ns and Ub governs the directionality of their enzymatic activity. The hydrolase (PR deubiquitinase) activity is favored by high affinity and longer residence time of the PR-ubiquitinated substrate to DupA/B, while the transferase (PR-Ub ligase) activity is dictated by lower-affinity interactions of the SidE family PDE domain with ADPR-Ub.

Identification of PR-Ubiquitinated Substrates upon *Legionella* Infection {#sec2.4}
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Until now, no general workflow for the specific enrichment of PR-ubiquitinated proteins has been presented. Initial concepts relied on the use of tagged Ub that lacks the C-terminal GG motif (Ub^ΔGG^) and can only be attached to other proteins by PR ubiquitination. However, this concept has several drawbacks and limitations, as it relies on overexpression of tagged Ub, which might stress the cell and is only usable in genetically engineered or transfected cells. Therefore, we aimed to establish a protocol that enables the efficient enrichment of PR-ubiquitinated proteins that do not rely on genetic perturbations.

Given that DupA displayed strong binding affinity to PR-ubiquitinated substrates, we hypothesized that catalytic inactive mutants of DupA (H67A or H189N) could be used as trapping mutants to enrich PR-ubiquitinated proteins from cellular lysates for subsequent proteomic analysis. To test this, we first co-expressed SdeA together with HA-tagged Ub 1-74 (HA-Ub^ΔGG^), which lacks two glycine residues at the carboxy terminus, thereby preventing canonical ubiquitination. SdeA utilizes HA-Ub^ΔGG^ to catalyze ubiquitination of cellular proteins, as demonstrated by a smear of PR-ubiquitinated proteins in cells ([Figure 4](#fig4){ref-type="fig"}A). Importantly, both DupA inactive mutants (H67A or DupA H189N) effectively bound and enriched PR-ubiquitinated substrates from cells co-expressing Ub^ΔGG^ and WT SdeA, but not canonical ubiquitinated proteins from cells expressing mutant SdeA (H277A or EE/AA, [Figures 4](#f