Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import (2020)
Figure 4

Figure 4. AK2 is target of the cytosolic dipeptidyl peptidases DPP8 and DPP9.

The N-terminus of AK2. Proteomic analysis of wildtype AK2 immunoprecipitated from HEK293 cells revealed the presence of four different N-termini: 'MAPS-', 'APS-', 'S-', and 'acetylated S-'. The processing from 'MAPS-' to 'APS-' is likely caused by methionine amino peptidase (MAP). The second processing step might be caused by the cytosolic DPP4 protease family members DPP8 and 9 that are known to cut at the N-terminus peptides of two amino acids length preferably after prolines. The N-terminus of AK2 is highly conserved as a sequence analysis of AK2 homologues from 163 species revealed (logo plot).

Steady state levels of AK2C40S,C42S,C92S-HA in presence and absence of DPP8 and DPP9. 72 h before experiments, HEK293 cells stably expressing AK2C40S,C42S,C92S-HA were transfected with control siRNA or siRNAs directed against DPP8 or/and DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. DPP9 depletion led to an increase in AK2C40S,C42S,C92S-HA levels, while DPP8 depletion did not. Combined depletion of DPP8 and DPP9 increased AK2C40S,C42S,C92S-HA levels even further. This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup. White and black arrow heads indicate the endogenous and HA-tagged AK2, respectively. The gray arrow head indicates the DPP9 band. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. * represents p<0.05, ,## represent p<0.01 and ***,### represent p<0.001.

Steady state levels of AK2C40S,C42S,C92S-HA upon incubation with the DPP9 inhibitor 1G244 (Wu et al., 2009). As (B) except that cells were treated for 16 h with inhibitor or DMSO as control. AK2C40S,C42S,C92S-HA levels were strongly increased upon DPP8/9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. *** represents p<0.001.

Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40. 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and/or DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. If import of endogenous AK2 was prevented by depletion of MIA40, then concomitant depletion of DPPs increased its levels. Strikingly, depletion of DPP9 alone also increased the levels of endogenous AK2. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01.

Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.

Steady state levels of AK2-HA variants with mutated N-termini upon incubation with either 1G244 or vehicle control. HEK293 cells stably expressing AK2-HA variants were lysed and subjected to immunoblot against the indicated proteins. Levels of DPP8/9-insensitive AK2 variants are strongly increased. Reported values are the mean of 4 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01 and ###, *** represents p<0.001. Asterisk denominate statistical comparison with respective control, and hash tag represents comparison to N-WT.