New insights of rotenone targeting cordycepin synthetase 1-cordycepin synthetase 2-glyceraldehyde-3-phosphate dehydrogenase (Cns1-Cns2-GAPDH) triple complex to enhance cordycepin synthesis in Cordyceps militaris.

From the paper

Abstract

Cordycepin isolated from Cordyceps militaris is an adenosine analogue with various biological activities, and its synthesis is dependent on cordycepin synthetase 1 (Cns1) and cordycepin synthetase 2 (Cns2). Our previous study showed that rotenone induction promotes cordycepin synthesis by regulating energy metabolism pathways such as Embden-Meyerhof-Parnas pathway (EMP). However, the exact molecular mechanism remains unclear. Here, we constructed a yeast hybrid library of Cordyceps militaris CM-001 strain and identified the Cns1/Cns2 interaction protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by yeast two-hybrid (Y2H) screening. Subsequently, the interaction between GAPDH and Cns1/2 was verified by Y2H validation, glutathione S-transferase (GST) pull-down, and Co-Immunoprecipitation (Co-IP). The Co-IP results showed that rotenone promoted the interaction of Cns1-Cns2-GAPDH complex. However, rotenone treatment did not affect the mRNA level of GAPDH but up-regulated the protein level and enzyme activity. In addition, rotenone treatment significantly increased the relative levels of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), but a GAPDH inhibitor partially offsets the promoting effect of rotenone. Our study shows that the Cns1-Cns2-GAPDH triple complex regulates cordycepin synthesis in Cordyceps militaris. Rotenone enhances the metabolic flux of cordycepin biosynthesis pathway by enhancing the Cns1-Cns2-GAPDH interaction and its induced aerobic glycolysis.