Leucine Carboxyl Methyltransferase 1 (LCMT1)-dependent Methylation Regulates the Association of Protein Phosphatase 2A and Tau Protein with Plasma Membrane Microdomains in Neuroblastoma Cells

Down-regulation of protein phosphatase 2A (PP2A) methylation occurs in Alzheimer disease (AD). However, the regulation of PP2A methylation remains poorly understood. We have reported that altered leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down-regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in AD. Here, we show that pools of LCMT1, methylated PP2A, and PP2A/Bα are co-enriched in cholesterol-rich plasma membrane microdomains/rafts purified from N2a cells. In contrast, demethylated PP2A is preferentially distributed in non-rafts wherein small amounts of the PP2A methylesterase PME-1 are exclusively present. A methylation-incompetent PP2A mutant is excluded from rafts. Enhanced methylation of PP2A promotes the association of PP2A and Tau with the plasma membrane. Altered PP2A methylation following expression of a catalytically inactive LCMT1 mutant, knockdown of LCMT1, or alterations in one-carbon metabolism all result in a loss of plasma membrane-associated PP2A and Tau in N2a cells. This correlates with accumulation of soluble phosphorylated Tau, a hallmark of AD and other tauopathies. Thus, our findings reveal a distinct compartmentalization of PP2A and PP2A regulatory enzymes in plasma membrane microdomains and identify a novel methylation-dependent mechanism involved in modulating the targeting of PP2A, and its substrate Tau, to the plasma membrane. We propose that alterations in the membrane localization of PP2A and Tau following down-regulation of LCMT1 may lead to PP2A and Tau dysfunction in AD.     

Age-related effects of methionine-enriched diet on plasma homocysteine concentration and methylation of hepatic DNA in rats

In the present study we determined the age-related effect of methionine-enriched diet, a model of hyperhomocysteinemia, on the level of plasma homocysteine and hepatic global DNA methylation in rats. Feeding methionine diet to middle-aged rats for only 14 days resulted in a significant increase in plasma homocysteine level and DNA hypomethylation. In contrast, feeding the methionine-containing diet for 2 weeks to juvenile or post-pubertal animals did not alter the level of plasma homocysteine or hepatic DNA methylation. Supplementation of the methionine-enriched diet with vitamins B6, B12 and folic acid prevented both hepatic DNA hypomethylation and an increase of plasma homocysteine concentration in the middle-aged rats. These findings indicate that the elevated level of plasma homocysteine may be indicative of much broader and deeper alterations in intracellular methylation dysfunction, and suggest that dietary enrichment with B-vitamins is essential for the metabolism of homocysteine, especially in adult animals.     

Physiological regulation of phospholipid methylation alters plasma homocysteine in mice

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.     

Betaine attenuates Alzheimer‐like pathological changes and memory deficits induced by homocysteine

Hyperhomocysteinemia (Hhcy) may induce memory deficits with β‐amyloid (Aβ) accumulation and tau hyperphosphorylation. Simultaneous supplement of folate and vitamin B12 partially restored the plasma homocysteine level and attenuated tau hyperphosphorylation, Aβ accumulation and memory impairments induced by Hhcy. However, folate and vitamin B12 treatment have no effects on Hhcy which has the methylenetetrahydrofolate reductase genotype mutation. In this study, we investigated the effects of simultaneous supplement of betaine on Alzheimer‐like pathological changes and memory deficits in hyperhomocysteinemic rats after a 2‐week induction by vena caudalis injection of homocysteine (Hcy). We found that supplementation of betaine could ameliorate the Hcy‐induced memory deficits, enhance long‐term potentiation (LTP) and increase dendritic branches numbers and the density of the dendritic spines, with up‐regulation of NR1, NR2A, synaptotagmin, synaptophysin, and phosphorylated synapsin I protein levels. Supplementation of betaine also attenuated the Hcy‐induced tau hyperphosphorylation at multiple AD‐related sites through activation protein phosphatase‐2A (PP2A) with decreased inhibitory demethylated PP2A_C at Leu309 and phosphorylated PP2A_C at Tyr307. In addition, supplementation of betaine also decreased Aβ production with decreased presenilin‐1 protein levels. Our data suggest that betaine could be a promising candidate for arresting Hcy‐induced AD‐like pathological changes and memory deficits.     

Detection of altered global DNA methylation in coronary artery disease patients

Epigenetic modifications, especially alteration in DNA methylation, are increasingly being recognized as a key factor in the pathogenesis of complex disorders, including atherosclerosis. However, there are limited data on the epigenetic changes in the coronary artery disease (CAD) patients. In the present study we evaluated the methylation status of genomic DNA from peripheral lymphocytes in a cohort of 287 individuals: 137 angiographically confirmed CAD patients and 150 controls. The differential susceptibility of genomic DNA to methylation-sensitive restriction enzymes was utilized to assess the methylation status of the genome. We observed that the genomic DNA methylation in CAD patients is significantly higher than in controls (p < 0.05). Since elevated homocysteine levels are known to be an independent risk factor for CAD and a key modulator of macromolecular methylation, we investigated the probable correlation between plasma homocysteine levels and global DNA methylation. We observed a significant positive correlation of global DNA methylation with plasma homocysteine levels in CAD patients (p = 0.001). Further, within a higher range of serum homocysteine levels (>/=12-50 muM), global DNA methylation was significantly higher in CAD patients than in controls. The alteration in genomic DNA methylation associated with cardiovascular disease per se appears to be further accentuated by higher homocysteine levels.     

Folate is related to phosphorylated neurofilament-H and P-tau (Ser396) in rat brain

Protein phosphatase PP2A dephosphorylates phosphorylated tau (P-tau) and neurofilaments (pNFs). PP2A is S-adenosylmethionine (SAM)-dependent and might thus link methylation with neurodegeneration. Low SAM and increased S-adenosylhomocysteine (SAH) can enhance the risk of dementia. We studied the effect of hyperhomocysteinemia on P-tau (Ser396), pNF-H (heavy chain), and PP2A-activity and level (the C subunit) in rat brain. Wistar rats (total n=55) were fed either on a standard, a homocystine 1.7% or a methionine 2.4%-rich diet for 5 months. P-tau was tested in 21 frontal cortex tissue slices using immuno-fluorescence. Concentrations of pNF-H and the activity and level of PP2A were measured in brain extracts. Concentrations of homocysteine, SAM and SAH strongly increased in plasma of rats on the modified diets. The diets caused lowering of plasma folate and vitamin B12 and a significant increase in P-tau (Ser396) in brain tissues but PP2A activity and level were unchanged. Plasma folate correlated to brain tissue PP2A activity (r=0.28), pNF-H (r=-0.30), and P-tau (Ser396) staining (r=-0.57) all p<0.05. Phosphorylation of brain functional proteins was related to folate. The effect of the diet on P-tau and pNF-H seemed not to be explained by a lower activity or protein level of PP2A. Folate might prove protective against multiple steps in the process of neurodegeneration.     

Why homocysteine-lowering therapy does not have beneficial effects on patients with cardiovascular disease?

Homocysteine is a sulfur-containing amino acid produced during the metabolism of methionine and elevated plasma levels of homocysteine have been linked to an increased risk of atherosclerosis and cardiovascular ischemic events by numerous authors. Several mechanisms by which elevated homocysteine impairs vascular function have been proposed including impairment of endothelial function and at least some of those mechanisms are induced via homocysteine-associated DNA hypomethylation. Oral administration of folic acid and B vitamins, required for remethylation of homocysteine to methionine, decreased plasma total homocysteine levels but clinical trials using folic acid and B vitamins did not confirm that the decreased plasma levels of homocysteine through diet or drugs may be paralleled by a reduction in cardiovascular risk. In our view a plausible explanation for the discordance between the epidemiologic studies and the results of the clinical trials may be related to the homocysteine-associated global DNA hypomethylation which cannot easily be reversed by homocysteine-lowering therapy.     

Homocysteine and Alzheimer's disease: a modifiable risk?

A hypothesis is proposed that reconciles the epidemiological observation of elevated homocysteine in Alzheimer's disease (AD) with clinical features of the disease, particularly evidence of increased oxidative stress. We propose homocysteine is involved in an iron dysregulation/oxidative stress cycle that has a central role in the pathogenesis of AD. The implications of the hypothesis and some strategies for testing it are discussed.     

Methylation demand and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate

S-adenosylmethionine, formed by the adenylation of methionine via S-adenosylmethionine synthase, is the methyl donor in virtually all known biological methylations. These methylation reactions produce a methylated substrate and S-adenosylhomocysteine, which is subsequently metabolized to homocysteine. The methylation of guanidinoacetate to form creatine consumes more methyl groups than all other methylation reactions combined. Therefore, we examined the effects of increased or decreased methyl demand by these physiological substrates on plasma homocysteine by feeding rats guanidinoacetate- or creatine-supplemented diets for 2 wk. Plasma homocysteine was significantly increased (~50%) in rats maintained on guanidinoacetate-supplemented diets, whereas rats maintained on creatine-supplemented diets exhibited a significantly lower (~25%) plasma homocysteine level. Plasma creatine and muscle total creatine were significantly increased in rats fed the creatine-supplemented or guanidinoacetate-supplemented diets. The activity of kidney L-arginine:glycine amidinotransferase, the enzyme catalyzing the synthesis of guanidinoacetate, was significantly decreased in both supplementation groups. To examine the role of the liver in mediating these changes in plasma homocysteine, isolated rat hepatocytes were incubated with methionine in the presence and absence of guanidinoacetate and creatine, and homocysteine export was measured. Homocysteine export was significantly increased in the presence of guanidinoacetate. Creatine, however, was without effect. These results suggest that homocysteine metabolism is sensitive to methylation demand imposed by physiological substrates.     

Plasma homocysteine is regulated by phospholipid methylation

Mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine, a non-protein amino acid, is formed from S-adenosylhomocysteine and partially secreted into plasma. A potential source for homocysteine is methylation of the lipid phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine N-methyltransferase in the liver. We show that mice that lack phosphatidylethanolamine N-methyltransferase have plasma levels of homocysteine that are approximately 50% of those in wild-type mice. Hepatocytes isolated from methyltransferase-deficient mice secrete approximately 50% less homocysteine. Rat hepatoma cells transfected with phosphatidylethanolamine N-methyltransferase secrete more homocysteine than wild-type cells. Thus, phosphatidylethanolamine N-methyltransferase is an important source of plasma homocysteine and a potential therapeutic target for hyperhomocysteinemia.     

Plasma total homocysteine is associated with DNA methylation in patients with schizophrenia

Schizophrenia (SCZ) is a devastating psychiatric disorder with a median lifetime prevalence rate of 0.7–0.8%. Elevated plasma total homocysteine has been suggested as a risk factor for SCZ, and various biological effects of hyperhomocysteinemia have been proposed to be relevant to the pathophysiology of SCZ. As increased attention is paid to aberrant DNA methylation in SCZ, homocysteine is attracting additional interest as a potential key substance. Homocysteine is formed in the methionine cycle, which is involved in one-carbon methyl group-transfer metabolism, and it acts as a methyl donor when it is converted to S-adenosyl-methionine. To date, no studies have examined the relationship between homocysteine and genome-wide DNA methylation in SCZ. We examined the relationship between plasma total homocysteine and DNA methylation patterns in the peripheral leukocytes of patients with SCZ (n = 42) using a quantitative high-resolution DNA methylation array (485,764 CpG sites). Significant homocysteine-related changes in DNA methylation were observed at 1,338 CpG sites that were located across whole gene regions, including promoters, gene bodies and 3′-untranslated regions. Of the 1,338 sites, 758 sites (56.6%) were located in the CpG islands (CGIs) and in the regions flanking CGIs (CGI: 15.8%; CGI shore: 28.2%; CGI shelf: 12.6%), and positive correlations between plasma total homocysteine and DNA methylation were observed predominantly at CpG sites in the CGIs. Our results suggest that homocysteine might play a role in the pathogenesis of SCZ via a molecular mechanism that involves alterations to DNA methylation.     

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.     

Daily variations of homocysteine concentration may influence methylation of DNA in normal healthy individuals

The regulation of genetic expression is tightly controlled and well balanced in the organism by different epigenetic mechanisms such as DNA methylation and histone modifications. DNA methylation occurring after embryogenesis is seen mainly as an irreversible event. Even small changes in genomic DNA methylation might be of biological relevance, and several factors influencing DNA methylation have been identified so far, one being homocysteine. In this study, genomic DNA methylation was analyzed and homocysteine plasma levels were measured over a 24 h period in 30 healthy students (15 males and 15 females) exposed to a standard 24 h regime of daytime activity alternating with nighttime sleep. Plasma homocysteine concentrations were measured using HPLC detection. DNA was extracted from whole EDTA blood, and genomic DNA methylation was assessed by fluorescently labeled cytosine extension assay. Both homocysteine and DNA methylation showed 24 h variation. Homocysteine showed a significant daily rhythm with an evening peak and nocturnal nadir in all subjects (p<0.001). Males showed higher overall homocysteine levels compared to females (p=0.002). Genomic DNA methylation showed a significant rhythm with increased levels at night (p=0.021), which was inverse to plasma homocysteine levels.     

Methylation demand: a key determinant of homocysteine metabolism

Elevated plasma homocysteine is a risk factor for cardiovascular disease and Alzheimer's disease. To understand the factors that determine the plasma homocysteine level it is necessary to appreciate the processes that produce homocysteine and those that remove it. Homocysteine is produced as a result of methylation reactions. Of the many methyltransferases, two are, normally, of the greatest quantitative importance. These are guanidinoacetate methyltransferase (that produces creatine) and phosphatidylethanolamine N-methyltransferase (that produces phosphatidylcholine). In addition, methylation of DOPA in patients with Parkinson's disease leads to increased homocysteine production. Homocysteine is removed either by its irreversible conversion to cysteine (transsulfuration) or by remethylation to methionine. There are two separate remethylation reactions, catalyzed by betaine:homocysteine methyltransferase and methionine synthase, respectively. The reactions that remove homocysteine are very sensitive to B vitamin status as both the transsulfuration enzymes contain pyridoxal phosphate, while methionine synthase contains cobalamin and receives its methyl group from the folic acid one-carbon pool. There are also important genetic influences on homocysteine metabolism.     

Daily Variations of Homocysteine Concentration May Influence Methylation of DNA in Normal Healthy Individuals

The regulation of genetic expression is tightly controlled and well balanced in the organism by different epigenetic mechanisms such as DNA methylation and histone modifications. DNA methylation occurring after embryogenesis is seen mainly as an irreversible event. Even small changes in genomic DNA methylation might be of biological relevance, and several factors influencing DNA methylation have been identified so far, one being homocysteine. In this study, genomic DNA methylation was analyzed and homocysteine plasma levels were measured over a 24 h period in 30 healthy students (15 males and 15 females) exposed to a standard 24 h regime of daytime activity alternating with nighttime sleep. Plasma homocysteine concentrations were measured using HPLC detection. DNA was extracted from whole EDTA blood, and genomic DNA methylation was assessed by fluorescently labeled cytosine extension assay. Both homocysteine and DNA methylation showed 24 h variation. Homocysteine showed a significant daily rhythm with an evening peak and nocturnal nadir in all subjects (p<0.001). Males showed higher overall homocysteine levels compared to females (p=0.002). Genomic DNA methylation showed a significant rhythm with increased levels at night (p=0.021), which was inverse to plasma homocysteine levels.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

Selection of protein phosphatase 2A regulatory subunits is mediated by the C terminus of the catalytic Subunit

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases all composed of a catalytic C, a structural A, and a regulatory B subunit. Assembly of the complex with the appropriate B subunit forms the key to the functional specificity and regulation of PP2A. Emerging evidence suggests a crucial role for methylation and phosphorylation of the PP2A C subunit in this process. In this study, we show that PP2A C subunit methylation was not absolutely required for binding the PR61/B' and PR72/B' subunit families, whereas binding of the PR55/B subunit family was determined by methylation and the nature of the C-terminal amino acid side chain. Moreover mutation of the phosphorylatable Tyr(307) or Thr(304) residues differentially affected binding of distinct B subunit family members. Down-regulation of the PP2A methyltransferase LCMT1 by RNA interference gradually reduced the cellular amount of methylated C subunit and induced a dynamic redistribution of the remaining methylated PP2A(C) between different PP2A trimers consistent with their methylation requirements. Persistent knockdown of LCMT1 eventually resulted in specific degradation of the PR55/B subunit and apoptotic cell death. Together these results establish a crucial foundation for understanding PP2A regulatory subunit selection.     

Structural mechanism of demethylation and inactivation of protein phosphatase 2A

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that plays a role in many biological processes. Reversible carboxyl methylation of the PP2A catalytic subunit is an essential regulatory mechanism for its function. Demethylation and negative regulation of PP2A is mediated by a PP2A-specific methylesterase PME-1, which is conserved from yeast to humans. However, the underlying mechanism of PME-1 function remains enigmatic. Here we report the crystal structures of PME-1 by itself and in complex with a PP2A heterodimeric core enzyme. The structures reveal that PME-1 directly binds to the active site of PP2A and that this interaction results in the activation of PME-1 by rearranging the catalytic triad into an active conformation. Strikingly, these interactions also lead to inactivation of PP2A by evicting the manganese ions that are required for the phosphatase activity of PP2A. These observations identify a dual role of PME-1 that regulates PP2A activation, methylation, and holoenzyme assembly in cells.     

Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase

Cobalamin-dependent methionine synthase (MetH) catalyzes the methylation of homocysteine using methyltetrahydrofolate as the methyl donor. The cobalamin cofactor serves as an intermediate carrier of the methyl group from methyltetrahydrofolate to homocysteine. In the two half-reactions that comprise turnover for MetH, the cobalamin is alternatively methylated by methyltetrahydrofolate and demethylated by homocysteine to form methionine. Upon binding to the protein, the usual dimethylbenzimidazole ligand is replaced by the imidazole side chain of His759 [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science 266, 1669-1674]. Despite the ligand replacement that accompanies binding of cobalamin to the holo-MetH protein, a MetH(2-649) fragment of methionine synthase that contains the regions that bind homocysteine and methyltetrahydrofolate utilizes exogenously supplied cobalamin in methyl transfer reactions akin to those of the catalytic cycle. However, the interactions of MetH(2-649) with endogenous cobalamin are first order in cobalamin, while the half-reactions catalyzed by the holoenzyme are zero order in cobalamin, so rate constants for reactions of bound and exogenous cobalamins cannot be compared. In this paper, we investigate the catalytic rate enhancements generated by binding cobalamin to MetH after dividing the protein in half and reacting MetH(2-649) with a second fragment, MetH(649-1227), that harbors the cobalamin cofactor. The second-order rate constant for demethylation of methylcobalamin by Hcy is elevated 60-fold and that for methylation of cob(I)alamin is elevated 120-fold. Thus, binding of cobalamin to MetH is essential for efficient catalysis.