Bi-substrate kinetic analysis of an E3-ligase-dependent ubiquitylation reaction.

Little is known about the kinetic mechanism of E3 ubiquitin ligases. This work describes basic methodology to investigate the kinetic mechanism of E3 ubiquitin ligases. The method used steady state, bi-substrate kinetic analysis of an E3 ligase-catalyzed monoubiquitylation reaction using ubiquitin-conjugated E2 (E2ub) and a mutant IkappaBalpha as substrates to evaluate whether the E3-catalyzed ubiquitin transfer from E2ub to protein substrate was sequential, meaning both substrates bound before products leaving, or ping pong, meaning that ubiquitin-conjugated E2 would bind, transfer ubiquitin to the E3, and debind before binding of protein substrate. The method requires the E3 reaction to be rate limiting and at steady state. This was accomplished through optimization of the conditions to ensure that the E3-dependent transfer of ubiquitin from E2ub to substrate was rate limiting. We observed a sequential bi-substrate E3-dependent ubiquitylation reaction on using E2UBCH7 and IkappaBalphaSS32/36EE (IkappaBalphaee as substrates and a partially purified Jurkat cell lysate as a source for the E3 ligase activity). The sequential bi-substrate kinetic mechanism is consistent with the formation of a ternary complex among E2UBCH7, IkappaBalphaSS32/36EE, and E3 before the transfer of ubiquitin from E2UBCH7 to IkappaBalphaSS32/36EE. The described method should be of use to characterize the kinetic mechanism of other E3 ligase-catalyzed ubiquitylation reactions.

A small molecule ubiquitination inhibitor blocks NF-kappa B-dependent cytokine expression in cells and rats.

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) alpha-induced IkappaB(alpha) degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFalpha, interleukin-1beta, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 microm. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP but associates with IkappaB(alpha) and will ubiquitinate IkappaB(alpha) S32E,S36E (IkappaB(alpha)(ee)) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IkappaB(alpha)(ee) ubiquitination with an IC(50) value of 2.3 +/- 0.09 microm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF(betaTRCP), the putative E3 for IkappaB(alpha) ubiquitination. Ro106-9920 was observed to be selective for IkappaB(alpha)(ee) ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFalpha-induced IkappaB(alpha) degradation and NF-kappaB activation.