journal contribution
posted on 2023-03-31, 01:44 authored by Hongzhuang Peng, Jeremy Prokop, Jayashree Karar, Kyewon Park, Li Cao, J. William Harbour, Anne M. Bowcock, S. Bruce Malkowicz, Mitchell Cheung, Joseph R. Testa, Frank J. Rauscher Introduced mutations in BAP1 that are predicted to disrupt the helix structure of the BAP1-ULD domain dramatically disassociate its binding to the ASXL2-AB box. The GST-UCH association assay was performed with highly purified recombinant ULD-WT, L-P, or F-P mutant proteins and/or ASXL2-AB box proteins.
Funding
NCI
Jayne Koskinas Ted Giovanis Foundation for Health and Policy
Palmira and James Nicolo Family Research Fund
International Association of Heat and Frost Insulators & Allied Workers
Ovarian Cancer Research Fund Alliance
Italian Association for Cancer Research
Samuel Waxman Cancer Research Foundation
Susan G. Komen
Office of the Assistant Secretary of Defense for Health Affairs
History
ARTICLE ABSTRACT
Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1–ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable.Significance: Combined computational and biochemical approaches demonstrate that the BAP1–ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1–ASXL2 binding. Cancer Res; 78(5); 1200–13. ©2017 AACR.
