Oral Presentation 9th General Meeting of the International Proteolysis Society 2015

Dipeptidyl peptidase 9 (DPP9): knockin mouse, novel natural substrates and association with cell adhesion, focal adhesion kinase and paxillin. (#27)

Hui Zhang 1 , Yiqian Chen 1 , Margaret G Gall 1 , Carol Wadham 2 , Geoff W McCaughan 1 , Ruth Geiss-Friedlander 3 , Denise Yu 2 , Andrew N Stephens 4 , Mark D Gorrell 1
  1. Centenary Institute, Sydney, NSW, Australia
  2. Childens Cancer Institute Australia, Sydney, NSW, Australia
  3. Georg-August-University of Goettingen, Goettingen, Germany
  4. Hudson Instiitute, Monash University, Melbourne, Vic, Australia
DPP9 is a ubiquitously expressed intracellular DPP having short (DPP9-S; cytoplasmic) and long (DPP9-L; nuclear) forms.  DPP9 is implicated in tumour biology, immunity, apoptosis, intracellular epidermal growth factor (EGF)-dependent signalling, cell adhesion and cell migration. Our homozygous mice lacking DPP9 enzyme activity (KI; knockin Ser-to-Ala) die soon after birth. The subcellular localisation in human hepatoma Huh7 cells of a DPP9-S - GFP chimeric fluorescent protein was visualised using confocal and super-resolution microscopy. While some DPP9 was associated with mitochondria, the strongest co-localisation was with microtubules. Upon EGF stimulation, some DPP9 re-distributed towards the ruffling membrane at the leading edge of the migrating cell, where it co-localised with the focal adhesion proteins integrin-β1 and talin. DPP9 gene silencing or enzyme inhibition reduced cell adhesion and migration and reduced expression of integrin-β1 and talin and the phosphorylation of focal adhesion kinase (FAK) and paxillin. Moreover, enzyme inhibition suppressed the viability of Huh7 cells (MTS assay). Mass spectrometry of 2D-DIGE spots identified 111 novel DPP9 substrates in KI mouse embryonic fibroblasts: 56 of these substrates contain an N-terminal Xaa-Pro and 55 contain Xaa-Ala. Xaa-Ala was not hydrolysed by DPP8. DPP9-mediated cleavage of nine peptides and full length proteins was confirmed by MALDI-TOF or immunoblotting. Those proteins are associated with immunity and cellular secretory processes.  PCR array analyses of neonate KI mice and follow-up in Huh7 cells suggested a defect in obtaining energy from lipid. These novel findings provide mechanistic insights into the regulatory role of DPP9 in hepatoma cell movement, and implicate DPP9 in tissue and tumour growth and adhesion. The degradomics data expand the potential roles of DPP9 and suggest different roles for DPP9 versus DPP8.

References:  1. Gall 2013 PLOS ONE 8:e0078378. 2. Zhang 2013 Molec Cancer Res 11:1487-96.  3. Zhang 2015 BBA-Molec Cell Res 1853:470-80.  4. Zhang 2015 FEBS Journal.