A simplified characterization of S-adenosyl-<scp>l</scp>-methionine-consuming enzymes with 1-Step EZ-MTase: a universal and straightforward coupled-assay for in vitro and in vivo setting

Burgos, Emmanuel S.; Walters, Ryan O.; Huffman, Derek M.; Shechter, David

doi:10.1039/c7sc02830j

Cited by 19 publications

(22 citation statements)

References 78 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GNMT activity within rat liver extracts was quantified using the 1-Step EZ-MTase coupled assay through its UV-mode of detection (Burgos et al, 2017). The methyltransfer from SAM onto the glycine substrate generates sarcosine and SAH.…”

Section: Star⋆methodsmentioning

confidence: 99%

“…Methyltransfer reactions started upon addition of SAM (1.76 mM). Initial rates were plotted against liver extract volumes to yield a linear activity curve; the slope of this representation (μM h −1 μL −1 ) was converted into GNMT activity and normalized to the amount of total protein originally present within liver extract (GNMT activity expressed in picomoles of sarcosine synthesized per minute and per milligram of total protein: pmol min −1 mg −1 ) with a Z’-factor of 0.75 (Burgos et al, 2017). …”

Section: Star⋆methodsmentioning

confidence: 99%

See 1 more Smart Citation

Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

et al. 2018

Self Cite

View full text Add to dashboard Cite

SUMMARY A hallmark of aging is a decline in metabolic homeostasis, which is attenuated by dietary restriction (DR). However, the interaction of aging and DR with the metabolome is not well understood. We report that DR is a stronger modulator of the rat metabolome than age in plasma and tissues. A comparative metabolomic screen in rodents and humans identified circulating sarcosine as being similarly reduced with aging and increased by DR, while sarcosine is also elevated in long-lived Ames dwarf mice. Pathway analysis in aged sarcosine-replete rats identify this biogenic amine as an integral node in the metabolome network. Finally, we show that sarcosine can activate autophagy in cultured cells and enhances autophagic flux in vivo, suggesting a potential role in autophagy induction by DR. Thus, these data identify circulating sarcosine as a biomarker of aging and DR in mammalians and may contribute to age-related alterations in the metabolome and in proteostasis.

show abstract

Section: Star⋆methodsmentioning

confidence: 99%

Section: Star⋆methodsmentioning

confidence: 99%

Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…High throughput screening (HTS) is a proven method to identify small molecule inhibitors as initial hits, which can be further modified to yield a cell-potent and selective inhibitor. , Current assays for MTase modulation either detect the production of methylated substrates or S-adenosyl- l -homocysteine (SAH). Methylation can be detected through radioisotope-labeled methyl group transfer from SAM to the substrates, mass spectrometry (MS), antibody-specific recognition in combination with fluorescence resonance energy transfer (FRET), or chemiluminescence. − Depending on the substrate of the MTase, the detection method for the methylated substrate often needs to be changed and/or reoptimized. These assays either have low throughput or require a specific assay kit, which limit their uses in high-throughput screening. , On the other hand, the detection of SAH is applicable to essentially any MTase because SAH is generated during the SAM-dependent methylation reaction.…”

Section: Introductionmentioning

confidence: 90%

Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors

et al. 2020

View full text Add to dashboard Cite

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

show abstract

“…The signal was amplified using the EN3HANCE spray (PerkinElmer) and detected following incubation with an x-ray film for 2 to 6 weeks. For fluorescence-based assays, the activity of GST-SETD2 (amino acids 1418 to 1714) and tSETD2-Flag (amino acids 1418 to 2564) was measured over 3 to 4 hours using a methyltransferase fluorescence assay kit (Cayman Chemical, 700150), a continuous enzyme-coupled assay that continuously monitors SAM-dependent methyltransferase activity ( 50 , 51 ). Readout fluorescence via resorufin was analyzed with an excitation wavelength of 540 nm and an emission wavelength of 590 nm using plate reader.…”

Section: Methodsmentioning

confidence: 99%

The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

et al. 2020

View full text Add to dashboard Cite

The methyltransferase SET domain–containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.

show abstract

A simplified characterization of S-adenosyl-l-methionine-consuming enzymes with 1-Step EZ-MTase: a universal and straightforward coupled-assay for in vitro and in vivo setting

Abstract: Methyltransferases use S-adenosyl-l-methionine (SAM) to deposit methyl marks. The 1-Step EZ-MTase coupled assay is a simple tool to study many of these epigenetic ‘writers’.

Cited by 19 publications

References 78 publications

Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

Sarcosine Is Uniquely Modulated by Aging and Dietary Restriction in Rodents and Humans

Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors

The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

Contact Info

Product

Resources

About