Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes

Abstract

Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12-ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.

Dihydromyricetin Ameliorates Cardiac Ischemia/Reperfusion Injury through Sirt3 Activation

“…In line with these observations, DHM decreased the infarction size, alleviated cardiac dysfunction, decreased cardiomyocyte oxidative stress and apoptosis, and improved mitochondrial function by activating Sirt3. Having demonstrated that the cardiac function is associated with the mitochondria biogenesis, and H/R induces the death of cardiomyocytes by damaging the mitochondria [25, 33], in the present study, DHM alleviated mitochondrial impairment in vitro study and this effect was abolished by Sirt3 knockdown. Furthermore, mitochondrial membrane potential was a marker of mitochondrial function, and it was decreased after H/R and increased after DHM treatment.…”

UHMK 1 promotes gastric cancer progression through reprogramming nucleotide metabolism

“…NCOA3 vector was from Dr. Bingbing Wang at the State University of New Jersey. Mutants or truncated fragments from different genes such as UHMK1 and NCOA3 were constructed as described previously (Song et al , ). And sequencing was used to verify the resulting mutants.…”

The cross-talk between methylation and phosphorylation in lymphoid-specific helicase drives cancer stem-like properties

“…In vitro methylation analysis was carried out as previously described. 69 In brief, a prokaryotic expression plasmid pGEX-4T-1-LSH was conducted, GST-LSH fusion protein was expressed in E. coli strain BL21 and later purified according to the manufacturer's directions. HA-PRMT5 fusion protein was obtained from 293T cells transfected with HA-PRMT5 plasmid and later immunoprecipitated with PRMT5 antibody and Dynabeads TM Protein G. GST-LSH fusion protein and immunoprecipitated HA-PRMT5 fusion protein were incubated in reaction buffer containing 1 μM methyl group donor SAM at 30°C for 60 min.…”