Tau K18 or full-length tau proteins were either mock acetylated with coenzyme A alone or modified in vitro using acetyl-CoA, butyryl-CoA, or propionyl-CoA (Sigma) in acetylation reaction buffer (50 mM Tris–HCl pH 8.0, 10% glycerol, 1 mM DTT, 100 μM EDTA, 1 mM PMSF, 0.4 mM CoA derivative) at 37°C for the indicated timepoints from 1 day—7 days. Purified recombinant CBP protein (Enzo Life Sciences) (0.5 ug) was incubated in the acetylation reactions, where indicated, leading to maximal tau acetylation. Reaction products were analyzed by resolving proteins on 15% SDS-PAGE gels followed by Coomassie staining or subsequent immunoblotting with the indicated tau antibodies, where indicated. For tau pharmacological inhibition studies, tau proteins were pre-incubated on ice with 100 μM chymostatin, 1 mM N-ethylmaleimide (NEM), 4mM iodoacetamide (IA), 10 μM ALLN, and 10 μM Antipain (Sigma) followed by the above described acetylation reactions. All reactions were performed with a minimum of N = 3 independent experimental replicates. For tau fragmentation studies, error bars represent standard deviation (SD) among replicates and statistical calculations were performed using student t-test with significant p-values ≤ 0.01.
Butyryl coa
Manufactured by Merck Group
Sourced in United States
Butyryl-CoA is a coenzyme A derivative that plays a role in the metabolism of fatty acids. It is an intermediate in the beta-oxidation pathway, where it is further converted to acetyl-CoA for use in the citric acid cycle.
Automatically generated - may contain errors
Lab products found in correlation
Acetyl coa, by Merck Group (9 mentions)
Octanoyl coa, by Merck Group (5 mentions)
Hexanoyl coa, by Merck Group (5 mentions)
Propionyl coa, by Merck Group (4 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Oleoyl coa, by Merck Group (3 mentions)
Antarctic phosphatase, by New England Biolabs (3 mentions)
Probond nickel chelating resin, by Thermo Fisher Scientific (2 mentions)
Kanamycin monosulfate, by GoldBio (2 mentions)
Decanoyl coa, by Merck Group (2 mentions)
Benzoyl coa, by Merck Group (2 mentions)
Xhoi, by New England Biolabs (2 mentions)
Pfuultra high fidelity dna polymerase, by Agilent Technologies (2 mentions)
Malonyl coa, by Merck Group (2 mentions)
Coenzyme a, by Merck Group (2 mentions)
Antipain, by Merck Group (1 mentions)
N acetylserotonin, by Merck Group (1 mentions)
Oligonucleotides, by Eurofins (1 mentions)
Cellytic b reagent, by Merck Group (1 mentions)
Caproyl coa, by Merck Group (1 mentions)
12 protocols using butyryl coa
1
Tau Acetylation Assay Protocol
2
Recombinant Protein Expression and Purification
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The
Ambion RETROscript Kit, ProBond nickel-chelating resin, and MicroPoly(A)
Purist were purchased from Invitrogen. NdeI, XhoI, Antarctic Phosphatase, and T4 DNA ligase were purchased
from New England Biolabs. BL21(DE3) E. coli cells,
XL10 E. coli cells, and the pET-28a(+) vector were purchased from Novagen. Kanamycin
monosulfate and isopropyl β-d -1-thiogalactopyranoside
were purchased from Gold Biotechnology. Oligonucleotides were purchased
from Eurofins MWG Operon, and PfuUltra High-Fidelity DNA polymerase
was purchased from Agilent. Benzoyl-CoA, acetyl-CoA, butyryl-CoA,
hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, oleoyl-CoA, and N-acetylserotonin were purchased from Sigma-Aldrich. All
other reagents were of the highest quality available from either Sigma-Aldrich
or Fisher Scientific.
Ambion RETROscript Kit, ProBond nickel-chelating resin, and MicroPoly(A)
Purist were purchased from Invitrogen. NdeI, XhoI, Antarctic Phosphatase, and T4 DNA ligase were purchased
from New England Biolabs. BL21(DE3) E. coli cells,
XL10 E. coli cells, and the pET-28a(+) vector were purchased from Novagen. Kanamycin
monosulfate and isopropyl β-
were purchased from Gold Biotechnology. Oligonucleotides were purchased
from Eurofins MWG Operon, and PfuUltra High-Fidelity DNA polymerase
was purchased from Agilent. Benzoyl-CoA, acetyl-CoA, butyryl-CoA,
hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, oleoyl-CoA, and N-acetylserotonin were purchased from Sigma-Aldrich. All
other reagents were of the highest quality available from either Sigma-Aldrich
or Fisher Scientific.
3
Enzymatic Assays of Strain CPB6
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Enzymatic assays with crude cell extract were performed to verify the existence of several key reactions and relative enzymes in the chain elongation pathways in strain CPB6. For each assay, cells of 1.5 mL preculture with an OD600 of 0.8 were washed with 1× PBS solution (pH 5.5) for three times, and crude cell extract was prepared from the cells using CelLytic B reagent (Sigma–Aldrich Corporation, St Louis, MO, USA) following the manufacturer’s instruction. The assays were conducted at 35 °C in 1.5-mL sealed centrifuge tubes containing substrates as listed below: caproyl-CoA (Sigma-Aldrich) + acetate, caproyl-CoA + butyrate, and butyryl-CoA (Sigma-Aldrich) + acetate. The final concentration of each substrate was 4.0 mM. Then ATP (Sigma-Aldrich) was added into the reaction with a final concentration of 0.2 mM. Control tests were also conducted at the same time following the same procedure but crude cell extract was replaced with the same amount of 1× PBS solution. All these tests were performed in triplicate in an anaerobic chamber (BactronEZ, Shellab, USA) which was kept under anaerobic condition all the time with mixed gases (CO2: H2: N2 = 5:5:90). Reaction rates for the tests were calculated based on the amount of CA/butyrate generated or the amount of butyrate/acetate consumed within 12 h.
4
Recombinant GLYAT Protein Expression
5
Thioesterase Activity Assay Protocol
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Thioesterase activity was measured by detection of the sulfhydryl group released as a product of the reaction as described previously (Yamada et al., 1999 ▶ ). The reaction mixture consisted of 100 mM phosphate buffer pH 8.0, 0.1 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and the enzyme (0.33 µg ml−1) in a final volume of 100 µl. The absorbance at 412 nm was measured for 20 min and the activity (expressed as moles of acyl-CoA hydrolysed per minute per milligram) was calculated using ∊412 = 13 600 M−1 cm−1. Substrates screened included acetyl-CoA (C2), malonyl-CoA (C3), butyryl-CoA (C4), hexanoyl-CoA (C6), octanoyl-CoA (C8), decanoyl-CoA (C10), lauroyl-CoA (C12), myristoyl-CoA (C14), palmitoyl-CoA (C16), stearoyl-CoA (C18) and arachidoyl-CoA (C20) sourced from Sigma–Aldrich.
6
Histone Acylation Assay with KAT6 Complexes
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The assays were performed as previously reported (18 (link), 21 (link)). Briefly, reconstituted KAT6 complexes were incubated with 0.2 to 1 μg of nucleosomes and 0.1 mM acetyl-CoA (Sigma-Aldrich, A2056), propionyl-CoA (Sigma-Aldrich, P5397), crotonyl-CoA (Sigma-Aldrich, 28007), or butyryl-CoA (Sigma-Aldrich, B1508) at 37°C for 1 hour. HeLa oligonucleosomes were purified as previously described (21 (link)), where recombinant mononucleosomes were purchased from EpiCypher (16-0009). Reactions were terminated by addition of a 3× reducing SDS sample buffer for separation by SDS–polyacrylamide gel electrophoresis (PAGE) (15%) and transferred to 0.22-μm nitrocellulose membrane (Pall Laboratory, 66485) and subsequent immunoblotting analysis to detect histone H3 or its acylation with site-specific antibodies as described below. The KAT6 complexes in the acylation reactions were verified by immunoblotting with anti-FLAG (Sigma-Aldrich, F3165) and anti-HA (BioLegend, 901515) antibodies.
7
Methylmalonic Acidemia Enzymatic Assay
8
Cloning and Expression of Acyl-CoA Thioesterases
9
Kinetic Analysis of Recombinant Acyl-CoA Dehydrogenases
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Recombinant human medium‐chain acyl‐CoA dehydrogenase (MCAD) was expressed and purified as described before,12 , 13 recombinant human long‐chain acyl CoA dehydrogenase (LCAD) as described by Eder et al. 1997 and short‐chain acyl CoA dehydrogenase (SCAD) as described in reference 14 butyryl‐CoA, propionyl‐CoA, hexanoyl‐CoA, octanoyl‐CoA, phenylpropionyl‐CoA, and palmitoyl‐CoA, glutaryl‐CoA were obtained from Sigma‐Aldrich (St. Louis, Missouri).
The activity of recombinant human acyl‐CoA dehydrogenases13 was measured at different substrate concentrations in Tris buffer with an assay using ferricenium hexafluorophosphate as an electron acceptor.15 Enzyme activity was additionally confirmed by identification of the product glutaconyl‐CoA by HPLC and analyzing it by mass spectrometry.11 The kinetic constants (Km and Vmax) were estimated by computer‐fitting of the data (n = 4) using an algorithm based on the Michaelis‐Menten equation. The turnover number (kcat) was calculated using the molecular mass of the acyl‐CoA dehydrogenases (43 kDa).16 , 17 In addition to glutaryl‐CoA, the activities of acyl‐CoA dehydrogenases toward their (optimal) substrates were determined as a control. The pH dependence of the activity toward the different substrates was measured in the range of pH 7.0‐8.5 in Tris buffer adjusted to the desired pH value with KOH or HCl.
The activity of recombinant human acyl‐CoA dehydrogenases
10
Characterizing Histone Modifications and Interactions
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Pan anti-Kbu antibody, anti-histone site-specific Kbu, and acetyllysine antibodies used were purchased from PTM Biolabs and anti-histone H3 and anti-histone H4 antibodies were from Abcam. Mouse monoclonal antibodies against H4K5ac and H4K8ac were a generous gift from Dr. H. Kimura. Anti-Brdt is a homemade antibody previously described (Gaucher et al., 2012 (link)). Butyryl-CoA and acetyl-CoA were purchased from Sigma-Aldrich. The modified porcine trypsin was purchased from Promega. HPLC-grade acetonitrile, water, and ethanol were purchased from EMD Chemicals. Peptides bearing one or a few acetyl and Kbu residues were custom synthesized and were verified by HPLC and mass spectrometry. JQ1 was synthesized by Charles McKenna and Elena Ferri (University of Southern California) (Emadali et al., 2013 (link)) and used as described in this reference.
The antigen recognition capacities of the antibodies used in ChIP-seq and ChIP-qPCR were determined using SPR as described in theSupplemental Information .
The antigen recognition capacities of the antibodies used in ChIP-seq and ChIP-qPCR were determined using SPR as described in the
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