bi9b00993_si_001.pdf (4.13 MB)
Comparison of Cross-Regulation by Different OTUB1:E2 Complexes
journal contribution
posted on 2020-02-21, 20:10 authored by Lauren
T. Que, Marie E. Morrow, Cynthia WolbergerOTUB1
is a highly expressed cysteine protease that specifically
cleaves K48-linked polyubiquitin chains. This unique deubiquitinating
enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes,
forming complexes in which the two enzymes can regulate one another’s
activity. OTUB1 can noncatalytically suppress the ubiquitin conjugating
activity of its E2 partners by sequestering the charged E2∼Ub
thioester and preventing ubiquitin transfer. The same E2 enzymes,
when uncharged, can stimulate the DUB activity of OTUB1 in
vitro, although the importance of OTUB1 stimulation in vivo remains unclear. To assess the potential balance
between these activities that might occur in cells, we characterized
the kinetics and thermodynamics governing the formation and activity
of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2
enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at
physiologically relevant concentrations of both partners. Whereas
E2 partners differ in their ability to stimulate OTUB1 activity, we
find that this variability is not correlated with the affinity of
each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we
find that OTUB1 inhibits the polyubiquitination activity of all three
UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although
OTUB1 also inhibits the auto-monoubiquitination and autopolyubiquitination
activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination
by UBE2E3. Our quantitative analysis provides a basis for further
exploring the biological roles of OTUB1:E2 complexes in cells.