Regulation of BAP1 activity by novel cancer-associated glutamate residues through ubiquitin binding and PARylation

BAP1 is a deubiquitinase with a ubiquitin C-terminal hydrolase domain that functions as a tumor suppressor and regulates key cellular processes, including DNA repair. Mutations in BAP1 are prevalent in cancers such as mesothelioma, uveal melanoma, and clear cell renal cell carcinoma, yet the functional consequences of individual residues remain incompletely defined. Here, using structural analysis, mutational screening, and cellular and in vitro assays, we identify three cancer-associated glutamate residues—Glu31, Glu200, and Glu201—that are also PARylation sites, as critical for BAP1's catalytic activity. Glu31 directly promotes ubiquitin binding through salt bridge formation with Arg72 of ubiquitin, whereas Glu200 and Glu201 contribute indirectly without direct ubiquitin engagement. Our data suggest that Glu200 and Glu201 help preserve the local structural environment—including the positioning of Glu31 through the β1–β2 loop—thereby supporting a conformation required for efficient catalysis. We further show that PARylation inhibits BAP1 activity, likely by disrupting these salt bridges, thereby providing a dynamic mode of enzymatic regulation. These findings define previously uncharacterized cancer-associated residues essential for BAP1 function, reveal a novel salt bridge–mediated mechanism that enhances ubiquitin binding, and suggest that PARylation serves as a regulatory switch for BAP1 activity with potential implications for tumorigenesis and therapeutic targeting.