U of New South Wales Wilkins Lab - PRMT6 substrate specificity analysis

PRMT6 substrate specificity analysis
Data License: CC BY 4.0 | ProteomeXchange: PXD016711
  • Organism: Homo sapiens
  • Instrument: Orbitrap Fusion Lumos
  • SpikeIn: No
  • Keywords: Arginine methyltransferase, enzyme specificity, synthetic peptides
  • Submitter: Joshua Hamey
Abstract
Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here we have systematically characterised the substrate recognition motif of PRMT6 in context of the histone H3 tail, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure the effect on PRMT6 in vitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an in vivo PRMT6 substrate. Then, using parallel reaction monitoring (PRM) with electron transfer dissociation (ETD), we quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 is capable of tolerating essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 prefers glycine in the position immediately following the target arginine, indicating that PRMT6 recognises the RG motif. We confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity, along with recognition of RG rather than RGG, is distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6.
Experiment Description
For initial analysis of PRMT6 activity towards the wild-type sequence H3 or H4 peptide, a synthetic H3 peptides corresponding to H3 (sequence ARTKQTARKSTGGKA, 2 μM) (ChinaPeptides) or H4 (sequence SGRGKGGKGLGKGGAK, 2 μM) (ChinaPeptides) wasere incubated with purified PRMT6 (1 μM) in in vitro methylation buffer (50 mM HEPES, 20 mM NaCl, 1 mM EDTA, pH 7.4) in the presence of S-Adenosyl-L-methionine-D3 (S-methyl-D3) tetra-(p-Toluenesulfonate) salt (500 μM) (Medical Isotopes) overnight at 37 °C. For analysis of PRMT6 specificity, H3 or H4 peptide sets (2 μM per peptide, 40 μM total) (ChinaPeptides) were incubated with or without purified PRMT6 (1 μM or 0.1 μM) in in vitro methylation buffer and 500 μM D3-AdoMet, as above. For confirmation of PRMT6 specificity, peptides carrying single or double amino acid substitutions (2 μM) (ChinaPeptides) were assayed with PRMT6 (1 μm) for 1 hr, as above. Histone H3 Ppeptides were analysed by LC-PRM-ETD on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Precursor scans were acquired in the Orbitrap (m/z 300 - 1500, resolution = 60,000 at m/z 200, maximum injection time = 50 ms, automated gain control (AGC) target = 4 × 105). Subsequently, ions at specified m/z values were sequentially isolated in the quadrupole (isolation window = 0.5 m/z), fragmented by ETD (using calibrated charge-dependent ETD parameters and no supplemental activation) and fragment ions detected in the Orbitrap (m/z 150 - 1750, resolution = 7500 at m/z 200, maximum injection time = 22 ms, AGC target = 5 × 104). In order to ensure short duty cycles and thus frequent sampling, peptides were analysed in groups of amino acid substitutions (e.g A+C+D+E or F+G+H+I/L or K+M+N+P or Q+R+S or T+V+W+Y), such that no more than 21 m/z values were analysed in a single run. For analysis of the wild-type H3 peptide assay the same parameters were used, except that fragment ion scans were acquired with a resolution of 30,000 and a maximum injection time of 54 ms. Histone H4 peptides, both wild-type and the G4X peptide set, were analysed by LC-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer.
Created on 2/23/21, 7:20 AM
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T11X_R2021-02-23 07:17:23177564
T11X_QS2021-02-23 07:17:23118181444
T6X_ACDE2021-02-23 07:17:23136362884
T6X_FGHIL2021-02-23 07:17:23136362884
A7X_KMNP2021-02-23 07:17:23136362884
A7X_QS2021-02-23 07:17:23118181444
R8X_ACDEFGH2021-02-23 07:17:23121211684
K9X_ACDE2021-02-23 07:17:23136362884
K9X_KMNP2021-02-23 07:17:23136362884
K9X_QS2021-02-23 07:17:22118181444
A1X_QS2021-02-23 07:17:22118181444
A1X_R2021-02-23 07:17:22177564
A1X_ACDE2021-02-23 07:17:22136362884
T3X_FGHIL2021-02-23 07:17:22136362884
T3X_R2021-02-23 07:17:22177564
R2X_RSTVWY2021-02-23 07:17:22124241924
R2X_ILKMNPQ2021-02-23 07:17:22118181444
K4X_TVWY2021-02-23 07:17:22136362884
A1X_KMNP2021-02-23 07:17:22136362884
A1X_1-10_KMNP2021-02-23 07:17:22136362864
K4X_KMNP2021-02-23 07:17:22136362884
K4X_QS2021-02-23 07:17:22127272164
K4X_ACDE2021-02-23 07:17:22136362884
A1X_FGHIL2021-02-23 07:17:22136362884
T3X_QS2021-02-23 07:17:22118181444
Q5X_FGHIL2021-02-23 07:17:21137372964
K9X_R2021-02-23 07:17:21177564
S10X_R2021-02-23 07:17:21177564
T11X_TVWY2021-02-23 07:17:21136362884
G12X_ACDE2021-02-23 07:17:21136362884
G12X_FGHIL2021-02-23 07:17:21136362884
G12X_QS2021-02-23 07:17:21118181444
R2X_ACDEFGH2021-02-23 07:17:21121211684
A1X_TVWY2021-02-23 07:17:21236362884
T3X_TVWY2021-02-23 07:17:21136362884
T3X_KMNP2021-02-23 07:17:21136362884
K4X_FGHIL2021-02-23 07:17:21136362884
K4X_R2021-02-23 07:17:21177564
Q5X_ACDE2021-02-23 07:17:21136362884
T6X_TVWY2021-02-23 07:17:20136362854
Q5X_R2021-02-23 07:17:20177564
Q5X_TVWY2021-02-23 07:17:20136362884
T6X_KMNP2021-02-23 07:17:20136362884
Q5X_QS2021-02-23 07:17:20127272164
R8X_ILKMNPQ2021-02-23 07:17:20118181444
R8X_RSTVWY2021-02-23 07:17:20124241924
Q5X_KMNP2021-02-23 07:17:20136362884
A7X_TVWY2021-02-23 07:17:20136362884
K9X_FGHIL2021-02-23 07:17:20136362864
S10X_ACDE2021-02-23 07:17:20136362884
S10X_KMNP2021-02-23 07:17:20136362864
T11X_ACDE2021-02-23 07:17:20136362884
T11X_FGHIL2021-02-23 07:17:20136362884
A1X_1-10_FGHIL2021-02-23 07:17:20136362884
T6X_QS2021-02-23 07:17:20118181444
T6X_R2021-02-23 07:17:19177564
A7X_ACDE2021-02-23 07:17:19136362884
A7X_FGHIL2021-02-23 07:17:19136362884
A7X_R2021-02-23 07:17:19177564
G12X_TVWY2021-02-23 07:17:19136362884
A1X_1-10_ACDE2021-02-23 07:17:19136362884
K9X_TVWY2021-02-23 07:17:19136362884
S10X_QS2021-02-23 07:17:19118181444
T11X_KMNP2021-02-23 07:17:19136362884
G12X_R2021-02-23 07:17:19177564
T3X_1-10_ACDE2021-02-23 07:17:19136362884
T3X_1-10_QS2021-02-23 07:17:19118181444
T3X_1-10_TVWY2021-02-23 07:17:19136362884
H3 WT peptide PRMT6 assay2021-02-23 07:17:19199721
S10X_TVWY2021-02-23 07:17:19136362874
G12X_KMNP2021-02-23 07:17:18136362884
A1X_1-10_QS2021-02-23 07:17:18118181444
T3X_1-10_FGHIL2021-02-23 07:17:18136362884
T3X_1-10_KMNP2021-02-23 07:17:18136362884
A1X_1-10_TVWY2021-02-23 07:17:18136362884
T3X_ACDE2021-02-23 07:17:18136362884
S10X_FGHIL2021-02-23 07:17:18136362884
K4D_1hr2021-02-23 07:17:18199724
K4Y_1hr2021-02-23 07:17:18199724
K9D_1hr2021-02-23 07:17:18199724
K9G_1hr2021-02-23 07:17:18199714
K9Y_1hr2021-02-23 07:17:18199724
S10G_1hr2021-02-23 07:17:18199724
T3D_1hr2021-02-23 07:17:18199724
T3G_1hr2021-02-23 07:17:18199724
T3Y_1hr2021-02-23 07:17:18199724
K4G_1hr2021-02-23 07:17:18199724
S10D_1hr2021-02-23 07:17:17199724
S10Y_1hr2021-02-23 07:17:17199724
WT_1hr2021-02-23 07:17:17199724
WT_time_series_assay2021-02-23 07:17:17199718
H4 1-16 PRMT6 assay2021-02-23 07:17:17136182
H3 T3G-K4G PRMT6 assay2021-02-23 07:17:17199724
H3 K9G-S10G PRMT6 assay2021-02-23 07:17:17199724
H4 G4X PRMT6 assay2021-02-23 07:17:171571143424