Reduced Expression of the PP2A Methylesterase, PME-1, or the PP2A Methyltransferase, LCMT-1, Alters Sensitivity to Beta-Amyloid-Induced Cognitive and Electrophysiological Impairments in Mice.

Beta-amyloid (Aß) is thought to play a critical role in Alzheimer's disease (AD), and application of soluble oligomeric forms of Aß produces AD-like impairments in cognition and synaptic plasticity in experimental systems. We found previously that transgenic overexpression of the PP2A methylesterase, PME-1, or the PP2A methyltransferase, LCMT-1, altered the sensitivity of mice to Aß-induced impairments, suggesting that PME-1 inhibition may be an effective approach for preventing or treating these impairments. To explore this possibility, we examined the behavioral and electrophysiological effects of acutely applied synthetic Aß oligomers in male and female mice heterozygous for either a PME-1 KO or an LCMT-1 gene-trap mutation. We found that heterozygous PME-1 KO mice were resistant to Aß-induced impairments in cognition and synaptic plasticity, whereas LCMT-1 gene-trap mice showed increased sensitivity to Aß-induced impairments. The heterozygous PME-1 KO mice produced normal levels of endogenous Aß and exhibited normal electrophysiological responses to picomolar concentrations of Aß, suggesting that reduced PME-1 expression in these animals protects against Aß-induced impairments without impacting normal physiological Aß functions. Together, these data provide additional support for roles for PME-1 and LCMT-1 in regulating sensitivity to Aß-induced impairments, and suggest that inhibition of PME-1 may constitute a viable therapeutic approach for selectively protecting against the pathologic actions of Aß in AD.SIGNIFICANCE STATEMENT Elevated levels of ß-amyloid (Aß) in the brain are thought to contribute to the cognitive impairments observed in Alzheimer's disease patients. Here we show that genetically reducing endogenous levels of the PP2A methylesterase, PME-1, prevents the cognitive and electrophysiological impairments caused by acute exposure to pathologic concentrations of Aß without impairing normal physiological Aß function or endogenous Aß production. Conversely, reducing endogenous levels of the PP2A methyltransferase, LCMT-1, increases sensitivity to Aß-induced impairments. These data offer additional insights into the molecular factors that control sensitivity to Aß-induced impairments, and suggest that inhibiting PME-1 may constitute a viable therapeutic avenue for preventing Aß-related impairments in Alzheimer's disease.

Methylation and subsequent glycosylation of 7,8-dihydroxyflavone.

An O-methyltransferase SpOMT2884, originating from Streptomyces peucetius ATCC 27952, was cloned, expressed, and applied for the production of target metabolite from Escherichia coli. Biochemical characterization of the 25kDa recombinant protein by in vitro and in vivo experiments showed that SpOMT2884 was an S-adenosyl-l-methionine-dependent O-methyltransferase. SpOMT2884 catalyzed O-methylation of different classes of flavonoids such as flavones (7,8-dihydroxyflavone (7,8-DHF), luteolin), flavonols (quercetin, rutin), flavanone (naringenin), and isoflavonoids (daidzein, formononetin). Biotransformation of 7,8-DHF, a preferred substrate of SpOMT2884, in a grown-induced culture of E. coli BL21 (DE3) harboring the recombinant pET-28a-SpOMT2884 stoichiometrically converted 7,8-DHF into 7-hydroxy-8-methoxyflavone, which was confirmed by liquid chromatography, mass spectrometry and various nuclear magnetic resonance (NMR) spectroscopy analyses. In order to improve the biotransformation substrate, time and media parameters were optimized and the production was scaled up using a 3-L fermentor. The maximum yield of 7-hydroxy-8-methoxyflavone was 192µM (52.57mg/L), representing almost 96% bioconversion within 12h, when 200µM of 7,8-DHF was supplemented in the culture. Further, the 7-hydroxy-8-methoxyflavone was purified in large scale and was used as a substrate separately for in vitro glycosylation to produce glucose, galactose and 2-deoxyglucose conjugated at 7th hydroxyl position of 7-hydroxy-8-methoxyflavone. Biological activity showed that 7-hydroxy-8-methoxyflavone had long term cytoprotective and antioxidant effects compared to 7,8-DHF suggesting that methylation enhances the stability of substrate and glycosylation has proved to increase the water solubility.

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.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.