The physiological roles and regulation of DPP9 - an intracellular prolyl dipeptidase

• Applicant: Privatdozentin Dr. Ruth Geiss-Friedlander
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2010 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 178975760

The focus of this proposal is DPP9, an intra-cellular amino-dipeptidase, which has the unique ability to cleave off dipeptides from the N-termini of proteins having a Pro (or Ala) residue in second position (NH2-Xaa-Pro/Ala). Previously, we showed that DPP9 localizes to the nucleus and to the cytosol, where it is rate limiting for cleavage of Pro-containing peptides, and is activated in an allosteric manner by SUMO1. Works from others show a role for DPP9 in cell migration, apoptosis, pyroptosis, cancer and neonatal survival. The underlying molecular mechanisms are poorly understood. For more insight into DPP9 functions, we searched for DPP9-interacting proteins, arguing that these include DPP9 substrates and regulators. One of the identified proteins was Filamin A (FLNA), a protein that cross-links actin filaments. In this funding period we showed that FLNA recruits DPP9 to Syk, a central kinase in B-cell signaling. This resulted in N-terminal processing of Syk by DPP9, demonstrating Syk as a novel DPP9 substrate. Cleavage by DPP9 produced a neo Syk N-terminus with a Ser in position 1. Pulse-chases combined with mutagenesis studies revealed that this Ser negatively influences Syk stability. Consistently, DPP9 silencing or inhibition stabilized Syk, thereby modulating Syk-mediated signaling, defining DPP9 as a novel negative regulator of Syk. Furthermore, our results show that DPP9 is an up-stream component of the N-end rule pathway, a route for regulated degradation of proteins based on their first N-terminal residue.Apart from actin, FLNA interacts with multiple proteins, including the Ser/Thr Kinases PKCs and proteins involved in DNA damage repair. We now report that DPP9 processes the N-terminus of PKCa and PKCg. Preliminary work suggests that the interaction of DPP9 with PKCs involves a second binding site (exosite) in addition to the active site. In the next funding period we aim to take a decisive step towards understanding the interactions of DPP9 with its substrates, and whether an exosite contributes to substrate specificity. We will investigate whether cleavage of PKCs by DPP9 is mediated by FLNA, and what the molecular and cellular outcomes of these cleavage events are. A second protein we will investigate is BRCA2, a central component in the repair of double strand DNA breaks (DSB). We show that DPP9 interacts with BRCA2 in cells, and demonstrate that it cleaves a peptide corresponding to BRCA2 N-terminus. Exposure of cells to Mitomycin C, which causes the formation of DSB, leads to an increase in the number of DPP9 - BRCA2 interactions, suggesting a role for DPP9 in regulating DSB repair. We plan to further analyze in detail the cleavage of BRCA2 by DPP9 in vitro, the cellular outcomes of BRCA2 processing by DPP9, and the possible involvement of DPP9 in regulating the repair of DSB. A special focus will be given to the N-end rule pathway, since DPP9 cleavage exposes a destabilizing residue in BRCA2 N-terminus.

• DFG Programme: Research Grants