purpose of this review is to summarize recent findings related to one-carbon metabolism and sulfur metabolites in COVID-19 and discuss how they inform strategies to combat the disease.

The Cbs Locus Affects the Expression of Senescence Markers and mtDNA Copy Number, but not Telomere Dynamics in Mice.

Cystathionine ß-synthase (CBS) is a housekeeping enzyme that catalyzes the first step of the homocysteine to cysteine transsulfuration pathway. Homozygous deletion of the Cbs gene in mice causes severe hyperhomocysteinemia and reduces life span. Here, we examined a possible involvement of senescence, mitochondrial DNA, and telomeres in the reduced life span of Cbs-/- mice. We found that senescence-related p21, Pai-1, Mcp1, and Il-6 mRNAs were significantly upregulated (2-10-fold) in liver, while p21 was upregulated in the brain of Cbs-/- mice (n = 20) compared with control Cbs+/- siblings (n = 20) in a sex- and age-dependent manner. Telomere length in blood (n = 80), liver (n = 40), and brain (n = 40) was not affected by the Cbs-/- genotype, but varied with sex and/or age. Levels of mitochondrial DNA tended to be reduced in livers, but not brains and blood, of Cbs-/- females (n = 20-40). The Cbs-/- genotype significantly reduced Tert mRNA expression in brain, but not liver, in a sex- and age-dependent manner. Multiple regression analysis showed that the senescence-related liver (but not brain) mRNAs and liver (but not brain or blood) mitochondrial DNA were associated with the Cbs genotype. In contrast, telomere length in blood, brain, and liver was not associated with the Cbs genotype or hyperhomocysteinemia, but was associated with sex (in brain and liver) and age (in brain and blood). Taken together, these findings suggest that the changes in senescence marker expression and mtDNA levels, but not telomere shortening, could account for the reduced life span of Cbs-/- mice.

Paraoxonase 1 Q192R genotype and activity affect homocysteine thiolactone levels in humans.

Genetic or nutritional deficiencies in 1 carbon and homocysteine (Hcy) metabolism elevate Hcy-thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy-thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio-protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy-thiolactone in vitro. However, whether Hcy-thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy-thiolactone levels in humans was unknown. Here we show that the PON1-192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy-thiolactone levels, normalized to creatinine. Carriers of the PON1-192R allele had significantly lower Hcy-thiolactone/creatinine levels than individuals carrying the PON1-192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy-thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy-thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy-thiolactone in humans and that the interindividual variations in PON1 genotype/activity can modulate the pathology of hyperhomocysteinemia.-Perla-Kaján, J., Borowczyk, K., Glowacki, R., Nygård, O., Jakubowski, H. Paraoxonase 1 Q192r genotype and activity affect homocysteine thiolactone levels in humans.

Dysregulation of Epigenetic Mechanisms of Gene Expression in the Pathologies of Hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) exerts a wide range of biological effects and is associated with a number of diseases, including cardiovascular disease, dementia, neural tube defects, and cancer. Although mechanisms of HHcy toxicity are not fully uncovered, there has been a significant progress in their understanding. The picture emerging from the studies of homocysteine (Hcy) metabolism and pathophysiology is a complex one, as Hcy and its metabolites affect biomolecules and processes in a tissue- and sex-specific manner. Because of their connection to one carbon metabolism and editing mechanisms in protein biosynthesis, Hcy and its metabolites impair epigenetic control of gene expression mediated by DNA methylation, histone modifications, and non-coding RNA, which underlies the pathology of human disease. In this review we summarize the recent evidence showing that epigenetic dysregulation of gene expression, mediated by changes in DNA methylation and histone N-homocysteinylation, is a pathogenic consequence of HHcy in many human diseases. These findings provide new insights into the mechanisms of human disease induced by Hcy and its metabolites, and suggest therapeutic targets for the prevention and/or treatment.

N-Homocysteinylation impairs collagen cross-linking in cystathionine ß-synthase-deficient mice: a novel mechanism of connective tissue abnormalities.

Cystathionine ß-synthase (CBS) deficiency, a genetic disorder in homocysteine (Hcy) metabolism in humans, elevates plasma Hcy-thiolactone and leads to connective tissue abnormalities that affect the cardiovascular and skeletal systems. However, the underlying mechanism of these abnormalities is not understood. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-homocysteinylated protein. Because lysine residues are involved in collagen cross-linking, N-homocysteinylation of these lysines should impair cross-linking. Using a Tg-I278T Cbs-/- mouse model of hyperhomocysteinemia (HHcy) which replicates the connective tissue abnormalities observed in CBS-deficient patients, we found that N-Hcy-collagen was elevated in bone, tail, and heart of Cbs-/- mice, whereas pyridinoline cross-links were significantly reduced. Plasma deoxypyridinoline cross-link and cross-linked carboxyterminal telopeptide of type I collagen were also significantly reduced in the Cbs-/- mice. Lysine oxidase activity and mRNA level were not reduced by the Cbs-/- genotype. We also showed that collagen carries S-linked Hcy bound to the thiol of N-linked Hcy. In vitro experiments showed that Hcy-thiolactone modifies lysine residues in collagen type I α-1 chain. Residue K160, located in the nonhelical N-telopeptide region and involved in pyridinoline cross-link formation, was also N-homocysteinylated in vivo Taken together, our findings showed that N-homocysteinylation of collagen in Cbs-/- mice impairs its cross-linking. These findings explain, at least in part, connective tissue abnormalities observed in HHcy.-Perla-Kajan, J., Utyro, O., Rusek, M., Malinowska, A., Sitkiewicz, E., Jakubowski, H. N-Homocysteinylation impairs collagen cross-linking in cystathionine ß-synthase-deficient mice: a novel mechanism of connective tissue abnormalities.

Diet-induced hyperhomocysteinemia causes sex-dependent deficiencies in offspring musculature and brain function.

Hyperhomocysteinemia (HHcy), characterized by elevated homocysteine (Hcy) levels, is a known risk factor for cardiovascular, renal, and neurological diseases, as well as pregnancy complications. Our study aimed to investigate whether HHcy induced by a high-methionine (high-Met) diet exacerbates cognitive and behavioral deficits in offspring and leads to other breeding problems. Dietary HHcy was induced four weeks before mating and continued throughout gestation and post-delivery. A battery of behavioral tests was conducted on offspring between postnatal days (PNDs) 5 and 30 to assess motor function/activity and cognition. The results were correlated with brain morphometric measurements and quantitative analysis of mammalian target of rapamycin (mTOR)/autophagy markers. The high-Met diet significantly increased parental and offspring urinary tHcy levels and influenced offspring behavior in a sex-dependent manner. Female offspring exhibited impaired cognition, potentially related to morphometric changes observed exclusively in HHcy females. Male HHcy pups demonstrated muscle weakness, evidenced by slower surface righting, reduced hind limb suspension (HLS) hanging time, weaker grip strength, and decreased activity in the beaker test. Western blot analyses indicated the downregulation of autophagy and the upregulation of mTOR activity in HHcy cortexes. HHcy also led to breeding impairments, including reduced breeding rate, in-utero fetal death, lower pups' body weight, and increased mortality, likely attributed to placental dysfunction associated with HHcy. In conclusion, a high-Met diet impairs memory and cognition in female juveniles and weakens muscle strength in male pups. These effects may stem from abnormal placental function affecting early neurogenesis, the dysregulation of autophagy-related pathways in the cortex, or epigenetic mechanisms of gene regulation triggered by HHcy during embryonic development.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Association of Metallic and Nonmetallic Elements with Fibrin Clot Properties and Ischemic Stroke.

Objectives-Metallic elements and fibrin clot properties have been linked to stroke. We examined metallic and nonmetallic elements, fibrin clot lysis time (CLT), and maximum absorbance (Absmax) in relation to ischemic stroke. Design-A case-control study of ischemic stroke patients vs. healthy individuals. Subjects and Methods-Plasma and serum were collected from 260 ischemic stroke patients (45.0% women; age, 68 ± 12 years) and 291 healthy controls (59.7% women; age, 50 ± 17 years). Fibrin CLT and Absmax were measured using a validated turbidimetric assay. Serum elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES). Data were analyzed by bivariate correlations and multiple or logistic regression. Results-In female stroke patients, copper, lithium, and aluminum were significantly lower compared with controls; in male stroke patients, potassium was lower, and beryllium was elevated. In female and male stroke patients, iron, zinc, nickel, calcium, magnesium, sodium, and silicon were significantly lower, while strontium was elevated. Positive correlations between fibrin clot properties and metals, observed in healthy controls, were lost in ischemic stroke patients. In multivariate regression analysis, fibrin CLT and/or Absmax was associated with zinc, calcium, potassium, beryllium, and silicon in stroke patients and with sodium, potassium, beryllium, and aluminum in controls. In logistic regression analysis, stroke was independently associated with lithium, nickel, beryllium, strontium, boron, and silicon and with sodium, potassium, calcium, and aluminum but not with fibrin CLT/Absmax. Conclusions-Various elements were associated with fibrin clot properties and the risk of ischemic stroke. Lithium, sodium, calcium, and aluminum abrogated the association of fibrin clot properties with ischemic stroke.

Paraoxonase 1 and homocysteine metabolism.

Paraoxonase 1 (PON1), a component of high-density lipoprotein (HDL), is a calcium-dependent multifunctional enzyme that connects metabolisms of lipoproteins and homocysteine (Hcy). Both PON1 and Hcy have been implicated in human diseases, including atherosclerosis and neurodegeneration. The involvement of Hcy in disease could be mediated through its interactions with PON1. Due to its ability to reduce oxidative stress, PON1 contributes to atheroprotective functions of HDL in mice and humans. Although PON1 has the ability to hydrolyze a variety of substrates, only one of them-Hcy-thiolactone-is known to occur naturally. In humans and mice, Hcy-thiolactonase activity of PON1 protects against N-homocysteinylation, which is detrimental to protein structure and function. PON1 also protects against neurotoxicity associated with hyperhomocysteinemia in mouse models. The links between PON1 and Hcy in relation to pathological states such as coronary artery disease, stroke, diabetic mellitus, kidney failure and Alzheimer's disease that emerge from recent studies are the topics of this review.

Homocysteine thiolactone contributes to the prognostic value of fibrin clot structure/function in coronary artery disease.

Fibrin clot structure/function contributes to cardiovascular disease. We examined sulfur-containing metabolites as determinants of fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to outcomes in coronary artery disease (CAD) patients. Effects of B-vitamin/folate therapy on CLT and Absmax were studied. Plasma samples were collected from 1,952 CAD patients randomized in a 2 x 2 factorial design to (i) folic acid, vitamins B12, B6; (ii) folic acid, vitamin B12; (iii) vitamin B6; (iv) placebo for 3.8 years in the Western Norway B-Vitamin Intervention Trial. Clot lysis time (CLT) and maximum absorbance (Absmax) were determined using a validated turbidimetric assay. Acute myocardial infarction (AMI) and mortality were assessed during a 7-year follow-up. Data were analyzed using bivariate and multiple regression. Survival free of events was studied using Kaplan Mayer plots. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazards models. Baseline urinary homocysteine (uHcy)-thiolactone and plasma cysteine (Cys) were significantly associated with CLT while plasma total Hcy was significantly associated with Absmax, independently of fibrinogen, triglycerides, vitamin E, glomerular filtration rate, body mass index, age, sex plasma creatinine, CRP, HDL-C, ApoA1, and previous diseases. B-vitamins/folate did not affect CLT and Absmax. Kaplan-Meier analysis showed associations of increased baseline CLT and Absmax with worse outcomes. In Cox regression analysis, baseline CLT and Absmax (>cutoff) predicted AMI (CLT: HR 1.58, 95% CI 1.10-2.28; P = 0.013. Absmax: HR 3.22, CI 1.19-8.69; P = 0.021) and mortality (CLT: HR 2.54, 95% CI 1.40-4.63; P = 0.002. Absmax: 2.39, 95% CI 1.17-4.92; P = 0.017). After adjustments for other prognostic biomarkers these associations remained significant. Cys and uHcy-thiolactone, but not tHcy, were significant predictors of AMI in Cox regression models that included CLT. Conclusions uHcy-thiolactone and plasma Cys are novel determinants of CLT, an important predictor of adverse CAD outcomes. CLT and Absmax were not affected by B-vitamin/folate therapy, which could account for the lack of efficacy of such therapy in CAD. Trial registration: URL: http://clinicaltrials.gov. Identifier: NCT00354081.

Paraoxonase 1 protects against protein N-homocysteinylation in humans.

Genetic or nutritional disorders in homocysteine (Hcy) or folate metabolism elevate plasma Hcy-thiolactone and lead to vascular and/or brain pathologies. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-Hcy-protein with autoimmunogenic and prothrombotic properties. Paraoxonase (PON1), carried on high-density lipoproteins (HDLs) in the blood, hydrolyzes Hcy-thiolactone and protects against the accumulation of N-Hcy-protein in vitro. To determine its role in vivo, we studied how natural variation in Hcy-thiolactonase activity of PON1 affects plasma N-Hcy-protein levels in cystathionine beta-synthase-deficient patients (n=28). We found that plasma N-Hcy-protein was negatively correlated with serum Hcy-thiolactonase activity (r=-0.43, P=0.01), i.e., the higher the Hcy-thiolactonase activity, the lower N-Hcy protein levels. This relation was faithfully replicated in vitro in experiments with radiolabeled Hcy-thiolactone. We also found that enzymatic activities of the PON1 protein measured with artificial substrates correlated less strongly (r=-0.36, P=0.025 for paraoxonase activity) or did not correlate at all (phenylacetate hydrolase and TBLase activities) with plasma N-Hcy protein. These findings provide evidence that the Hcy-thiolactonase activity of PON1 is a determinant of plasma N-Hcy-protein levels and that Hcy-thiolactonase/PON1 protects proteins against N-homocysteinylation in vivo, a novel mechanism likely to contribute to atheroprotective roles of HDL in humans.-Perla-Kaján, J., Jakubowski, H. Paraoxonase 1 protects against protein N-homocysteinylation in humans.

Cation exchange HPLC analysis of desmosines in elastin hydrolysates.

Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft(-/-) mice deficient in intestinal folate transport compared with wild-type Pcft(+/+) animals.

Anti-N-homocysteine-protein autoantibodies are associated with impaired cognition.

INTRODUCTION: Elevated homocysteine (Hcy) and related metabolites accelerate Alzheimer's disease. Hcy-lowering B vitamins slow brain atrophy/cognitive decline in mild cognitive impairment (MCI). Modification with Hcy-thiolactone generates auto-immunogenic N-Hcy-protein. We tested a hypothesis that anti-N-Hcy-protein autoantibodies predict cognition in individuals with MCI participating in a randomized, double-blind, placebo-controlled VITACOG trial of B vitamins. METHODS: Participants with MCI (n = 196, 76.8 years old, 60% women) were randomly assigned to receive a daily dose of folic acid (0.8 mg), vitamin B12 (0.5 mg), and B6 (20 mg) (n = 98) or placebo (n = 98) for 2 years. Cognition was analyzed by neuropsychological tests. Brain atrophy was quantified in a subset of patients (n = 167) by magnetic resonance imaging. Anti N-Hcy-protein auto-antibodies were quantified by enzyme-linked immunosorbent assay. Associations among anti-N-Hcy-protein autoantibodies, cognition, and brain atrophy were examined by multiple regression analysis. RESULTS: At baseline, anti-N-Hcy-protein autoantibodies were significantly associated with impaired global cognition (Mini-Mental State Examination [MMSE]), episodic memory (Hopkins Verbal Learning Test-revised), and attention/processing speed (Map Search). At the end of the study, anti-N-Hcy-protein autoantibodies were associated with impaired global cognition (MMSE) and attention/processing speed (Trail Making A). In the placebo group, baseline anti-N-Hcy-protein autoantibodies predicted, independently of Hcy, global cognition (Telephone Inventory for Cognitive Status modified [TICS-m]; MMSE) and attention/processing speed (Trail Making A) but not brain atrophy, at the end of study. B-vitamin treatment abrogated association of anti-N-Hcy-protein autoantibodies with cognition. DISCUSSION: These findings suggest that anti-N-Hcy-protein autoantibodies can impair functional (attention/processing speed and global cognition), but not structural (brain atrophy), aspects of cognition. Anti-N-Hcy-protein autoantibodies are a new factor associated with impaired cognition, which could be ameliorated by B vitamins.

Paraoxonase 1, B Vitamins Supplementation, and Mild Cognitive Impairment.

BACKGROUND: Identification of modifiable risk factors that affect cognitive decline is important for the development of preventive and treatment strategies. Status of paraoxonase 1 (PON1), a high-density lipoprotein-associated enzyme, may play a role in the development of neurological diseases, including Alzheimer's disease. OBJECTIVE: We tested a hypothesis that PON1 status predicts cognition in individuals with mild cognitive impairment (MCI). METHODS: Individuals with MCI (n = 196, 76.8-years-old, 60% women) participating in a randomized, double-blind placebo-controlled trial (VITACOG) were assigned to receive a daily dose of folic acid (0.8 mg), vitamin B12 (0.5 mg) and B6 (20 mg) (n = 95) or placebo (n = 101) for 2 years. Cognition was analyzed by neuropsychological tests. Brain atrophy was quantified in a subset of participants (n = 168) by MRI. PON1 status, including PON1 Q192R genotype, was determined by quantifying enzymatic activity of PON1 using paraoxon and phenyl acetate as substrates. RESULTS: In the placebo group, baseline phenylacetate hydrolase (PhAcase) activity of PON1 (but not paraoxonase activity or PON1 Q192R genotype) was significantly associated with global cognition (Mini-Mental State Examination, MMSE; Telephone Inventory for Cognitive Status-modified, TICS-m), verbal episodic memory (Hopkins Verbal Learning Test-revised: Total Recall, HVLT-TR; Delayed Recall, HVLT-DR), and attention/processing speed (Trail Making A and Symbol Digits Modalities Test, SDMT) at the end of study. In addition to PhAcase, baseline iron and triglycerides predicted MMSE, baseline fatty acids predicted SDMT, baseline anti-N-Hcy-protein autoantibodies predicted TICS-m, SDMT, Trail Making A, while BDNF V66M genotype predicted HVLT-TR and HVLT-DR scores at the end of study. B-vitamins abrogated associations of PON1 and other variables with cognition. CONCLUSION: PON1 is a new factor associated with impaired cognition that can be ameliorated by B-vitamins in individuals with MCI.

Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice.

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy-thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine beta-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N-Hcy-protein levels. Liver N-Hcy-protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N-Hcy-protein is an important metabolite associated with Hcy pathophysiology in the mouse.

Telomere length and mtDNA copy number in human cystathionine ß-synthase deficiency.

Telomere shortening and mitochondrial DNA (mtDNA) copy number are associated with human disease and a reduced life span. Cystathionine ß-synthase (CBS) is a housekeeping enzyme that catalyzes the first step in metabolic conversion of homocysteine (Hcy) to cysteine. Mutations in the CBS gene cause CBS deficiency, a rare recessive metabolic disease, manifested by severe hyperhomocysteinemia (HHcy) and thromboembolism, which ultimately reduces the life span. However, it was not known whether telomere shortening or mtDNA is involved in the pathology of human CBS deficiency. We quantified leukocyte telomere length (TL), mtDNA copy number, and plasma Hcy levels in CBS-/- patients (n = 23) and in sex- and age-matched unaffected CBS+/+ control individuals (n = 28) 0.08-57 years old. We found that TL was significantly increased in severely HHcy CBS-/- female patients but unaffected in severely HHcy CBS-/- male patients, relative to the corresponding CBS+/+ controls who had normal plasma Hcy levels. In multiple regression analysis TL was associated with CBS genotype in women but not in men. MtDNA copy number was not significantly affected by the CBS-/- genotype. Taken together, these findings identify the CBS gene as a new locus in human DNA that affects TL in women and illustrate a concept that a housekeeping metabolic gene can be involved in telomere biology. Our findings suggest that neither telomere shortening nor reduced mtDNA copy number contribute to the reduced life span in CBS-/- patients.

Genetic Attenuation of Paraoxonase 1 Activity Induces Proatherogenic Changes in Plasma Proteomes of Mice and Humans.

High-density lipoprotein (HDL), in addition to promoting reverse cholesterol transport, possesses anti-inflammatory, antioxidative, and antithrombotic activities. Paraoxonase 1 (PON1), carried on HDL in the blood, can contribute to these antiatherogenic activities. The PON1-Q192R polymorphism involves a change from glutamine (Q variant) to arginine (R variant) at position 192 of the PON1 protein and affects its enzymatic activity. The molecular basis of PON1 association with cardiovascular and neurological diseases is not fully understood. To get insight into the function of PON1 in human disease, we examined how genetic attenuation of PON1 levels/activity affect plasma proteomes of mice and humans. Healthy participants (48.9 years old, 50% women) were randomly recruited from the Poznan population. Four-month-old Pon1-/- (n = 17) and Pon1+/+ (n = 8) mice (50% female) were used in these experiments. Plasma proteomes were analyzed using label-free mass spectrometry. Bioinformatics analysis was carried out using the Ingenuity Pathway Analysis (IPA) resources. PON1-Q192R polymorphism and Pon1-/- genotype induced similar changes in plasma proteomes of humans and mice, respectively. The top molecular network, identified by IPA, affected by these changes involved proteins participating in lipoprotein metabolism. Other PON1 genotype-dependent proteomic changes affect different biological networks in humans and mice: "cardiovascular, neurological disease, organismal injury/abnormalities" in PON1-192QQ humans and "humoral immune response, inflammatory response, protein synthesis" and "cell-to-cell signaling/interaction, hematological system development/function, immune cell trafficking" in Pon1-/- mice. Our findings suggest that PON1 interacts with molecular pathways involved in lipoprotein metabolism, acute/inflammatory response, and complement/blood coagulation that are essential for blood homeostasis. Modulation of those interactions by the PON1 genotype can account for its association with cardiovascular and neurological diseases.

Sex affects N-homocysteinylation at lysine residue 212 of albumin in mice.

The modification of protein lysine residues by the thioester homocysteine (Hcy)-thiolactone has been implicated in cardiovascular and neurodegenerative diseases. However, only a handful of proteins carrying Hcy on specific lysine residues have been identified and quantified in humans or animals. In the present work, we developed a liquid chromatography/mass spectrometry targeted assay, based on multiple reaction monitoring, for quantification of N-Hcy-Lys212 (K212Hcy) and N-Hcy-Lys525 (K525Hcy) sites in serum albumin in mice. Using this assay, we found that female (n = 20) and male (n = 13) Cbs-/- mice had significantly elevated levels of K212Hcy and K525Hcy modifications in serum albumin relative to their female (n = 19) and male (n = 17) Cbs+/- littermates. There was significantly more K212Hcy modification in Cbs-/- males than in Cbs-/- females (5.78 ± 4.21 vs. 3.15 ± 1.38 units, P = 0.023). Higher K212Hcy levels in males than in females were observed also in Cbs+/- mice (2.72 ± 0.81 vs. 1.89 ± 1.07 units, P = 0.008). In contrast, levels of the K525Hcy albumin modification were similar between males and females, both in Cbs-/- and Cbs+/- mice. These findings suggest that the sex-specific K212Hcy modification in albumin might have an important biological function in mice that is not affected by the Cbs genotype.

Immunohistochemical detection of N-homocysteinylated proteins in humans and mice.

N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.