10 research outputs found
IDH1/2 mutants inhibit TET-promoted oxidation of RNA 5mC to 5hmC.
<p>(A) HEK293 cells were transfected with/without pS-Flag-SBP (control), pS-Flag-SBP TET2, IDH1 WT, IDH1 (R132H), IDH2 WT, IDH2 (R140Q) and/or IDH2 (R172K) as noted. After 48 hrs, 2-HG was extracted and analyzed by mass spectrometry. The cells transfected with pS-Flag-SBP were taken as control (Ctrl). (B) LC-MS/MS analysis of the DNA from (A). (C) LC-MS/MS analysis of the mRNA from (A). (D) LC-MS/MS analysis of the total RNA from (A). (E)HEK293 cells were transfected with/without pCDNA3, pCDNA3B-Flag-TET2 CD, pCDNA3B-Flag-TET2 CD mut, IDH1 WT, IDH1 (R132H), IDH2 WT, IDH2 (R140Q) and/or IDH2 (R172K) as noted. After 48 hrs, 2-HG was extracted and analyzed by mass spectrometry. The cells transfected with pCDNA3 were taken as control (Ctrl). (F) LC-MS/MS analysis of the DNA from (E). (G) LC-MS/MS analysis of the mRNA from (E). (H) LC-MS/MS analysis of the total RNA from (E).</p
Overexpression of TETs significantly increases RNA 5hmC in human HEK293 cells.
<p>(A) HEK293 cells were transfected with pS-Flag-SBP (control), pS-Flag-SBP TET1, pS-Flag-SBP TET2 or pS-Flag-SBP TET3. After 48 hrs, DNA was extracted, digested and analyzed by mass spectrometry as described in Methods and Materials. The 5mdC level (5mdC/dC) and 5hmdC level (5hmdC/dC) were normalized. The 5mdC and 5hmdC levels in the cells transfected with pS-Flag-SBP were taken as control (Ctrl) and considered as 1. In this study, n = 3, ± SEM, and *<i>p</i>< 0.05 were applied unless otherwise stated. (B) The total ion chromatogram (TIC) of RNA 5hmC. Both mRNA and total RNA from (A) were extracted and digested. Resultant 6 μg of mRNA (or 6 μg of total RNA) were analyzed by LC-MS/MS as noted. TIC of RNA 5hmC represents the signal intensity normalized to RNA C level in the cells transfected with pS-Flag-SBP. RT: retention time. More details are described in Methods and Materials. (C) LC-MS/MS analysis of the mRNA (6μg) from (A). (D) LC-MS/MS analysis of the total RNA (60 μg) from (A).(E) HEK293 cells were transfected with pCDNA3 (control), pCDNA3B-Flag-TET1 CD, pCDNA3B-Flag-TET1 CD mut, pCDNA3B-Flag-TET2 CD, pCDNA3B-Flag-TET2 CD mut, pCDNA3B-Flag-TET3 CD or pCDNA3B-Flag-TET3 CD mut. After 48 hrs, DNA was extracted, digested and analyzed by LC-MS/MS. The cells transfected with pCDNA3 were taken as control (Ctrl). (F) LC-MS/MSanalysis of the mRNA from (E). (G) LC-MS/MS analysis of the total RNA from (E).</p
Triple quadrupole mass spectrometer was applied to assess DNA and RNA modifications.
<p>(A) Scheme of oxidation of DNA and RNA by TETs. (B) Base ion mass transitions for LC-MS/MS analysis of DNA nucleosides (dC, 5mdC and 5hmdC) and RNA nucleosides (C, 5mC and 5hmC) as noted. The nucleosides(C, 5mC, 5hmC,dC, 5mdC and 5hmdC) were separated by reverse phase ultra-performance liquid chromatography on a C18 column, with online mass spectrometry detection using triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside to base ion mass transitions of 228 to 112 (dC), 242 to 126 (5mdC), 258 to 142 (5hmdC), 244 to 112 (C), 258 to 126 (5mC), and 274 to 142 (5hmC). More details are described in Methods and Materials. (C) DNA (dC, 5mdC and 5hmdC) and RNA (C, 5mC and 5hmC) nucleoside standard curves as noted. Standard curves were built by serial dilutions. Y-axis: normalized peak area of nucleoside standards as noted; X-axis: concentration of nucleoside standards as noted. More details are described in Methods and Materials.</p
LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
<div><p>Fat mass and obesity-associated protein (FTO) is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A) in mRNA, 3-methylthymine (m3T) in single-stranded DNA (ssDNA) and 3-methyluracil (m3U) in single-stranded RNA (ssRNA). Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC) modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function.</p></div
Immunofluorescence and LC-MS-MS experiments measure the demethylation activity of FTO in vivo.
<p>(a) Immunofluorescence analysis of 5hmdC level generated from 5mdC in FTO or TET2 overexpressed Hela cells. Cells were stained with anti-5hmdC antibody (green), showed that 5hmdC signal is obvious in the TET2 overpexpressed cells, instead of TET2 mutant or FTO gene transfected cells. Nuclei are stained by DAPI. Scale bar: 0–50 μm. (b) LC-MS-MS quantification analysis showed percentage of m6A/A in mRNA and total RNA isolated from control and FTO overexpressed cells. (c) LC-MS-MS quantification analysis showed percentage of 5hmdC/dC, 5hmdC/5mdC in DNA isolated from control, FTO and TET2 overexpressed cells. *p < 0.05; N.S.: No Significance. Error bars, mean ± S.E.M. for triplicate experiments.</p
LC-MS-MS assay measures the demethylation activity of FTO in vitro.
<p>(a) (left) LC-MS-MS profiles of nucleosides derived from enzymatic hydrolysis of RNA samples containing m6A upon treatment with the FTO protein. The upper LC-MS-MS profile shows nucleoside A and m6A standards. (Right) Quantitation of m6A in vitro reaction system of FTO and synthetic RNA substrates. (b) (left) LC-MS-MS profiles of nucleosides derived from enzymatic hydrolysis of DNA samples containing 5mdC upon treatment with the FTO proteins. The upper LC-MS-MS profile shows nucleoside 5hmdC, dC and 5mdC standards. (Right) Quantitation of 5mdC in vitro reaction system of FTO and synthetic DNA. (c) FTO catalyzes the demethylation of m6A in a dose- and time-dependent manner. (d) FTO shows no obvious converting 5mdC to 5hmdC in DNA with the increase of the dose of FTO and reaction time. *p < 0.05; N.S.: No Significance. Error bars, mean ± S.E.M. for triplicate experiments.</p
Kinetic constants for FTO demethylation of m6A in ssRNA and 5mdC in DNA.
<p>Kinetic constants for FTO demethylation of m6A in ssRNA and 5mdC in DNA.</p
Characterization of several kinds of nucleoside modification in DNA and RNA.
<p>(a) Proposed oxidative demethylation of m6A to N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) in RNA by FTO and oxidation of 5mdC to 5hmdC and 5-formylcytosine (5fC) in DNA by TET2. (b) Coomassie staining and western blot of His-tagged full-length human FTO proteins purified from BL21(DE3)-PlysS <i>E</i>. <i>coli</i>. (c) Base ion mass transitions for LC-MS-MS analysis of A, m6A, dC, 5mdC and 5hmdC standard. The MRM transitions were monitored as follows: 267.9 to 136.1 (A); 282.1 to 150.1 (m6A); 228.0 to 112.0 (dC); 258.0 to 142.0 (5hmdC); 242.1 to 126.1 (5mdC). (d) LC-MS-MS standards curves of A, m6A, dC, 5mdC and 5hmdC.</p
Discovery of a benzo[e]pyrimido-[5,4-b][1,4]diazepin-6(11H)-one as a Potent and Selective Inhibitor of Big MAP Kinase 1
Kinome-wide selectivity profiling of a collection of 2-amino-pyrido[2,3-d]pyrimidines followed by cellular structure−activity relationship-guided optimization resulted in the identification of moderately potent and selective inhibitors of BMK1/ERK5 exemplified by <b>11</b>, <b>18</b>, and <b>21</b>. For example, <b>11</b> possesses a dissociation constant (<i>K</i><sub>d</sub>) for BMK1 of 19 nM, a cellular IC<sub>50</sub> for inhibiting epidermal growth factor induced BMK1 autophosphorylation of 0.19 ± 0.04 μM, and an Ambit KINOME<i>scan</i> selectivity score (<i>S</i><sub>5</sub>) of 0.035. Inhibitors <b>18</b> and <b>21</b> are also potent BMK1 inhibitors and possess favorable pharmacokinetic properties which enable their use as pharmacological probes of BMK1-dependent phenomena as well as starting points for further optimization efforts
Discovery of a benzo[e]pyrimido-[5,4-b][1,4]diazepin-6(11H)-one as a Potent and Selective Inhibitor of Big MAP Kinase 1
Kinome-wide selectivity profiling of a collection of 2-amino-pyrido[2,3-d]pyrimidines followed by cellular structure−activity relationship-guided optimization resulted in the identification of moderately potent and selective inhibitors of BMK1/ERK5 exemplified by <b>11</b>, <b>18</b>, and <b>21</b>. For example, <b>11</b> possesses a dissociation constant (<i>K</i><sub>d</sub>) for BMK1 of 19 nM, a cellular IC<sub>50</sub> for inhibiting epidermal growth factor induced BMK1 autophosphorylation of 0.19 ± 0.04 μM, and an Ambit KINOME<i>scan</i> selectivity score (<i>S</i><sub>5</sub>) of 0.035. Inhibitors <b>18</b> and <b>21</b> are also potent BMK1 inhibitors and possess favorable pharmacokinetic properties which enable their use as pharmacological probes of BMK1-dependent phenomena as well as starting points for further optimization efforts