Abstract
BRISC (Brcc36-containing isopeptidase complex) is a four-subunit deubiquitinating (DUB) enzyme that has a catalytic subunit, called Brcc36, that is a member of the JAMM/MPN(+) family of zinc metalloproteases. A notable feature of BRISC is its high specificity for cleaving Lys(63)-linked polyubiquitin. Here, we show that BRISC selectivity is not due to preferential binding to Lys(63)-linked polyubiquitin but is instead dictated by how the substrate isopeptide linkage is oriented within the enzyme active site. BRISC possesses a high affinity binding site for the ubiquitin hydrophobic surface patch that accounts for the bulk of the affinity between enzyme and substrate. Although BRISC can interact with either subunit of a diubiquitin conjugate, substrate cleavage occurs only when BRISC is bound to the hydrophobic patch of the distal (i.e. the "S1") ubiquitin at a ubiquitin-ubiquitin cleavage site. The importance of the Lys(63)-linked proximal (S1') ubiquitin was underscored by our finding that BRISC could not cleave the isopeptide bond joining a ubiquitin to a non-ubiquitin substrate. Finally, we also show that Abro1, another BRISC subunit, binds directly to Brcc36 and that the Brcc36-Abro1 heterodimer includes a minimal complex with Lys(63)-specific DUB activity.
Highlights
Versatility in ubiquitin signaling is due partly to the large number of cellular proteins that interact with mono- or polyubiquitin to mediate downstream events and partly to the variety of ubiquitin polymers that can be attached to substrate proteins
Lys63-linked polyubiquitin chains mediate nonproteolytic events that include some forms of the DNA damage response
The Amsh-like protein (Amsh-LP) JAMM/MPNϩ domain contains two insertions that are absent from the Brcc36 JAMM/ MPNϩ domain, our results indicate that the Lys63 linkage preference of this family of DUBs derives from a similar mechanism whereby a high affinity interaction between the enzyme and the distal ubiquitin at the scissile bond of a dior polyubiquitin substrate is required for cleavage
Summary
Of Polyubiquitin Chains—Polyubiquitin (polyUb) dimers and tetramers were made as described [27] using E2-25K or Ubc13/Mms to generate Lys or Lys63-linked chains, respectively. All reactions contained 20 mM Hepes, pH 7.3, 1 mM DTT, and 1 mg/ml bovine serum albumin, were incubated at 37 °C, and stopped at the appropriate time (after 20 min, unless otherwise indicated) by the addition of Laemmli gel loading buffer. The proteins were exchanged into HDE buffer (20 mM Hepes, pH 7.3, 1 mM DTT, 0.1 mM EDTA) supplemented with 0.5 mg/ml bovine serum albumin and bound to Q-Sepharose, which was equilibrated with HDE buffer. The Q-Sepharose was washed with HDE buffer containing 20 mM NaCl, and the E2-25K-(Lys48-linked)Ub4 conjugates were eluted with HDE buffer containing 300 mM NaCl. To generate 125I-Lys63-Ub4-Ubc13, 5 M 125I-labeled Lys63Ub4 was incubated with 15 M Ubc13/Mms2-His heterodimer and 0.1 M E1 for 90 min at 37 °C in 50 mM Tris, pH 8, 5 mM.
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