scholarly journals A Continuous Protein Methyltransferase (G9a) Assay for Enzyme Activity Measurement and Inhibitor Screening

2009 ◽  
Vol 14 (9) ◽  
pp. 1129-1133 ◽  
Author(s):  
Arunkumar Dhayalan ◽  
Emilia Dimitrova ◽  
Philipp Rathert ◽  
Albert Jeltsch
Keyword(s):  
Scintillation Counting ◽  
Standard Errors ◽  
Activity Measurement ◽  
Protein Methylation ◽  
Substrate Protein ◽  
Zinc Finger Motifs ◽  
Methylation Assay ◽  
Lysine Methyltransferase ◽  
Protein Methyltransferase ◽  
Nonhistone Substrate

The authors describe a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs). The assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate. The reaction progress is monitored continuously by proximity scintillation counting. The assay is very accurate, convenient, well suited for automation, and highly reproducible with standard errors in the range of 5%. Because of few pipetting steps and continuous data readout, it is ideal for high-throughput applications such as screening of inhibitors, testing many enzyme variants, or analyzing differences in methylation rates of different substrates under various conditions. By using this new assay, the IC 50 of AdoHcy and the G9a inhibitor BIX-01294 were determined for methylation of the G9a nonhistone substrate WIZ. ( Journal of Biomolecular Screening 2009:1129-1133)

scholarly journals SET8 prevents excessive DNA methylation by methylation-mediated degradation of UHRF1 and DNMT1

2019 ◽  
Author(s):  
Huifang Zhang ◽  
Qinqin Gao ◽  
Shuo Tan ◽  
Jia You ◽  
Cong Lyu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Cell Division ◽  
Global Dna Methylation ◽  
Protein Methylation ◽  
Accessory Factor ◽  
Protein Methyltransferase ◽  
A Cell ◽  
Do So

Abstract Faithful inheritance of DNA methylation across cell division requires DNMT1 and its accessory factor UHRF1. However, how this axis is regulated to ensure DNA methylation homeostasis remains poorly understood. Here we show that SET8, a cell-cycle-regulated protein methyltransferase, controls protein stability of both UHRF1 and DNMT1 through methylation-mediated, ubiquitin-dependent degradation and consequently prevents excessive DNA methylation. SET8 methylates UHRF1 at lysine 385 and this modification leads to ubiquitination and degradation of UHRF1. In contrast, LSD1 stabilizes both UHRF1 and DNMT1 by demethylation. Importantly, SET8 and LSD1 oppositely regulate global DNA methylation and do so most likely through regulating the level of UHRF1 than DNMT1. Finally, we show that UHRF1 downregulation in G2/M by SET8 has a role in suppressing DNMT1-mediated methylation on post-replicated DNA. Altogether, our study reveals a novel role of SET8 in promoting DNA methylation homeostasis and identifies UHRF1 as the hub for tuning DNA methylation through dynamic protein methylation.

scholarly journals Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Maren Kirstin Schuhmacher ◽  
Serap Beldar ◽  
Mina S. Khella ◽  
Alexander Bröhm ◽  
Jan Ludwig ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Histone H3 ◽  
Protein Methylation ◽  
Sequence Specificity ◽  
Sequence Design ◽  
Substrate Peptide ◽  
Lysine Methyltransferase ◽  
In Cells

AbstractSETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

scholarly journals Characterization of SETD3 methyltransferase–mediated protein methionine methylation

2020 ◽  
Vol 295 (32) ◽  
pp. 10901-10910
Author(s):  
Shaobo Dai ◽  
Matthew V. Holt ◽  
John R. Horton ◽  
Clayton B. Woodcock ◽  
Anamika Patel ◽  
...  
Keyword(s):  
Active Site ◽  
Side Chain ◽  
Protein Methylation ◽  
Aromatic Rings ◽  
Set Domain ◽  
Histone Lysine ◽  
Histone Lysine Methyltransferase ◽  
Close Proximity ◽  
Lysine Methyltransferase

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain–containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310. Our results suggest that placing methionine properly in the active site—within close proximity to and in line with the incoming methyl group of SAM—would allow some SET domain proteins to selectively methylate methionine in proteins.

scholarly journals Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity

2014 ◽  
Vol 25 (25) ◽  
pp. 4174-4186 ◽  
Author(s):  
Bridget T. Jacques-Fricke ◽  
Laura S. Gammill
Keyword(s):  
Gene Expression ◽  
Neural Crest ◽  
Dominant Negative ◽  
Methyltransferase Activity ◽  
Lysine Methyltransferase ◽  
Neural Plate Border ◽  
Protein Methyltransferase ◽  
And Migration ◽  
Protein Methyltransferases ◽  
Neural Crest Migration

Neural crest precursors express genes that cause them to become migratory, multipotent cells, distinguishing them from adjacent stationary neural progenitors in the neurepithelium. Histone methylation spatiotemporally regulates neural crest gene expression; however, the protein methyltransferases active in neural crest precursors are unknown. Moreover, the regulation of methylation during the dynamic process of neural crest migration is unclear. Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells. NSD3 expression commences before up-regulation of neural crest genes, and NSD3 is necessary for expression of the neural plate border gene Msx1, as well as the key neural crest transcription factors Sox10, Snail2, Sox9, and FoxD3, but not gene expression generally. Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation. In addition, by temporally limiting expression of a dominant negative to migratory stages, we identify a novel, direct requirement for NSD3-related methyltransferase activity in neural crest migration. These results identify NSD3 as the first protein methyltransferase essential for neural crest gene expression during specification and show that NSD3-related methyltransferase activity independently regulates migration.

A Rapid Psychrometric Procedure for Water Activity Measurement of Foods in the Intermediate Moisture Range1

1981 ◽  
Vol 44 (12) ◽  
pp. 892-895 ◽  
Author(s):  
H. H. WIEBE ◽  
R. N. KIDAMBI ◽  
G. H. RICHARDSON ◽  
C. A. ERNSTROM
Keyword(s):  
Water Activity ◽  
Milk Powder ◽  
Standard Errors ◽  
Activity Measurement ◽  
Milk Chocolate ◽  
Step Procedure ◽  
Coefficients Of Variation ◽  
Sample Chamber ◽  
Juice Concentrate ◽  
Luncheon Meat

Commercial vapor pressure thermocouple psychrometers (hygrometers) are now generally accepted for measuring water activity, aw (water potential) in plants and soils, and commercial instruments are available. We have adapted them for aw measurements in the 0.99 to 0.60 range using a two-step procedure. Water is first condensed on the thermocouple; then the sample is inserted in the thermocouple chamber and the psychrometric cooling of the wet thermocouple measured. The procedure is calibrated with a series of saturated salt slurries of known aw values. Typical aw values (with standard deviations) for a variety of foods were: Cheddar cheese, 0.95 ± 0.03; Parmesan cheese, 0.76 ± 0.03; milk powder, 0.75 ± 0.02; milk chocolate, 0.60 ± 0.04; luncheon meat, 0.96 ± 0.03; bread, 0.95 ± 0.03; dried raisins, 0.82 ± 0.02; corn syrup, 0.60 ± 0.02; and orange juice concentrate, 0.80 ± 0.03. The coefficients of variation ranged from 1.9 to 5.8%. When compared with published values obtained by other methods, these figures were within the standard errors of measurement. The thermocouple detector did not foul since it had only vapor contact with the sample. An economical sample chamber and instrument is described. Analysis time is 4 to 8 min. The procedure is accurate, convenient and rapid.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

Sign in / Sign up

Export Citation Format

Share Document