Zoledronic acid-induced IPP/ApppI production in vivo

Bisphosphonates are currently the most important class of anti-resorptive drugs used for the treatment of diseases involving excess bone resorption. Recently we discovered a new mechanism of action for bisphosphonates. Previously it has been shown that nitrogen-containing bisphosphonates (N-BPs) are not metabolized. However, our studies revealed that N-BPs induce formation of a novel pro-apoptotic ATP analog (ApppI), as a consequence of the inhibition of FPP synthase in the mevalonate pathway, and the subsequent accumulation of isopentenyl pyrophosphate (IPP) in vitro. The primary aim of the current study was to determine whether zoledronic acid (a N-BP) induces IPP/ApppI formation in vivo. Mass spectrometry was used to identify whether in vivo administration of zoledronic acid-induced IPP/ApppI production by mouse peritoneal macrophages or bone marrow cells. IPP/ApppI could be detected in extracts from peritoneal macrophages isolated from zoledronic acid-treated animals. Increasing IPP/ApppI accumulation was determined up to 7 days after drug injection, indicating prolonged FPP synthase inhibition by zoledronic acid. Importantly, this is the first report of in vivo production of ApppI, supporting the biological significance of this molecule.     

Multi-target-directed design,syntheses, and characterization of fluorescent bisphosphonate derivatives as multifunctional enzyme inhibitors in mevalonate pathway

Background

Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important regulatory roles in cholesterol biosynthesis and cell proliferation.

Methods

The following methods were used: cloning, expression and purification of enzymes in mevalonate pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC₅₀ values for above four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluorescence microscopy.

Results and conclusions

We report our multi-target-directed design, syntheses, and characterization of two blue fluorescent bisphosphonate derivatives compounds 15 and 16 as multifunctional enzyme inhibitors in mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC₅₀ values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two compounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that these two compounds could easily get into cancer cells.

General significance

Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibitors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead compounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.     

Syntheses and characterization of non-bisphosphonate quinoline derivatives as new FPPS inhibitors

Background

Farnesyl pyrophosphate synthase (FPPS) is a key regulatory enzyme in the biosynthesis of cholesterol and in the post-translational modification of signaling proteins. It has been reported that non-bisphosphonate FPPS inhibitors targeting its allosteric binding pocket are potentially important for the development of promising anti-cancer drugs.

Methods

The following methods were used: organic syntheses of non-bisphosphonate quinoline derivatives, enzyme inhibition studies, fluorescence titration assays, synergistic effect studies of quinoline derivatives with zoledronate, ITC studies for the binding of FPPS with quinoline derivatives, NMR-based HAP binding assays, molecular modeling studies, fluorescence imaging assay and MTT assays.

Results

We report our syntheses of a series of quinoline derivatives as new FPPS inhibitors possibly targeting the allosteric site of the enzyme. Compound 6b showed potent inhibition to FPPS without significant hydroxyapatite binding affinity. The compound showed synergistic inhibitory effect with active-site inhibitor zoledronate. ITC experiment confirmed the good binding effect of compound 6b to FPPS, and further indicated the binding ratio of 1:1. Molecular modeling studies showed that 6b could possibly bind to the allosteric binding pocket of the enzyme. The fluorescence microscopy indicated that these compounds could get into cancer cells.

Conclusions

Our results showed that quinoline derivative 6b could become a new lead compound for further optimization for cancer treatment.

General significance

The traditional FPPS active-site inhibitors bisphosphonates show poor membrane permeability to tumor cells, due to their strong polarity. The development of new non-bisphosphonate FPPS inhibitors with good cell membrane permeability is potentially important.     

Expression of farnesyl pyrophosphate synthase is increased in diabetic cardiomyopathy

Farnesyl pyrophosphate synthase (FPPS)-catalyzed isoprenoid intermediates are involved in diabetic cardiomyopathy. This study investigated the specific role of FPPS in the development of diabetic cardiomyopathy. We demonstrated that FPPS expression was elevated in both in vivo and in vitro models of diabetic cardiomyopathy. FPPS inhibition decreased the expression of proteins related to cardiac fibrosis and cardiomyocytic hypertrophy, including collagen I, collagen III, connective tissue growth factor, natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Furthermore, FPPS inhibition and knockdown prevented phosphorylated c-Jun N-terminal kinase 1/2 (JNK1/2) activation in vitro. In addition, a JNK1/2 inhibitor downregulated high-glucose-induced responses to diabetic cardiomyopathy. Finally, immunofluorescence revealed that cardiomyocytic size was elevated by high glucose and was decreased by zoledronate, small-interfering farnesyl pyrophosphate synthase (siFPPS), and a JNK1/2 inhibitor. Taken together, our findings indicate that FPPS and JNK1/2 may be part of a signaling pathway that plays an important role in diabetic cardiomyopathy.     

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM⁻¹ s⁻¹), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C_MDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C_MDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.     

Biochemical characterization of a protein tyrosine phosphatase from Trypanosoma cruzi involved in metacyclogenesis and cell invasion

Protein tyrosine phosphatases (PTPs) form a large family of enzymes involved in the regulation of numerous cellular functions in eukaryotes. Several protein tyrosine phosphatases have been recently identified in trypanosomatides. Here we report the purification and biochemical characterization of TcPTP1, a protein tyrosine phosphatase from Trypanosoma cruzi, the causing agent of Chagas’ disease. The enzyme was cloned and expressed recombinantly in Escherichia coli and purified by Ni-affinity chromatography. Biochemical characterization of recombinant TcPTP1 with the PTP pseudo-substrate pNPP allowed the estimation of a Michaelis–Menten constant K_m of 4.5 mM and a k_cat of 2.8 s⁻¹. We were able to demonstrate inhibition of the enzyme by the PTP1b inhibitor BZ3, which on its turn was able to accelerate the differentiation of epimastigotes into metacyclic forms of T. cruzi induced by nutritional stress. Additionally, this compound was able to inhibit by 50% the infectivity of T. cruzi trypomastigotes in a separate cellular assay. In conclusion our results indicate that TcPTP1 is of importance for cellular differentiation and invasivity of this parasite and thus is a valid target for the rational drug design of potential antibiotics directed against T. cruzi.     

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for β-carotene production in recombinant Escherichia coli harboring β-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of β-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and β-carotene synthesis genes produced high amount of β-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains – MG1655, DH5α, S17-1, XL1-Blue and BL21 – the DH5α was found to be the best β-carotene producer. Using glycerol as the carbon source for β-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5α harboring the whole MVA pathway and β-carotene synthesis genes produced β-carotene of 465 mg/L at glycerol concentration of 2% (w/v).     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

Biosynthesis of artemisinin – revisited

Artemisinin is a well-known antimalarial drug isolated from the Artemisia annua plant. The biosynthesis of this well-known molecule has been reinvestigated by using [1-¹³C]acetate, [2-¹³C]acetate, and [1,6-¹³C₂]glucose. The ¹³C peak enrichment in artemisinin was observed in six and nine carbon atoms from [1-¹³C]acetate and [2-¹³C]acetate, respectively. The ¹³C NMR spectra of ¹³C-enriched artemisinin suggested that the mevalonic acid (MVA) pathway is the predominant route to biosynthesis of this sesquiterpene. On the other hand, the peak enrichment of five carbons of ¹³C-artemisinin including carbon atoms originating from methyls of dimethylallyl group of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) was observed from [1,6-¹³C₂]glucose. This suggested that GPP which is supposed to be biosynthesized in plastids travels from plastids to cytosol through the plastidial wall and combines with isopentenyl pyrophosphate (IPP) to form the (E,E)-FPP which finally cyclizes and oxidizes to artemisinin. In this way the DXP pathway also contributes to the biosynthesis of this sesquiterpene.     

Crystal structures of human IPP isomerase: new insights into the catalytic mechanism

Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.     

Translocation of isopentenyl pyrophosphate for Taxol biosynthesis in suspension cultures of Taxus chinensis var. mairei

Two inhibitors of metabolite translocators, sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), respectively, were added into suspension cultures of Taxus chinensis var. mairei at the early and late growth phases to investigate the translocation of isopentenyl pyrophosphate (IPP) from cytoplasm to plastids for Taxol biosynthesis. NaPP and DLG hardly affected cell biomass and viability regardless of the phases of their introduction. The Taxol content varied less when NaPP or DLG was added at day 7 but decreased obviously when NaPP or DLG was introduced at day 14. It is thus inferred that NaPP and DLG inhibited Taxol biosynthesis by blocking IPP translocation at the late growth phase, suggesting that the translocation of IPP be involved only at the late growth phase of Taxus cells.     

Prenyl lipid formation in spinach chloroplasts and in a cell-free system of Synechococcus (Cyanobacteria): polyprenols,chlorophylls, and fatty acid prenyl esters

Isolated chloroplasts from spinach leaf cells, chloroplast subfractions, and a cell-free system of the cyanobacterium Synechococcus CCAP 6312 incorporated [1-¹⁴C]isopentenyl pyrophosphate in high yields into prenyl lipids. Products were polyprenols (C₂₀, C₄₅) chlorophylls, quinoid compounds, and fatty acid prenyl esters; prenyl pyrophosphates occurred in trace amounts, and carotenes were only formed to a limited extent in the Synechococcus system. The formation of fatty acid prenyl esters, which is described here for the first time, was found to occur in two different ways in the chloroplast system; by an acyl-CoA: polyprenol acyltransferase reaction associated with the envelope membranes and by a transesterification reaction from chlorophyll associated with the thylakoids. Endogenous fatty acid prenyl esters made up about 3% by weight of total lipids in spinach chloroplasts and were also found to be natural constituents of the cyanobacterial cells.Abbreviations Chl chlorophyll - Chl_GG chlorophyll a containing a geranylgeranyl side chain - IPP isopentenyl pyrophosphate     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

Phosphoribosyl pyrophosphate and the measurement on inorganic pyrophosphate in plant tissues

This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.Abbreviations EDTA ethylenediaminetetraacetic acid - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate - PPRibP phosphoribosyl pyrophosphate     

Upregulation of endogenous farnesyl diphosphate synthase overcomes the inhibitory effect of bisphosphonate on protein prenylation in Hela cells

Nitrogen-containing bisphosphonates (N-BPs) such as zoledronic acid (ZOL) are the gold standard treatment for diseases of excessive bone resorption. N-BPs inactivate osteoclasts via inhibition of farnesyl diphosphate synthase (FPPS), thereby preventing the prenylation of essential small GTPases. Not all patients respond to N-BP therapy to the same extent, and some patients, for example with tumour-associated bone disease or Paget's disease, appear to develop resistance to N-BPs. The extent to which upregulation of FPPS might contribute to these phenomena is not clear. Using quantitative PCR and western blot analysis we show that levels of FPPS mRNA and protein can be upregulated in HeLa cells by culturing in lipoprotein deficient serum (LDS) or by over-expression of SREBP-1a. Upregulated, endogenous FPPS was predominantly localised to the cytosol and did not co-localise with peroxisomal or mitochondrial markers. Upregulation of endogenous FPPS conferred resistance to the inhibitory effect of low concentrations of ZOL on the prenylation of the small GTPase Rap1a. These observations suggest that an increase in the expression of endogenous FPPS could confer at least partial resistance to the pharmacological effect of N-BP drugs such as ZOL in vivo.     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Enhancing epi‐cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi‐cedrol synthase

Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi‐cedrol synthase (AaECS). The stop codon at the N‐terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐epi‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K_m values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.     

Identification of an artificial peptide motif that binds and stabilizes reduced human DJ-1

Although the precise biochemical function of DJ-1 remains unclear, it has been found to exert cytoprotective activity against oxidative stress. Cys106 is central to this function since it has a distinctly low pK_a rendering it extremely susceptible for oxidation. This characteristic, however, also poses a severe hindrance to obtain reduced DJ-1 for in vitro investigation. We have developed an approach to produce recombinant human DJ-1 in its reduced form as a bona fide basis for exploring the redox capacities of the protein. We solved the crystal structure of this DJ-1 at 1.56 Å resolution, allowing us to capture Cys106 in the reduced state for the first time. The dimeric structure reveals one molecule of DJ-1 in its reduced state while the other exhibits the characteristics of a mono-oxygenated cysteine. Comparison with previous structures indicates the absence of redox dependent global conformational changes in DJ-1. The capture of reduced Cys106 is facilitated by stabilization within the putative active site achieved through a glutamate side chain. This side chain is provided by a crystallographic neighbor as part of a ‘Leu–Glu’ motif, which was added to the C-terminus of DJ-1. In the structure this motif binds DJ-1 in close proximity to Cys106 through extended hydrophilic and hydrophobic interactions depicting a distinct binding pocket, which can serve as a basis for compound development targeting DJ-1.     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.