Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin’s Gly⁷⁶. Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD⁺. Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD⁺ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.

泛素化和其他翻译后修饰之间的细胞串扰有助于调节许多过程。一个例子是 E3 DTX3L/ADP-核糖基转移酶 PARP9 异二聚体对泛素羧基末端的 ADP-核糖基化，但其机制仍然难以捉摸。在这里，我们表明独立于 PARP9，DTX3L 和其他人类 Deltex 蛋白（DTX1 到 DTX4）的保守羧基末端环和 DTC（Deltex 羧基末端）结构域催化泛素 Gly 的 ADP 核糖基化⁷⁶。结构研究揭示了 DTC 结构域在结合 NAD ⁺中的未知功能。Deltex RING 结构域招募了用泛素硫酯化的 E2，并将其与 NAD ^+并列与 DTC 结构域结合以促进泛素的 ADP 核糖基化。这种泛素修饰阻止了它的激活，但被连接的非特异性去泛素酶逆转。我们的研究提供了 Deltex E3s 对泛素 ADP 核糖基化的机制见解，并将使未来的研究能够旨在了解日益复杂的泛素串扰网络。