Human DDI2 is a ubiquitin-directed endo-protease

The Ddi1/DDI2 proteins are classified as ubiquitin shuttling factors and are implicated in a wide range of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, these proteins contain a conserved region with similarity to retroviral proteases, but whether and how they function as proteases has remained unknown. Here we show that human DDI2 knock-out cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. Thus far, no evidence for DDI2 acting as a de-ubiquitylating enzyme has been uncovered, suggesting that it might cleave the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is capable of cleaving NRF1 in vitro, but only when NRF1 protein is poly-ubiquitylated. Together, these data suggest that DDI2 represents the first example of a ubiquitin-directed endo-protease.