Citrullination of pyruvate kinase by PADI1 and PADI3 regulates glycolysis and cancer cell proliferation

Abstract

CHD3 and CHD4 are mutually exclusive ATPase subunits of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of three arginines of the allostericallyregulated glycolytic enzyme pyruvate kinase M2 (PKM2). PKM2 citrullination lowers sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine shifting the equilibrium towards the activator Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis defining a novel pathway regulating proliferation of melanoma and other cancer cells. We provide unique insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 reprogramming its activity as an additional layer of complexity to the mechanisms regulating this important enzyme.Significance. This study describes the mechanism by which citrullination of key arginines in pyruvate kinase M2 reprograms its allosteric regulation by the activating amino acid serine.PKM2 citrullination defines a novel pathway regulating glycolysis and cancer cell proliferation.3 Introduction.

Enabling Global Analysis of Protein Citrullination via Biotin Thiol Tag-Assisted Mass Spectrometry

“…In accordance with this, we also identified four citrullination sites on an essential glycolytic enzyme pyruvate kinase (PKM). Interestingly, many of these sites on PKM are located at critical positions (Figure S17a), which raises the likelihood that citrullination can influence the kinase activity and supports a recent study concluding that citrullination regulates glycolysis . For instance, R455 is located in the proximity of one allosteric center (Figure S17b), which was known to regulate enzyme activity .…”

“…Interestingly, many of these sites on PKM are located at critical positions (Figure S17a), which raises the likelihood that citrullination can influence the kinase activity and supports a recent study concluding that citrullination regulates glycolysis. 61 For instance, R455 is located in the proximity of one allosteric center (Figure S17b), which was known to regulate enzyme activity. 62 Another position R399 which we found to be citrullinated was also shown to be very important in stabilizing the highly active tetrameric form (Figure S17c, d).…”

Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

“…Interestingly, many of these sites on PKM are located at critical positions (Supplementary Fig. 15a), which raises the likelihood that citrullination can influence the kinase activity and supports a recent study concluding that citrullination regulates glycolysis 60 . For instance, R455 is located in the proximity of one allosteric center (Supplementary Fig.…”

Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

“…Interestingly, many of these sites on PKM are located in the proximity of its substrate binding pockets ( Supplementary Fig. 11a), which raises the likelihood that citrullination can in uence the kinase activity and supports a recent study concluding that citrullination regulates glycolysis 43 . For instance, R120 and R294 are located near the catalytic pocket ( Supplementary Fig.…”