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Updated: Apr 30, 2026
Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates
Published on: February 27, 2016
Oxidative stress activates SIRT2 to deacetylate and stimulate phosphoglycerate mutase
Yanping Xu1, Fulong Li1, Lei Lv2
1Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, School of Life Sciences, Fudan University, Shanghai, PR China;
Abstract:
Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.
Insights
Phosphoglycerate mutase (PGAM) acetylation at K100 regulates its activity, impacting NADPH production and cell proliferation. SIRT2 deacetylates and activates PGAM, a process influenced by oxidative stress.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Glycolytic enzyme phosphoglycerate mutase (PGAM) is crucial for energy production and biosynthesis.
- PGAM activity is elevated in tumors, but its in vivo regulation is unclear.
- PGAM is vital for cell proliferation and tumor growth.
Purpose of the Study:
- To investigate the in vivo regulation of phosphoglycerate mutase (PGAM) activity.
- To identify post-translational modifications affecting PGAM function.
- To understand PGAM's role in cellular metabolism and tumor growth.
Main Methods:
- Site-directed mutagenesis to create acetylation mimetic PGAM2 (K100Q).
- Western blotting and mass spectrometry to detect PGAM acetylation.
- Enzyme activity assays and NADPH production measurements.
- Cell proliferation and tumor growth assays.
Main Results:
- PGAM is acetylated at conserved active site residue K100 in diverse species and tissues.
- K100 acetylation decreases PGAM2 activity.
- Sirtuin 2 (SIRT2) deacetylates and activates PGAM2.
- Oxidative stress promotes PGAM2 deacetylation and activity via SIRT2 interaction.
- PGAM2 K100Q mutant reduces NADPH production, cell proliferation, and tumor growth.
Conclusions:
- PGAM2 activity is regulated by acetylation at K100, impacting cellular metabolism.
- SIRT2-mediated deacetylation of PGAM2 is a key regulatory mechanism.
- This regulatory pathway is responsive to oxidative stress and influences cell proliferation and tumor growth.
- PGAM2 acetylation represents a potential therapeutic target for cancer.
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