Preparation Method

The reaction was run in 8 complete rows (half plate) with or without a known inhibitor. S-adenosylhomocysteine was used as an inhibitor for METTL3-14.

Reaction Conditions

0-100?M S-adenosylhomocysteine

Applications

S-adenosylhomocysteine showed strong inhibitory effects on METTL3-14 activity with an IC50 value of 0.9±0.1?M.

Cell lines

WY2 and WY35(cys4? strains)

Preparation Method

Saturated 2-day-old cultures of the WY2 and WY35 were diluted to an OD600 of approximately 0.05. S-adenosylhomocysteine was added at concentration at 25, 50, 100, 600?M. Growth was monitored by observing OD600 at specific time points during the course of 24h

Reaction Conditions

25, 50, 100, 600?M S-adenosylhomocysteine, 24h

Applications

As little as 25?M S-adenosylhomocysteine showed measurable growth inhibition with the doubling time increasing about 15% from 222 to 256min. Exposure to 600?M S-adenosylhomocysteine increased the doubling time 206%, from 222 to 680min.

Animal models

zebrafishes (F0) carrying the ahcyl1-KO allele

Preparation Method

The identified chimeras were raised and crossed to wild-type zebrafish. The fertilized eggs were collected and injected with 0 or 5mM S-adenosylhomocysteine and RFP-LC3 mRNA at one-cell stage. For the injection of 5mM S-adenosylhomocysteine, the final concentration of injected S-adenosylhomocysteine was about 5?M. 26h after fertilization, the embryos were fixed by 8% paraformaldehyde with DAPI overnight.

Dosage form

0 or 5mM S-adenosylhomocysteine, the final concentration of injected S-adenosylhomocysteine was about 5?M, embryo injection, 26h

Applications

Less LC3 puncta was observed when injected with S-adenosylhomocysteine. When one allele of ahcyl1 was knocked out, a mild increase of LC3 puncta was observed and the decrease of LC3 puncta by S-adenosylhomocysteine was significantly weakened. These results demonstrated that AHCYL1 senses the increased S-adenosylhomocysteine to inhibit autophagy in zebrafish.

S-adenosylhomocysteine (SAH), an amino acid derivative, is a key intermediate metabolite in

methionine metabolism^[1]. It is an intermediate in the synthesis of cysteine and adenosine^[2]. S-adenosylhomocysteine inhibited METTL3-14 activity with an IC50 value of 0.9 ± 0.1 µM^[3].

S-adenosylhomocysteine(25 µM) inhibited the growth of CBS deficient yeast, but had no effect on wild-type yeast. Growth inhibition by S-adenosylhomocysteine in CBS deficient yeast can be totally reversed by addition of SAM to the media^[4]. High S-adenosylhomocysteine levels inhibited NFκB-mediated gene expression and sensitized primary hepatocytes and HepG2 cells to the cytotoxic effects of TNF^[5]. Increased adipose S-adenosylhomocysteine levels generate methylation defects that promote lipolysis. Alcohol-induced increases in hepatocellular S-adenosylhomocysteine and the resultant lowering of SAM/SAH ratio lead to the pathogenesis and progression of ALD^[6].

S-adenosylhomocysteine enhances the interaction between AHCYL1 and PIK3C3. When cells are in the presence of S-adenosylhomocysteine, the enhanced interaction suppresses the production of PtdIns3P, which blocks the autophagy initiation. When in the absence of S-adenosylhomocysteine, the decreased interaction releases PIK3C3 to produce PtdIns3P, eventually promotes autophagy^[1]. CKD was associated with a low SAM level and SAM/SAH ratio in urine. The use of the SAM level or the SAM/SAH ratio in urine could be considered as a promising, noninvasive indicator of renal dysfunction^[7].

References:
[1] Huang W, Li N, et al. AHCYL1 senses SAH to inhibit autophagy through interaction with PIK3C3 in an MTORC1-independent manner. Autophagy. 2022;18(2):309-319.
[2] DE LA HABA G, et al. The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. J Biol Chem. 1959;234(3):603-608.
[3] Li F, Kennedy S, et al. A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5. J Biomol Screen. 2016;21(3):290-297.
[4] Christopher SA, Melnyk S, et al. S-adenosylhomocysteine, but not homocysteine, is toxic to yeast lacking cystathionine beta-synthase. Mol Genet Metab. 2002;75(4):335-343.
[5] Watson WH, Burke TJ, et al. S-adenosylhomocysteine inhibits NF-κB-mediated gene expression in hepatocytes and confers sensitivity to TNF cytotoxicity. Alcohol Clin Exp Res. 2014;38(4):889-896.
[6] Arumugam MK, Chava S, et al. Elevated S-adenosylhomocysteine induces adipocyte dysfunction to promote alcohol-associated liver steatosis. Sci Rep. 2021;11(1):14693. Published 2021 Jul 19.
[7] Kruglova MP, Grachev SV, et al. Low S-adenosylmethionine/ S-adenosylhomocysteine Ratio in Urine is Associated with Chronic Kidney Disease. Lab Med. 2020;51(1):80-85.