Author: Dono Indarto
Indarto, Dono, 2013 Validating dipeptidyl peptidase (DP) 8 and DP9 potential substrates and investigating the effects of DP8 and DP9 overexpression and silencing on adenylate kinase (AK) 2 in ovarian cancer cells, Flinders University, School of Biological Sciences
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The dipeptidyl peptidase (DP) 4 gene family is a serine protease family that cleaves various biopeptides at the post-prolyl bond. DP4 is the most studied member of the family and is involved in regulating metabolism, the immune system and cell signaling. Fibroblast activation protein (FAP) is another member of the DP4 gene family that contributes to tissue proliferation during cell malignancy and wound healing. In contrast to DP4 and FAP, the biological functions of DP8 and DP9 are still under investigation although their mRNA and protein are ubiquitously expressed in human tissues. Previous work in the laboratory using a proteomics approach identified adenylate kinase 2 (AK2) and other proteins as potential in vivo DP8 and DP9 substrates and revealed a role for DP8 and DP9 in cell metabolism. AK2 is an enzyme that is localized in the intermembrane space of mitochondria. This enzyme has a pivotal role in catalyzing reversibly ATP and AMP to 2ADP, maintaining cellular energy homeostasis. The major aim of this study was to further validate the substrates identified and to investigate the role of these proteases in cellular metabolism, in particular in regulating the function of AK2. Immunocytochemistry and western blotting were used for studying DP8 and DP9 co-localization with AK2 and calreticulin in ovarian cancer cell lines (SKOV3) overexpressing DP8 and DP9, which were tagged with enhanced green fluorescent protein (EGFP). MALDI-TOF mass spectrometry (MS) was then used to test a further 12 substrates for cleavage by DP8 and DP9 that were previously identified using a recent proteomics approach. Cleavage activity of DP8 and DP9 towards AK2 in these SKOV3 cells was evaluated using proliferation rate and AK assays. Changes in adenine nucleotide levels (ATP, ADP and AMP) and cellular energy charge were assessed by using high performance liquid chromatography (HPLC). DP8 and DP9 small interfering RNA (siRNA) were used to silence these proteases in OVCA 429, OVCA 432 and SKOV3 cell lines in order to test the effects of each DP gene on AK2 protein and enzyme activity. Immunofluorescence confocal microscopy was used to demonstrate that DP8 and DP9 and their substrates AK2 and calreticulin are in close proximity. No difference in substrate localization was observed in cells expressing wt or mt proteases. The work also revealed that N-terminal oligopeptides of seven of 12 potential substrates are cleaved by DP8, DP9 and DP4. However cleavage rates differed between the three enzymes, reflecting differences in their active sites. These differences reflect different amino acids that line entry to the catalytic site and the site itself between the three proteases. In the first six hrs after sub culture in a 96 well plate cells expressing wt and mt DP8 had increased viability compared to vector expressing cells, while the proliferation rate of SKOV3 cells with wt and mt DP9 overexpression was lower. In contrast in a T75 flask it was observed that both wt and mt DP8 took longer to reach confluence and that vector and wt and mt DP9 expressing cells (four days compared to seven). In these SKOV3 cells overexpressing wt and mt DP9, ATP levels and adenylate energy charge decreased significantly, compared to those in SKOV3 cells overexpressing vector control. Meanwhile, ADP and AMP levels in SKOV3 cells overexpressing wt and mt DP9 increased but a significant increase in AMP level was only observed in SKOV3 cells overexpressing wt DP9. ADP/ATP and AMP/ATP ratios also increased in wt and mt DP9 overexpression. This data combined suggests opposite roles for both DP8 and DP9 in cell growth and proliferation that are independent of their enzyme activity. DP8 and DP9 were silenced in three different ovarian cancer cell lines. The results were more similar in OVCA 423 and SKOV3 cells than in OVCA 429 cells, this difference probably reflects the different phenotypes of these cells to start with. Intriguing data was obtained using silencing that suggests that in vivo in SKOV3 cells that AK2 is a substrate of DP8 but not DP9. DP8 silencing led to a decrease in AK specific activity and an increase in AMPK phosphorylation. A similar result was observed when AK2 was silenced in these cells. In contrast DP9 silencing had no significant effect on AK specific activity but did appear to increase the expression of AK2 and had no effect on AMPK phosphorylation In summary this work has provided additional evidence for the role of DP8 and DP9 in maintaining cellular metabolism. However, further work is required. Important questions that need to be answered is what effect does DP9 and or DP8 overexpression have on activated AMPK and the pathways downstream including glucose uptake and lactate production. In addition more work needs to be performed to investigate the effect of DP8 and DP9 silencing on genes downstream of AMPK. DP8 and DP9 may modulate AMPK an important player in both cellular metabolism and cancer growth. Further understanding of this role may lead to development of DP8/DP9 inhibitors or activators that may be used to treat cancers such as ovarian cancer.
Keywords: DP8,DP9,AK2,ovarian cancer cell
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Ass. Prof. Catherine A. Abbott