Physiologische Aufgaben und Regulation von DPP9, einer intrazellulären Prolyl-Dipeptidase

• Antragstellerin: Dr. Ruth Geiss-Friedlander
• Fachliche Zuordnung: Biochemie ; Zellbiologie
• Förderung: Förderung von 2010 bis 2022
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 178975760

• Erstellungsjahr: 2023

Zusammenfassung der Projektergebnisse

Serine proteases of the S9B/DPPIV family share the rare ability to cleave a peptide bond following proline. The overall goal of this project was to gain more insight into the physiological roles and regulation of a DPP9, an intracellular member of the S9B family, which was poorly characterized at the time this project was initiated. The starting point was the identification of two novel binding partners of DPP9: the small-ubiquitin like protein modifier (SUMO1) and the scaffold protein Filamin A (FLNA). Analysis of the DPP9-SUMO1 interaction revealed a novel binding surface on SUMO1, the E67 interacting loop (EIL). We further showed that the binding site for SUMO1 overlaps with an area important for DPP9 activity, and that SUMO1 is an allosteric activator for DPP9. We then developed peptide variants of the EIL that inhibit DPP9 with Ki values in the nanomolar range. Additionally, when analyzing the cellular localization of DPP9, FLNA and SUMO1, we noticed a nuclear localization of DPP9, in addition to its previously characterized cytosolic localization. We showed that this nuclear localization is due to the expression of a second DPP9 isoform which contains an N-terminal nuclear localization signal. We identified Syk, a tyrosine kinase important in B cell receptor mediated signaling (BCR), as a novel DPP9 substrate. DPP9 cleaves Syk N-terminus and targets the kinase to the N-degron pathway, thereby regulating the duration and strength of BCR-mediated signaling. Importantly, the interaction of DPP9 with Syk is promoted by FLNA. In collaboration with Prof. Dr Robert Huber, we presented first crystallographic structures of DPP8 and DPP9 revealing an extensive rearrangement in their active site upon ligand binding. We identified BRCA2, a key player in homologous recombination (HR) as a novel DPP9 substrate. we show that DPP9 targets BRCA2 for degradation by the N-degron pathway in response to DNA damage. Consistently, DPP9 depleted cells show defect in repair of DNA double strand breaks (DSBs) by HR, and are hypersensitive to genotoxic agents. In a collaborative project we contributed to the development of selective DPP8/9 inhibitors, providing essential tools for future research and potential therapeutic applications. Taken together, we showed for the first time that DPP9 is an upstream component in the N-degron pathway. We further showed the physiological consequences of these events, demonstrating a role for DPP9 in regulating BCR signaling and repair of DSBs. Finally, we showed that SUMO1 is an allosteric activator of DPP9 and that FLNA acts as a recruitment factor linking DPP9 to its substrates.

Projektbezogene Publikationen (Auswahl)

• A Novel SUMO1-specific Interacting Motif in Dipeptidyl Peptidase 9 (DPP9) That Is Important for Enzymatic Regulation. Journal of Biological Chemistry, 287(53), 44320-44329.
  Pilla, Esther; Möller, Ulrike; Sauer, Guido; Mattiroli, Francesca; Melchior, Frauke & Geiss-Friedlander, Ruth
  
• The SUMO1-E67 Interacting Loop Peptide Is an Allosteric Inhibitor of the Dipeptidyl Peptidases 8 and 9. Journal of Biological Chemistry, 288(45), 32787-32796.
  Pilla, Esther; Kilisch, Markus; Lenz, Christof; Urlaub, Henning & Geiss-Friedlander, Ruth
  
• The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus. Cellular and Molecular Life Sciences, 71(18), 3611-3626.
  Justa-Schuch, Daniela; Möller, Ulrike & Geiss-Friedlander, Ruth
  
• Identification of novel dipeptidyl peptidase 9 substrates by two‐dimensional differential in‐gel electrophoresis. The FEBS Journal, 282(19), 3737-3757.
  Zhang, Hui; Maqsudi, Sadiqa; Rainczuk, Adam; Duffield, Nadine; Lawrence, Josie; Keane, Fiona M.; Justa‐Schuch, Daniela; Geiss‐Friedlander, Ruth; Gorrell, Mark D. & Stephens, Andrew N.
  
• DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk. eLife, 5(2016, 9, 10).
  Justa-Schuch, Daniela; Silva-Garcia, Maria; Pilla, Esther; Engelke, Michael; Kilisch, Markus; Lenz, Christof; Möller, Ulrike; Nakamura, Fumihiko; Urlaub, Henning & Geiss-Friedlander, Ruth
  
• Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants. The EMBO Journal, 35(23), 2519-2535.
  Strenzke, Nicola; Chakrabarti, Rituparna; Al‐Moyed, Hanan; Müller, Alexandra; Hoch, Gerhard; Pangrsic, Tina; Yamanbaeva, Gulnara; Lenz, Christof; Pan, Kuan‐Ting; Auge, Elisabeth; Geiss‐Friedlander, Ruth; Urlaub, Henning; Brose, Nils; Wichmann, Carolin & Reisinger, Ellen
  
• Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer. Proceedings of the National Academy of Sciences, 115(7).
  Ross, Breyan; Krapp, Stephan; Augustin, Martin; Kierfersauer, Reiner; Arciniega, Marcelino; Geiss-Friedlander, Ruth & Huber, Robert
  
• DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages. Life Science Alliance, 2(1),e201900313.
  de Vasconcelos, Nathalia M; Vliegen, Gwendolyn; Gonçalves, Amanda; De Hert, Emilie; Martín-Pérez, Rosa; Van Opdenbosch, Nina; Jallapally, Anvesh; Geiss-Friedlander, Ruth; Lambeir, Anne-Marie; Augustyns, Koen; Van Der Veken, Pieter; De Meester, Ingrid & Lamkanfi, Mohamed
  
• Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP). ACS Medicinal Chemistry Letters, 10(8), 1173-1179.
  De Decker, An; Vliegen, Gwendolyn; Van Rompaey, Dries; Peeraer, Anke; Bracke, An; Verckist, Line; Jansen, Koen; Geiss-Friedlander, Ruth; Augustyns, Koen; De Winter, Hans; De Meester, Ingrid; Lambeir, Anne-Marie & Van der Veken, Pieter
  
• Dipeptidyl peptidase 9 triggers BRCA2 degradation by the N-degron pathway to promote DNA-damage repair. (2020, 8, 25). Wallstein Verlag.
  Silva-Garcia, Maria; Bolgi, Oguz; Ross, Breyan; Pilla, Esther; Kari, Vijayalakshmi; Killisch, Markus; Stark, Nadine; Lenz, Christof; Spitzner, Melanie; Gorrell, Mark D.; Grade, Marian; Urlaub, Henning; Dobbelstein, Matthias; Huber, Robert & Geiss-Friedlander, Ruth
  
• Aerosol-based ligand soaking of reservoir-free protein crystals. Journal of Applied Crystallography, 54(3), 895-902.
  Ross, Breyan; Krapp, Stephan; Geiss-Friedlander, Ruth; Littmann, Walter; Huber, Robert & Kiefersauer, Reiner
  
• A Photo-Crosslinking Approach to Identify Class II SUMO-1 Binders. Frontiers in Chemistry, 10(2022, 5, 30).
  Brüninghoff, Kira; Wulff, Stephanie; Dörner, Wolfgang; Geiss-Friedlander, Ruth & Mootz, Henning D.
  
• Chemoproteomics‐Enabled Identification of 4‐Oxo‐β‐Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9. Angewandte Chemie International Edition, 61(47).
  Carvalho, Luís A. R.; Ross, Breyan; Fehr, Lorenz; Bolgi, Oguz; Wöhrle, Svenja; Lum, Kenneth M.; Podlesainski, David; Vieira, Andreia C.; Kiefersauer, Reiner; Félix, Rita; Rodrigues, Tiago; Lucas, Susana D.; Groß, Olaf; Geiss‐Friedlander, Ruth; Cravatt, Benjamin F.; Huber, Robert; Kaiser, Markus & Moreira, Rui
  
• Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA damage repair. EMBO reports, 23(10).
  Bolgi, Oguz; Silva‐Garcia, Maria; Ross, Breyan; Pilla, Esther; Kari, Vijayalakshmi; Killisch, Markus; Spitzner, Melanie; Stark, Nadine; Lenz, Christof; Weiss, Konstantin; Donzelli, Laura; Gorrell, Mark D; Grade, Marian; Riemer, Jan; Urlaub, Henning; Dobbelstein, Matthias; Huber, Robert & Geiss‐Friedlander, Ruth
  
• The amino-dipeptidyl peptidases DPP8 and DPP9: Purification and enzymatic assays. Methods in Enzymology (2023), 289-323. Elsevier.
  Donzelli, Laura; Bolgi, Oguz & Geiss-Friedlander, Ruth