Cellular Proteases as molecular switches
Cellular proteases play essential roles for cellular homeostasis. The best-studied function of proteases is the degradation of unwanted, damaged or regulatory proteins. Additionally proteases act as non-reversible post-translational protein modifiers in a process termed limited proteolysis. By acting as sharp scissors proteases can remove defined parts of a protein/polypeptide resulting in altered N- or C- termini of their targets (neo-termini). The exposure or removal of parts of a polypeptide chain can then lead to a variety of outcomes, including changes in stability, protein interactions, catalytic activity or even cellular localization of the processed target. Consequently, proteases are involved in numerous physiological processes including cell metabolism, apoptosis, immunity and development. Deregulation of protease activity, localization or expression is linked to pathophysiological processes such as cancer, auto-immunological and neurodegenerative disorders. Work in the lab focuses on a small family of proteases from the dipeptidyl family. DPP8 and DPP9 are two widely distributed intracellular proteases. On the molecular level, DPP8 and DPP9 are unique because they can remove dipeptides from the N-terminus of their substrates, if the residue in second position is an alanine or a proline (Xaa-Pro/Ala) (MEROPS - the Peptidase Database). Inhibition of DPP8 and DPP9 impair cell adhesion and migration, and result in increased cell death. Knock-in mice expressing an inactive variant of DPP9 die shortly after birth, demonstrating the importance of this protease for neonatal survival. The molecular mechanisms leading to these outcomes are poorly understood.
To uncover the physiological roles of DPP8 and DPP9, we combine biochemical approaches with cell-based assays.
DPP9 functions in the immune system
Previously, we demonstrated that DPP9 is the rate-limiting enzyme for cleavage of proline-containing peptides in the cytosol, and demonstrated a role for DPP9 in antigen maturation for presentation on MHC class I alleles.
Importantly, we recently identified a novel substrate of DPP9, the tyrosine kinase Syk which is a central kinase in B cell-receptor (BCR) mediated signalling. We find that DPP9 preferentially cleaves Syk in its activated state, thus acting as a negative regulator for Syk signalling. Consequently, DPP9 influences both the strength and the duration of the cellular response to stimulation of the B cell receptor.
Previously we identified the small ubiquitin like protein modifier SUMO1 as an allosteric regulator of DPP9. Ongoing projects in the lab further address the role of the DPP9-SUMO interaction, especially in screening for sumoylated substrates and regulators of DPP9.
The N-end rule pathway
Mutagenesis analysis combined with siRNA studies and in vitro processing demonstrate that cleavage of Syk N-terminus by DPP9 leads to the exposure of a Neo N-terminus with a serine in first position. Surprisingly, the serine acts as a destabilizing residue targeting Syk for degradation by the N-end rule pathway. Current work aims at identifying further DPP9-substartes which are targeted to the N-end rule, as well as studying the role of the unusual serine as a novel N-degron.
DPP8 and DPP9 inhibitors
We developed DPP8 and DPP9 specific inhibitors, showing a high selectivity over DPPIV: 'Specific DPP8 and/or DPP9 inhibitors.' Inventors: Ruth Geiss-Friedlander and Esther Pilla. WO2014068023, US9593148B2., and Pilla, E., et al (2013) J Biol Chem 288: 32787-32796. Enzyme kinetics assays with the peptide inhibitor SLRFLYEG reveal a cooperative allosteric mechanism of these inhibitors. Furthermore, the structure of DPP8 with SLRFLYEG is now solved, revealing an extensive rearrangement at the active site (DOI: /10.1073/pnas.1717565115).