Novel Mechanism By Which Flavonoid Compounds Exert Anti-inflammatory Activities

Ubiquitination,particularly the ubiquitin-conjugating enzyme Ubcl 3/Uevl-mediated K63 polyubiquitination,plays critically regulatory roles in proinflammatory and innate immune responses.Natural compounds flavonoids such as quercetin and luteolin exhibit beneficial health effects showing anti-inflammatory,anti-tumor and antioxidant activities.Our studies have been focusing on elucidating the novel biochemical mechanism by which flavonoids exert their anti-inflammatory activity.We firstly determined that two typical flavonoid compounds,quercetin and luteolin,effectively inhibited the activation of TAK1-IKK-NF-?B signaling and downstream gene induction in cells in response to IL-1? and LPS stimulation.Further analysis revealed that K63 polyubiquitin chain assembly and polyubiquitination of key components IRAK1 and TRAF6 in the signaling pathways were severely impaired by the flavonoid compounds.Reconstitution of ubiquitination reactions using recombinant proteins demonstrated that flavonoids specifically inhibited Ubc13/Uev2-mediated K63 polyubiquitination but not other E2-mediated polyubiquitination.Mechanistic studies revealed that flavonoids inhibited Ub discharge from Ub-Ubcl3/Uev2 intermediate via directly binding to Ubc13/Uev2,The KD values of binding were determined to be 24?M for luteolin-Ubc13 and 14?M for morin-Ubc13,respectively.Structure-activity relationship analysis demonstrated that the hydroxyl groups of flavonoid's rings A and B and the aromaticity of ring C are indispensable for their inhibitory effect.Our studies demonstrated that flavonoids are potent Ubc 13-specific inhibitors,which provides a novel molecular mechanism to understand how flavonoids exhibit clinical beneficial effects.Our studies also illustrate that flavonoids can be unique lead compounds for further development as a Ubc 13-specific inhibitor for clinical applications against inflammatory and autoimmune diseases as well as for cancer treatment.