Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA-damage repair (2022)
Figure 2

Figure 2: The BRCA2 N-terminal dipeptide Met-Pro is processed by DPP9

A. Surface Plasmon Resonance (SPR) data showing a direct interaction of purified DPP9 with a BRCA21-40 peptide and a truncated BRCA23-40 peptide, which lacks the N-terminal dipeptide Met-Pro. A serial dilution of BRCA2-derived peptides was injected over a surface covered with DPP9. Equilibrium binding isotherms obtained for interactions measured between DPP9 and BRCA21-40 (black line) and BRCA23-40 (red line). Data were fitted to a sigmoidal dose-response curve fit. Mean ± SEM of technical triplicates of a representative experiment out of 3.

B. Pull-down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N-terminal1-40 fragment immobilized on HA-beads. The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21-40HA for interaction with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.

C. Quantification of PLAs showing fewer MMC-induced DPP9-BRCA2 PLA events in HeLa cells treated with 10µM 1G244. Each dot represents the number of PLA events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001).

D. Representative images of DPP9-BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071 (these images, along with the corresponding controls can be found in Appendix Figure S1C).

E. In vitro processing of BRCA21-40 synthetic peptide by purified recombinant DPP9. Samples were analysed by high-resolution liquid chromatography/tandem mass spectrometry, in quadruplicate. The panels show extracted MS1 ion chromatograms for both substrate BRCA21-40 peptide (MPIGSKERPT…) (labelled S, [M+5H]5+ m/z 917.8637; retention time 17.7 min) and product BRCA23-40 peptide (IGSKERPT…) (labelled P, [M+5H]5+ m/z 872.2451; retention time 17.6 min). The identity of the product peak was established both by accurate mass measurement to within 5 ppm and by product ion spectra.

F. Pull-down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N-terminus. While each protein can bind to the BRCA2 N-terminal peptide, in the presence of DPP9, the PALB2-BRCA2 binding is negatively affected. Representative data of three technical replicates is shown. Anti-PALB2: RRID:AB_890607, anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.

G. Peptide sequences used in the DPP9 competition assays shown in (H).

H. DPP9 activity assays showing different competitive effects of BRCA2 peptides (G), hydrolysis of GP-AMC is used as read out. Shown are % of inhibition in GP-AMC processing normalised to the control reactions (DPP9 without competing peptides). Syk1-31 was used as a positive control. n=3 independent experiments. Data were analysed by a one-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001).