Blockage Of Protein Neddylation Alters Glycolysis:Mechanistic And Functional Studies

Neddylation is one type of post-translational modifications,which only modulates protein activity and function,not mediates protein degradation through 26S proteasome.Like the process of ubiquitination,neddylation is catalyzed by an E1 NEDD8-activating enzyme(NAE),an E2 NEDD8-conjugating enzyme,and an E3 ligase,to tag ubiquitin-like protein NEDD8 to a substrate protein.Among the substrates of neddylation,Cullin-RING ligases(CRLs),the largest E3 ubiquitin ligase family,are best studied as the physiological substrates.MLN4924,an adenosine sulfamate derivative,is a small molecule inhibitor of NAE.By forming a covalent MLN4924-NEDD8 adduct catalyzed by NAE,which cannot be further utilized in subsequent intraenzyme reactions,MLN4924 inhibits the entire neddylation modification.Thus,MLN4924 is widely used in studying the biochemical function and biological process of neddylation pathway.Many cancer cells have an increased rate of glycolysis to meet their energy needs for accelerated growth and proliferation.Pyruvate kinase(PK)is an important rate-limiting enzyme in glycolysis,catalyzing the conversion of phosphoenolpyruvate(PEP)to pyruvate.As a result,PK affects the rate of glycolysis.PKM2,a variable splice form of PKM,is the major form found in cancer cells.More and more studies showed that PKM2 effectively promotes the reprogramming of energy metabolism in cancer cells.Many types of human cancer cells have overactivated neddylation modification and dysregulated energy metabolism.It remains largely uncleared,however,whether and how neddylation modification affects cellular metabolism.To this end,we explored the effect of neddylation blockage on overall cellular energy metabolism.MDA-MB-231 breast cancer cells were treated with MLN4924 for 24 hours,and subjected to Mass-Spectrometry-based metabolic profiling.We found that MLN4924 treatment caused a dramatical increases in the levels of 3-phosphoglyceric acid and pyruvic acid,strongly suggesting an enhancement of glycolysis.We also found increased glucose consumption,pyruvate and lactate production,further supporting the notion that MLN4924 promotes glycolysis.What is the mechanism of MLN4924 action in promoting glycolysis?We focused on PK,and found that MLN4924 treatment remarkably increase PK activity in time-and dose-dependent manners.This phenomenon was almost disappeared when PKM2 was knocked down via siRNA silencing,indicating that PKM2 plays a major role in mediating MLN4924 effect.We further performed glutaraldehyde cross-linking coupled western blot assay,and found that the level of PKM2 homotetramers,an active form of PKM2 was significantly increased after MLN4924 treatment in a dose-dependent manner,indicating an enhancement of PKM2 activity.To further confirm this,we expressed and purified PKM2 protein in bacteria.Through an in vitro biochemical assay,we observed MLN4924 increases PKM2 activity in a dose-dependent manner.We further tested biological significance of PKM2 activation by MLN4924,and found that inactivaiton of PKM2,by genetic(siRNA knockdown)or pharmacological(shikonin,a small molecular inhibitor of PKM2)approach,significantly enhanced anti-tumor activity of MLN4924,as evidenced by remarkable reduction of cell growth and colony formation,when used in combination.Taken together,we made a novel observation that blockage of neddylation modification by MLN4924 promotes glycolysis by activating PKM2,and PKM2 inhibitor shikonin sensitizes breast cancer to MLN4924.Our study made a novel linkage between neddylation pathway and glycolysis,and provides a sound rationale for combinational therapy of MLN4924-Shikonin against breast cancer.