Oxygen-dependent asparagine hydroxylation of the ubiquitin-associated (UBA) domain in Cezanne regulates ubiquitin binding.

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain-containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn³⁵ within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBA^Cez), which lacks conserved UBA domain properties. We show that UBA^Cez binds Lys¹¹-, Lys⁴⁸-, Lys⁶³-, and Met¹-linked ubiquitin chains in vitro, establishing UBA^Cez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBA^Cez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn³⁵ inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBA^Cez Our results indicate that UBA^Cez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.

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