# Proximity labeling reveals non-catalytic interactions between DPP9 and ubiquitin signaling complexes

## TL;DR

This study uses proximity labeling to identify new interactions of DPP9 with ubiquitin signaling complexes, revealing its non-catalytic roles in cellular processes.

## Contribution

The study identifies novel non-catalytic interactions of DPP9 with ubiquitin signaling components using TurboID-based proximity labeling.

## Key findings

- DPP9 interacts with ubiquitin signaling complexes like BRISC and CYLD-SPATA2.
- DPP9 disrupts interactions between BRCC36/BRCC3 and ABRO1/ABRAXAS2, and between CYLD and SPATA2.
- The findings suggest DPP9 has regulatory roles in ubiquitin signaling beyond its catalytic activity.

## Abstract

Dipeptidyl peptidase 9 (DPP9) is an amino-peptidase with roles in immunity, DNA-repair, cell signaling, memory and neonatal survival; its dysregulation is linked to cancer and immune-related disorders. While many studies focus on its catalytic activity, scaffolding functions of DPP9 are emerging. Here, we mapped the DPP9 interactome using TurboID-based proximity labeling in DPP9 knock-out HEK293 cells reconstituted with doxycycline-inducible miniTurboID-DPP9, allowing fine-tuned expression that approximates physiological levels. Besides known partners, proteins involved in autophagy, mRNA decay and ubiquitin signaling along with DPP8, were strongly enriched among the identified DPP9 binding partners. Notably, we validated DPP8, the E3 ligase CBL, the deubiquitinase complex CYLD-SPATA2 and the BRISC complex components BRCC36/BRCC3 and ABRO1/ABRAXAS2 as novel DPP9 interactors. Furthermore, NanoBRET assays in living cells demonstrated that DPP9 disrupts the binding between BRCC36/BRCC3 and ABRO1/ABRAXAS2, and the interaction of CYLD with SPATA2, thereby compromising these protein-protein interactions. Mechanistically, these findings reveal physical and potentially regulatory interactions between DPP9 and components of the ubiquitin system and provide a basis for dissecting the non-catalytic functions of DPP9.

The online version contains supplementary material available at 10.1007/s00018-025-06021-z.