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Biochemical Inhibition of the Acetyltransferases ATase1 and ATase2 Reduces β-Secretase (BACE1) Levels and Aβ Generation*

  • Yun Ding
    Footnotes
    Affiliations
    Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53705

    Neuroscience Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Mi Hee Ko
    Footnotes
    Affiliations
    Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Mariana Pehar
    Affiliations
    Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Frank Kotch
    Footnotes
    Affiliations
    Small Molecule Screening Facility, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Noel R. Peters
    Affiliations
    Small Molecule Screening Facility, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Yun Luo
    Affiliations
    Small Molecule Screening Facility, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Shahriar M. Salamat
    Affiliations
    Department of Pathology, University of Wisconsin-Madison, Madison, Wisconsin 53705
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  • Luigi Puglielli
    Correspondence
    To whom correspondence should be addressed: Department of Medicine, University of Wisconsin-Madison, VAH-GRECC, 2500 Overlook Terrace, Madison, WI 53705. Tel.: 608-256-1901 (ext. 11569); Fax: 608-280-7291
    Affiliations
    Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53705

    Neuroscience Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705

    Geriatric Research Education Clinical Center, VA Medical Center, Madison, Wisconsin 53705
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  • Author Footnotes
    * This research was supported, in whole or in part, by National Institutes of Health (NIH)/NIA Grants AG028569 and AG033514, Department of Veterans Affairs, Rotary Coins for Alzheimer Research Trust Fund, and UW Institute for Clinical and Translational Research (funded through an NCRR/NIH Clinical and Translational Science Award, 1UL1RR025011).
    This article contains supplemental Tables SI–SIII, Figs. S1–S8, and experimental methods.
    1 Both authors contributed equally to this work.
    2 Present address: Korea National Institute of Health, Chungcheongbuk-do, South Korea.
    3 Present address: Pfizer Worldwide Research and Development, Pearl River, NY.
Open AccessPublished:January 20, 2012DOI:https://doi.org/10.1074/jbc.M111.310136
      The cellular levels of β-site APP cleaving enzyme 1 (BACE1), the rate-limiting enzyme for the generation of the Alzheimer disease (AD) amyloid β-peptide (Aβ), are tightly regulated by two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2. Here we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of first and second generation compounds that inhibit ATase1/ATase2 and down-regulate the expression levels as well as activity of BACE1. The mechanism of action involves competitive and non-competitive inhibition as well as generation of unstable intermediates of the ATases that undergo degradation.

      Introduction

      The membrane protein β-site APP-cleaving enzyme 1 (BACE1)
      The abbreviations used are: BACE1
      β-site APP-cleaving enzyme 1
      αsAPP
      α-secreted APP
      amyloid β-peptide
      AD
      Alzheimer disease
      AICD
      APP intracellular domain
      APP
      amyloid precursor protein
      ATase
      acetyl-CoA lysine acetyltransferase
      βsAPP
      β-secreted APP
      ER
      endoplasmic reticulum
      ERGIC
      ER Golgi intermediate compartment.
      is responsible for the β cleavage of the amyloid precursor protein (APP). The cleavage, which has been linked to the pathogenesis of Alzheimer disease (AD), results in the generation of a small APP fragment (commonly referred to as C99) acting as the immediate substrate for γ secretase (
      • Puglielli L.
      Aging of the brain, neurotrophin signaling, and Alzheimer disease: is IGF1-R the common culprit?.
      ). The sequential β/γ processing of APP results into two small fragments, the amyloid β-peptide (Aβ), and the APP intracellular domain (AICD). Both have neurotoxic properties and both have been linked to the pathogenesis of AD (
      • Lambert M.P.
      • Barlow A.K.
      • Chromy B.A.
      • Edwards C.
      • Freed R.
      • Liosatos M.
      • Morgan T.E.
      • Rozovsky I.
      • Trommer B.
      • Viola K.L.
      • Wals P.
      • Zhang C.
      • Finch C.E.
      • Krafft G.A.
      • Klein W.L.
      Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins.
      ,
      • Lansbury Jr., P.T.
      Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis and disease.
      ,
      • Klein W.L.
      • Krafft G.A.
      • Finch C.E.
      Targeting small Aβ oligomers: the solution to an Alzheimer disease conundrum?.
      ,
      • Haass C.
      • Steiner H.
      Protofibrils, the unifying toxic molecule of neurodegenerative disorders?.
      ,
      • Cleary J.P.
      • Walsh D.M.
      • Hofmeister J.J.
      • Shankar G.M.
      • Kuskowski M.A.
      • Selkoe D.J.
      • Ashe K.H.
      Natural oligomers of the amyloid-β protein specifically disrupt cognitive function.
      ,
      • Puzzo D.
      • Privitera L.
      • Leznik E.
      • Fà M.
      • Staniszewski A.
      • Palmeri A.
      • Arancio O.
      Picomolar amyloid-β positively modulates synaptic plasticity and memory in hippocampus.
      ,
      • Giliberto L.
      • Zhou D.
      • Weldon R.
      • Tamagno E.
      • De Luca P.
      • Tabaton M.
      • D'Adamio L.
      Evidence that the Amyloid β Precursor Protein-intracellular domain lowers the stress threshold of neurons and has a “regulated” transcriptional role.
      ,
      • Ghosal K.
      • Vogt D.L.
      • Liang M.
      • Shen Y.
      • Lamb B.T.
      • Pimplikar S.W.
      Alzheimer disease-like pathological features in transgenic mice expressing the APP intracellular domain.
      ). Importantly, BACE1 acts as the rate-limiting enzyme. As a result, genetic disruption of BACE1 in the mouse abolishes both β and γ cleavage of APP and prevents AD neuropathology (
      • Cai H.
      • Wang Y.
      • McCarthy D.
      • Wen H.
      • Borchelt D.R.
      • Price D.L.
      • Wong P.C.
      BACE1 is the major β-secretase for generation of Aβ peptides by neurons.
      ,
      • Luo Y.
      • Bolon B.
      • Kahn S.
      • Bennett B.D.
      • Babu-Khan S.
      • Denis P.
      • Fan W.
      • Kha H.
      • Zhang J.
      • Gong Y.
      • Martin L.
      • Louis J.C.
      • Yan Q.
      • Richards W.G.
      • Citron M.
      • Vassar R.
      Mice deficient in BACE1, the Alzheimer β-secretase, have normal phenotype and abolished β-amyloid generation.
      ). Therefore, mechanisms that regulate levels and/or activity of BACE1 could serve for therapeutic purposes. Unfortunately, biochemical design of BACE1 inhibitors has proven to be challenging due to the rather large size of the catalytic pocket of the enzyme (
      • Gravitz L.
      Drugs: a tangled web of targets.
      ). Therefore, approaches that affect expression levels rather than catalytic activity of BACE1 are being actively sought.
      We recently reported that nascent BACE1 is transiently acetylated in the lumen of the endoplasmic reticulum (ER) (
      • Costantini C.
      • Ko M.H.
      • Jonas M.C.
      • Puglielli L.
      A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1.
      ) by two ER-based acetyl-CoA:lysine acetyltransferases, which we named ATase1 (also known as camello-like 2 and N-acetyltransferase 8B) and ATase2 (also known as camello-like 1 and N-acetyltransferase 8) (
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ). The Nϵ-lysine acetylation regulates the ability of nascent BACE1 to complete maturation. In fact, the acetylated intermediates of the nascent protein are able to reach the Golgi apparatus and complete maturation while the non-acetylated intermediates are retained and degraded in the ER Golgi intermediate compartment (ERGIC) (
      • Costantini C.
      • Ko M.H.
      • Jonas M.C.
      • Puglielli L.
      A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1.
      ,
      • Jonas M.C.
      • Costantini C.
      • Puglielli L.
      PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1.
      ). Ex vivo studies show that the levels of BACE1 are tightly regulated by the ATases. Specifically, up-regulation of ATase1 and ATase2 increases the levels of BACE1 and the generation of Aβ while siRNA-mediated down-regulation of either transferase achieves the opposite effects (
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ).
      Here, we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of novel biochemical inhibitors of ATase1 and ATase2 that significantly reduce the levels of BACE1 and the generation of Aβ in cellular systems. The biochemical properties of first and second generation compounds as well as mechanisms of inhibition are also described.

      EXPERIMENTAL PROCEDURES

      The complete description of the compounds used in this study can be found under supplemental experimental materials.

      Antibodies

      The following antibodies were used in this study: anti-acetylated lysine (ab409; Abcam); anti-BACE1 (ab2077; Abcam); anti-Myc (sc-40; Santa Cruz Biotechnology); anti-ATases/NAT8 (AP4957c; Abgent); anti-actin (A1978; Sigma); anti-C99 (M066-3; MBL); anti-acetylated H3 (06-599, Millipore); anti-acetylated H4 (06-866, Millipore); anti-αPCNA (AP2835b, Abgent).

      Western Blot Analysis

      Western blotting was performed on a 4–12% Bis-Tris SDS-PAGE system (NuPAGE; Invitrogen) as described previously (
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ,
      • Jonas M.C.
      • Costantini C.
      • Puglielli L.
      PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1.
      ,
      • Costantini C.
      • Scrable H.
      • Puglielli L.
      An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation.
      ,
      • Ko M.H.
      • Puglielli L.
      The sterol carrier protein SCP-x/pro-SCP-2 gene has transcriptional activity and regulates the Alzheimer disease γ-secretase.
      ,
      • Jonas M.C.
      • Pehar M.
      • Puglielli L.
      AT-1 is the ER membrane acetyl-CoA transporter and is essential for cell viability.
      ,
      • Pehar M.
      • O'Riordan K.J.
      • Burns-Cusato M.
      • Andrzejewski M.E.
      • del Alcazar C.G.
      • Burger C.
      • Scrable H.
      • Puglielli L.
      Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death.
      ). Samples were imaged with classical chemiluminescence or with the LiCor Odyssey Infrared Imaging System (LI-COR Biosciences). For chemiluminescent detection, HRP-conjugated anti-mouse or anti-rabbit secondary antibodies were used at 1:6000 dilution in 3% BSA/TBST (GE Healthcare). Detection was performed with either Lumiglo (KPL) or ECL Plus (GE Healthcare). For infrared imaging, goat anti-rabbit Alexa Fluor 680- or anti-mouse Alexa Fluor 800-conjugated secondary antibodies were used. For quantification, values were normalized to the appropriate loading control (shown in the figures).

      Cell Cultures and Animals

      Immortalized cell lines (CHO, H4, SH-SY5Y, SHEP, PC-12) were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Mediatech) as described before (
      • Costantini C.
      • Ko M.H.
      • Jonas M.C.
      • Puglielli L.
      A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1.
      ,
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ,
      • Costantini C.
      • Scrable H.
      • Puglielli L.
      An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation.
      ). Mouse primary neurons were prepared as described (
      • Costantini C.
      • Scrable H.
      • Puglielli L.
      An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation.
      ) and plated on poly-(l-lysine)-coated 6-well plates (Becton Dickinson Labware) for 2 h. Neurons were then changed to Neurobasal medium containing 2% B27 supplement (Invitrogen) in the absence of serum or antibiotics. Cultures grown in serum-free media yielded 99.5% neurons and 0.5% glia. Microscopically, glial cells were not apparent in cultures at the time they were used for experimental analysis.
      Non-transgenic C57B6/6J and p44+/+ transgenic mice were euthanized according to the NIH Guide for the Care and Use of Laboratory Animals. For Western blot analysis, brains were immediately removed for the isolation of neocortex and hippocampus. Tissue was processed for protein extraction in GTIP buffer (100 mm Tris, pH 7.6, 20 mm EDTA, 1.5 m NaCl) supplemented with 1% Triton X-100 (Roche), 0.25% Nonidet P-40 (Roche), and a complete protease inhibitors mixture (Roche), as described before (
      • Jonas M.C.
      • Costantini C.
      • Puglielli L.
      PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1.
      ,
      • Costantini C.
      • Scrable H.
      • Puglielli L.
      An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation.
      ,
      • Pehar M.
      • O'Riordan K.J.
      • Burns-Cusato M.
      • Andrzejewski M.E.
      • del Alcazar C.G.
      • Burger C.
      • Scrable H.
      • Puglielli L.
      Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death.
      ). For histology and immunostaining, brains were immediately processed as described below.
      All animal experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison.

      Human Brain Tissue

      Brain tissue from late-onset AD patients and age-matched controls was kindly provided by the Brain Bank of the Neuropathology Core of the Wisconsin Alzheimer Disease Research Center (established by grant P50-AG033514 from NIH/NIA). The use of human brain tissue was approved by the University of Wisconsin-Madison Institutional Review Board in accordance with US federal regulations (as defined under 45 CFR 46.102(f)). Pathological grading of AD patients is found in supplemental Table SI.

      Histology and Immunostaining

      Mouse brains were removed, fixed overnight in 10% neutral buffered formalin, and paraffin-embedded using standard techniques. Coronal tissue sections (5 μm) were prepared using a microtome. Paraffin-embedded tissue sections from AD patients and age-matched controls were obtained from the Wisconsin Alzheimer Disease Research Center, as described above. Following standard deparaffinization and rehydration, the tissue sections were processed for immunofluorescence. Antigen retrieval was performed in 100 mm citrate buffer (pH 6) heated in an autoclave. After washing with PBS, tissue sections were permeabilized with 0.1% Triton X-100 in PBS and blocked for 2 h with 10% goat serum, 2% bovine serum albumin, and 0.1% Triton X-100 in PBS. Sections were then incubated with primary antibodies (diluted in blocking solution) overnight at 4°C. The following primary antibodies were used: mouse anti-NeuN (clone A60; 1:100; Chemicon-Millipore) and rabbit anti-ATases/NAT8 (1:50; Abgent). After washing with PBS, they received Alexa 594-goat anti-mouse (5 μg/ml; Molecular Probes-Invitrogen) and biotin-conjugated goat anti-rabbit (5 μg/ml; Molecular Probes-Invitrogen) for 1 h at room temperature; followed by Alexa 488-conjugated streptavidin (5 μg/ml; Molecular Probes-Invitrogen) for 1 h at room temperature. Controls were performed by omitting the primary antibody. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes-Invitrogen, Carlsbad, CA). Slides were mounted using Gel/Mount aqueous mounting medium (Electron Microscopy Sciences, Hatfield, PA) and imaged on a Zeiss Axiovert 200 inverted fluorescent microscope.

      Preparation of Cytosolic and Nuclear Fractions

      Cytosolic and nuclear extracts were prepared as described before (
      • Ko M.H.
      • Puglielli L.
      The sterol carrier protein SCP-x/pro-SCP-2 gene has transcriptional activity and regulates the Alzheimer disease γ-secretase.
      ). For cytosolic extracts, cells were homogenized in homogenization buffer containing 25 mm Tris-HCl, pH 7.4, 0.5 mm EDTA, 0.5 mm EGTA, and a protease inhibitor mixture. The homogenates were centrifuged at 14,000 × g for 15 min, and supernatants were collected as cytosolic proteins.
      For nuclear extracts cells were scraped into ice-cold phosphate-buffered saline and collected by centrifugation. The cell pellets were suspended in 3 volumes of lysis buffer (20 mm Hepes, pH 7.9, 10 mm KCl, 1 mm EDTA, pH 8.0, 0.2% Nonidet P-40, 10% glycerol, and a protease inhibitor mixture) followed by incubation on ice for 10 min. Cell suspensions were gently pipetted up and down; the lysates were then centrifuged at 14,000 × g for 5 min at 4 °C to obtain nuclear pellets. Nuclear pellets were washed twice with cell lysis buffer (lacking Nonidet P-40 and protease inhibitor mixture) and then resuspended in 2 volumes of nuclear extract buffer (20 mm Hepes, pH 7.9, 10 mm KCl, 1 mm EDTA, pH 8.0, 420 mm NaCl, 20% glycerol, and a protease inhibitor mixture). The nuclei were extracted by incubation at 4 °C for 30 min with gentle agitation followed by centrifugation at 14,000 × g at 4 °C for 5 min; the resultant supernatant fraction was used as a nuclear extract.

      AcetylCoA:Lysine Acetyltransferase Assay

      The acetyl:CoA lysine acetyltransferase activity of ATase1 and ATase2 was assayed as described before (
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ). Briefly, we adapted a commercially available fluorescent kit (cat. no. 10006515, Cayman Chemicals) as following: as source of the enzymatic activity, we used affinity purified ATase1-myc and ATase2-myc at the final concentration of 300 ng/μl; as donor of the acetyl group, we used acetyl-CoA at the final concentration of 12.5 μm. ATase1 and ATase2 were purified with the ProFound c-myc-Tag IP/Co-IP kit (Pierce) as suggested by the manufacturer and already described in our previous work (
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ). The acetyltransferase assay was performed as recommended by the manufacturer.
      For kinetic analysis of ATase1 and ATase2 inhibition, the assays were performed in the presence of the indicated concentrations of compound 9, 19, and 9.I. The concentration of acetyl-CoA was varied from 0.5 to 15 μm. Values were plotted as a Lineweaver-Burk plot using Graphpad Prism software.

      Real Time PCR

      Real-time PCR was carried out as described before (
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ). The cycling parameters were: 95 °C, 10 s; 55 °C, 60 °C, or 61 °C, 10 s; 72 °C, 15 s, for a maximum of 40 cycles. Controls without reverse transcription were included in each assay. PCR primers specific to each gene were: ATase1/NAT8B-human, 5′-AGACGGGCCAGTCCTTCTTC-3′ and 5′-GCACAGAAAGGTCATTGCAGTCAG-3′; ATase2/NAT8-human, 5′-TCCTTGCCAAAAAACCCTGG-3′ and 5′-ATGCCCACCACCTTCTCTTCA-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-human, 5′-TTTGTCAAGCTCATTTCCTGGTA-3′ and 5′-TTCAAGGGGTCTACATGGCAACTG-3′. ATase1 and ATase2 expression levels were normalized against GAPDH levels and are expressed as percent of control.

      Aβ Determination

      For Aβ determinations in the conditioned media, H4 cells were plated in 6-well Petri dishes. When 80–90% confluent, cells were washed in PBS and incubated in 1 ml of fresh medium for 48 h in the presence or absence of inhibitors. Secreted Aβ was determined by standard sandwich ELISA as described before (
      • Costantini C.
      • Ko M.H.
      • Jonas M.C.
      • Puglielli L.
      A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1.
      ,
      • Ko M.H.
      • Puglielli L.
      Two endoplasmic reticulum (ER)/ER Golgi intermediate compartment-based lysine acetyltransferases post-translationally regulate BACE1 levels.
      ,
      • Jonas M.C.
      • Costantini C.
      • Puglielli L.
      PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1.
      ,
      • Costantini C.
      • Scrable H.
      • Puglielli L.
      An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation.
      ,
      • Jonas M.C.
      • Pehar M.
      • Puglielli L.
      AT-1 is the ER membrane acetyl-CoA transporter and is essential for cell viability.
      ,
      • Pehar M.
      • O'Riordan K.J.
      • Burns-Cusato M.
      • Andrzejewski M.E.
      • del Alcazar C.G.
      • Burger C.
      • Scrable H.
      • Puglielli L.
      Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death.
      ). For each sample, the levels of Aβ40, Aβ42, and Aβtotal were quantified as triplicate based upon standard curves run (on every ELISA plate) and then expressed as pmol Aβ/mg of protein. Aβ42 was constantly found to be 20–25% of total Aβ values.

      Circular Dichroism

      The experiments with circular dichroism (CD) were carried out at the Biophysics Instrumentation Facility (Department of Biochemistry, University of Wisconsin-Madison), which was established by funding from NSF (BIR-9512577), NIH (S10 RR13790) and the University of Wisconsin. Briefly, ATase1 and ATase2 were purified as for the acetyl:CoA lysine acetyltransferase assay and then incubated with 10 μm compound 9 or 19. The difference in the absorbances of left- and right-handed circularly polarized light impinging on the solution was measured with a 202SF CD Spectrophotometer (Aviv Biomedical) in PBS and at room temperature. Appropriate controls included PBS alone and compound 9 or 19 in the absence of the ATases.

      Statistical Analysis

      Data were analyzed by ANOVA and Student's t test comparison, using GraphPad InStat3 software. Statistical significance was reached at p ≤ 0.05.

      Acknowledgments

      We thank Dr. James Malter for critical reading of an early version of this manuscript. The human brain tissue was provided by the Neuropathology Core of the Wisconsin ADRC established by NIH/NIA (P50-AG033514). We are grateful to Dr. Darrell R. McCaslin for help with circular dichroism.

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