A Binary Arginine Methylation Switch on Histone H3 Arginine 2 Regulates Its Interaction with WDR5.

Histone H3 arginine 2 (H3R2) is post-translationally modified in three different states by "writers" of the protein arginine methyltransferase (PRMT) family. H3R2 methylarginine isoforms include PRMT5-catalyzed monomethylation (me1) and symmetric dimethylation (me2s) and PRMT6-catalyzed me1 and asymmetric dimethylation (me2a). WD-40 repeat-containing protein 5 (WDR5) is an epigenetic "reader" protein that interacts with H3R2. Previous studies suggested that H3R2me2s specified a high-affinity interaction with WDR5. However, our prior biological data prompted the hypothesis that WDR5 may also interact with H3R2me1. Here, using highly accurate quantitative binding analysis combined with high-resolution crystal structures of WDR5 in complex with unmodified (me0) and me1/me2s l-arginine amino acids and in complex with the H3R2me1 peptide, we provide a rigorous biochemical study and address long-standing discrepancies of this important biological interaction. Despite modest structural differences at the binding interface, our study supports an interaction model regulated by a binary arginine methylation switch: H3R2me2a prevents interaction with WDR5, whereas H3R2me0, -me1, and -me2s are equally permissive.

N-Acetylcysteine Resolves Placental Inflammatory-Vasculopathic Changes in Mice Consuming a High-Fat Diet.

The mechanism by which poor maternal nutrition can affect the long-term health of offspring is poorly understood. In mice, we previously found that maternal high-fat diet (HFD) exposure results in reduced fetal growth regardless of maternal genotype. We tested our hypothesis that maternal HFD-induced inflammation contributes to metabolic disease susceptibility of the offspring via alterations in the placenta. The effect of maternal genotype, diet, and treatment with the anti-inflammatory compound N-acetylcysteine (NAC) on placental morphologic features was investigated. Placentas from wild-type dams maintained on a HFD but not those heterozygous (+/-) for Glut4 (Slc2a4) on the same diet had an increase in decidual inflammation and vasculopathy occurring together. NAC administration resulted in amelioration of HFD-induced decidual vasculopathy independent of offspring genotype and sex. Consistent with these morphologic improvements, placentas from HFD dams treated with NAC had decreased mRNA and immunostaining of IL-1ß and monocyte chemoattractant protein-1, decreased mRNA of inflammatory genes, and increased mRNA of Vegfa. These results strongly suggest consumption of an HFD results in vascular changes in placenta reflected by alterations in expression of pivotal vascular developmental markers and inflammatory genes all of which are ameliorated by NAC. These placental changes play a key role in the increased programed metabolic disease of HFD-exposed offspring.

Boosting NAD+ with a small molecule that activates NAMPT.

Pharmacological strategies that boost intracellular NAD+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+. These effects of SBI-797812 turn NAMPT into a "super catalyst" that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+. Dosing of mice with SBI-797812 elevates liver NAD+. Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects.

Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans.

A hallmark of aging is a decline in metabolic homeostasis, which is attenuated by dietary restriction (DR). However, the interaction of aging and DR with the metabolome is not well understood. We report that DR is a stronger modulator of the rat metabolome than age in plasma and tissues. A comparative metabolomic screen in rodents and humans identified circulating sarcosine as being similarly reduced with aging and increased by DR, while sarcosine is also elevated in long-lived Ames dwarf mice. Pathway analysis in aged sarcosine-replete rats identify this biogenic amine as an integral node in the metabolome network. Finally, we show that sarcosine can activate autophagy in cultured cells and enhances autophagic flux in vivo, suggesting a potential role in autophagy induction by DR. Thus, these data identify circulating sarcosine as a biomarker of aging and DR in mammalians and may contribute to age-related alterations in the metabolome and in proteostasis.

A simplified characterization of S-adenosyl-l-methionine-consuming enzymes with 1-Step EZ-MTase: a universal and straightforward coupled-assay for in vitro and in vivo setting.

Methyltransferases use S-adenosyl-l-methionine (SAM) to deposit methyl marks. Many of these epigenetic 'writers' are associated with gene regulation. As cancer etiology is highly correlated with misregulated methylation patterns, methyltransferases are emerging therapeutic targets. Successful assignment of methyltransferases' roles within intricate biological networks relies on (1) the access to enzyme mechanistic insights and (2) the efficient screening of chemical probes against these targets. To characterize methyltransferases in vitro and in vivo, we report a highly-sensitive one-step deaminase-linked continuous assay where the S-adenosyl-l-homocysteine (SAH) enzyme-product is rapidly and quantitatively catabolized to S-inosyl-l-homocysteine (SIH). To highlight the broad capabilities of this assay, we established enzymatic characteristics of two protein arginine methyltransferases (PRMT5 and PRMT7), a histone-lysine N-methyltransferase (DIM-5) and a sarcosine/dimethylglycine N-methyltransferase (SDMT). Since the coupling deaminase TM0936 displays robust activity over a broad pH-range we determined the pH dependence of SDMT reaction rates. TM0936 reactions are monitored at 263 nm, so a drawback may arise when methyl acceptor substrates absorb within this UV-range. To overcome this limitation, we used an isosteric fluorescent SAM-analog: S-8-aza-adenosyl-l-methionine. Most enzymes tolerated this probe and sustained methyltransfers were efficiently monitored through loss of fluorescence at 360 nm. Unlike discontinuous radioactive- and antibody-based assays, our assay provides a simple, versatile and affordable approach towards the characterization of methyltransferases. Supported by three logs of linear dynamic range, the 1-Step EZ-MTase can detect methylation rates as low as 2 µM h-1, thus making it possible to quantify low nanomolar concentrations of glycine N-methyltransferase within crude biological samples. With Z'-factors above 0.75, this assay is well suited to high-throughput screening and may promote the identification of novel therapeutics.

Active site and remote contributions to catalysis in methylthioadenosine nucleosidases.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition.

Nucleoplasmin (Npm) is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs) specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg.

Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.

Transition-state analysis of 2-O-acetyl-ADP-ribose hydrolysis by human macrodomain 1.

Macrodomains, including the human macrodomain 1 (MacroD1), are erasers of the post-translational modification of monoadenosinediphospho-ribosylation and hydrolytically deacetylate the sirtuin product O-acetyl-ADP-ribose (OAADPr). OAADPr has been reported to play a role in cell signaling based on oocyte microinjection studies, and macrodomains affect an array of cell processes including transcription and response to DNA damage. Here, we investigate human MacroD1 by transition-state (TS) analysis based on kinetic isotope effects (KIEs) from isotopically labeled OAADPr substrates. Competitive radiolabeled-isotope effects and mass spectrometry were used to obtain KIE data to yield intrinsic KIE values. Intrinsic KIEs were matched to a quantum chemical structure of the TS that includes the active site residues Asp184 and Asn174 and a structural water molecule. Transition-state analysis supports a concerted mechanism with an early TS involving simultaneous nucleophilic water attack and leaving group bond cleavage where the breaking C-O ester bond=1.60 Å and the C-O bond to the attacking water nucleophile=2.30 Å. The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry.

Potent inhibition of the C-P lyase nucleosidase PhnI by Immucillin-A triphosphate.

The C-P lyase complex in bacteria catalyzes the transformation of phosphonates to orthophosphate under conditions of phosphate starvation. The first committed step in the C-P lyase-catalyzed reaction is the displacement of adenine from MgATP by phosphonate substrates, yielding ribose-1-phosphonate-5-triphosphate. In the C-P lyase complex, this reaction is catalyzed by the nucleosidase PhnI and modulated by the addition of PhnG, PhnH, and PhnL. Here we describe the synthesis of Immucillin-A triphosphate, a mimic of the transition state structure for the nucleosidase reaction catalyzed by PhnI. This compound inhibits PhnI with a dissociation constant of 20 nM at pH 7.5.

Recycling nicotinamide. The transition-state structure of human nicotinamide phosphoribosyltransferase.

Human nicotinamide phosphoribosyltransferase (NAMPT) replenishes the NAD pool and controls the activities of sirtuins, mono- and poly-(ADP-ribose) polymerases, and NAD nucleosidase. The nature of the enzymatic transition-state (TS) is central to understanding the function of NAMPT. We determined the TS structure for pyrophosphorolysis of nicotinamide mononucleotide (NMN) from kinetic isotope effects (KIEs). With the natural substrates, NMN and pyrophosphate (PPi), the intrinsic KIEs of [1'-(14)C], [1-(15)N], [1'-(3)H], and [2'-(3)H] are 1.047, 1.029, 1.154, and 1.093, respectively. A unique quantum computational approach was used for TS analysis that included structural elements of the catalytic site. Without constraints (e.g., imposed torsion angles), the theoretical and experimental data are in good agreement. The quantum-mechanical calculations incorporated a crucial catalytic site residue (D313), two magnesium atoms, and coordinated water molecules. The TS model predicts primary (14)C, α-secondary (3)H, ß-secondary (3)H, and primary (15)N KIEs close to the experimental values. The analysis reveals significant ribocation character at the TS. The attacking PPi nucleophile is weakly interacting (r(C-O) = 2.60 Å), and the N-ribosidic C1'-N bond is highly elongated at the TS (r(C-N) = 2.35 Å), consistent with an A(N)D(N) mechanism. Together with the crystal structure of the NMN·PPi·Mg2·enzyme complex, the reaction coordinate is defined. The enzyme holds the nucleophile and leaving group in relatively fixed positions to create a reaction coordinate with C1'-anomeric migration from NAM to the PPi. The TS is reached by a 0.85 Å migration of C1'.

Exploring inhibition of Pdx1, a component of the PLP synthase complex of the human malaria parasite Plasmodium falciparum.

Malaria tropica is a devastating infectious disease caused by Plasmodium falciparum. This parasite synthesizes vitamin B6 de novo via the PLP (pyridoxal 5'-phosphate) synthase enzymatic complex consisting of PfPdx1 and PfPdx2 proteins. Biosynthesis of PLP is largely performed by PfPdx1, ammonia provided by PfPdx2 subunits is condensed together with R5P (D-ribose 5-phosphate) and G3P (DL-glyceraldehyde 3-phosphate). PfPdx1 accommodates both the R5P and G3P substrates and intricately co-ordinates the reaction mechanism, which is composed of a series of imine bond formations, leading to the production of PLP. We demonstrate that E4P (D-erythrose 4-phosphate) inhibits PfPdx1 in a dose-dependent manner. We propose that the acyclic phospho-sugar E4P, with a C1 aldehyde group similar to acyclic R5P, could interfere with R5P imine bond formations in the PfPdx1 reaction mechanism. Molecular docking and subsequent screening identified the E4P hydrazide analogue 4PEHz (4-phospho-D-erythronhydrazide), which selectively inhibited PfPdx1 with an IC50 of 43 µM. PfPdx1 contained in the heteromeric PLP synthase complex was shown to be more sensitive to 4PEHz and was inhibited with an IC50 of 16 µM. Moreover, the compound had an IC50 value of 10 µM against cultured P. falciparum intraerythrocytic parasites. To analyse further the selectivity of 4PEHz, transgenic cell lines overexpressing PfPdx1 and PfPdx2 showed that additional copies of the protein complex conferred protection against 4PEHz, indicating that the PLP synthase is directly affected by 4PEHz in vivo. These PfPdx1 inhibitors represent novel lead scaffolds which are capable of targeting PLP biosynthesis, and we propose this as a viable strategy for the development of new therapeutics against malaria.

Femtomolar inhibitors bind to 5'-methylthioadenosine nucleosidases with favorable enthalpy and entropy.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolytic cleavage of adenine from methylthioadenosine (MTA). Inhibitor design and synthesis informed by transition state analysis have developed femtomolar inhibitors for MTANs, among the most powerful known noncovalent enzyme inhibitors. Thermodynamic analyses of the inhibitor binding reveals a combination of highly favorable contributions from enthalpic (-24.7 to -4.0 kcal mol(-1)) and entropic (-10.0 to 6.4 kcal mol(-1)) interactions. Inhibitor binding to similar MTANs from different bacterial species gave distinct energetic contributions from similar catalytic sites. Thus, binding of four transition state analogues to EcMTAN and SeMTAN is driven primarily by enthalpy, while binding to VcMTAN is driven primarily by entropy. Human MTA phosphorylase (hMTAP) has a transition state structure closely related to that of the bacterial MTANs, and it binds tightly to some of the same transition state analogues. However, the thermodynamic signature of binding of an inhibitor to hMTAP differs completely from that with MTANs. We conclude that factors other than first-sphere catalytic residue contacts contribute to binding of inhibitors because the thermodynamic signature differs between bacterial species of the same enzyme.

Luciferase-based assay for adenosine: application to S-adenosyl-L-homocysteine hydrolase.

S-Adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (K(m), k(cat)) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10(-7) unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.

A high-affinity adenosine kinase from Anopheles gambiae.

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K(m) = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap(4)A (2.0 Å resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg(2+) ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer α/ß/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT.

Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD(+)) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF(3)(-) as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.

Weak coupling of ATP hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyltransferase.

Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PP i) from nicotinamide (NAM) and alpha- d-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at product:substrate ratios thermodynamically forbidden in the absence of ATP. With ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases ( K m (NAM) from 855 to 5 nM), and the K eq shifts -2.1 kcal/mol toward NMN formation. ADP-ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalytic efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a nonoptimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD (+)). NMN synthesis by NAMPT is powerfully inhibited by both NAD (+) ( K i = 0.14 muM) and NADH ( K i = 0.22 muM), an apparent regulatory feedback mechanism.