Consistent with a clear separation of biological function be

Consistent with a clear separation of biological function between ARISC and BRISC in cells, mutations in BRCC36 and Abraxas are prevalent in cancer genomes whereas mutations in KIAA0157 are rare. A survey of the Catalogue of Somatic Mutations in Cancer (COSMIC) consortium, revealed that to date, 37 substituting mutations were mapped to BRCC36 (>15,000 samples sequenced) and 35 to Abraxas (>19,000 samples sequenced), while only 1 mutation was mapped to KIAA0157 (>19,000 samples sequenced) (Table S3). The mutations in BRCC36 and Abraxas map to both MPN and CCHB domains (and additionally for Abraxas, the regulatory C-terminal tail, deleted in the KIAA0157 crystallization construct) and encode both single site amino Caspase-4 Colorimetric Assay Kit substitutions and truncations, many of which would be predicted to cause loss of protein function. In contrast, only one mutation mapped to KIAA0157, and it resided outside the MPN and CCHB domain without a predictable effect on function. These observations demonstrate a strong selection for the loss of ARISC function in cancers and conversely, no specific selection against BRISC function. In terms of a broader perspective on DUB regulation, it is fascinating that many multi-subunit DUBs contain two different MPN domain proteins. This feature is reminiscent of a subset of eukaryotic protein kinases, which employ dimerization to allosterically regulate their enzyme activity. Considerable diversity is displayed in how different protein kinase families dimerize, ranging from side-to-side, back-to-back, and head-to-tail configurations of kinase domains displayed by the RAF, eIF2α, and EGFR families (Lavoie et al., 2014). This diversity reflects differences in the underlying modes of catalytic regulation. In the case of the RAF family and EGFR family kinases, specific members like KSR and HER2, respectively, have dispensed with catalytic function altogether, while maintaining the ability to transactivate other family members through dimerization (Lavoie et al., 2014). These specific examples parallel the operation of MPN+–MPN– paired DUBs. We see that BRCC36 (MPN+) heterodimerizes with the MPN– pseudo DUBs KIAA0157 or Abraxas using a mode of interaction that is very different from that of the CSN5–CSN6 and RPN8–RPN11 heterodimers. This likely reflects the fact that the mechanism by which KIAA0157/Abraxas support BRCC36 will differ from how CSN6 and RPN8 support/regulate the function of CSN5 and RPN11. As approximately 10% of DUBs are predicted to be pseudo DUBs (Nijman et al., 2005), more examples of DUBs regulated by inactive pseudo DUBs are likely to emerge. Understanding the scope of DUB–pseudo DUB pairings may reveal new insights into how these important enzymes are regulated and how they achieve isopeptide hydrolysis.
Experimental Procedures
Acknowledgments This work is based upon research conducted at the Advanced Photon Source and the Northeastern Collaborative Access Team beam lines, supported by a grant from the National Institute of General Medical Sciences (P41 GM103403) from the National Institutes of Health and by the U.S. DOE under contract No. DE-AC02-06CH11357. The Pilatus 6M detector on 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). We are grateful to Zhiqin Li for baculovirus/insect cell support and to Alessandro Datti, Frederick Vizeacoumar, and Thomas Sun (LTRI SMART facility) for providing robotics. This work was supported by a Human Frontiers Science Program fellowship and a Sir Henry Wellcome postdoctoral fellowship (to E.Z.); Canadian Institutes of Health Research grants MOP-126129, MOP-57795 and Foundation grant (to F.S.), and MOP-67189 (to A.G.); Canada research chairs in structural biology (to F.S.); and by NIH grants CA138835, CA17494, GM101149 (to RAG) who is also supported by a Harrington Discovery Institute Scholar-Innovator Award, and funds from the Abramson Family Cancer Research Institute and Basser Research Center for BRCA.