Proteolytic enzymes (peptidases) participate in and regulate an increasing number of important cellular processes by specifically cleaving their target proteins. Although structurally and biochemically very well characterized, many peptidases still lack information about their specific substrates and physiological roles.

Dipeptidyl peptidase III (DPP III) is the cytosolic metallopeptidase that is well characterized at the structural and enzymatic level, however its physiological roles are still largely unknown. DPP III peptidase activity towards four to ten amino acid long oligopeptides indicates its role in the terminal stages of protein turnover, while its activity, and affinity towards certain biologically active peptides indicates its role in the pain modulation, blood pressure control, and inflammation.

Furthermore, its involvement in the oxidative stress response through the interaction with Keap1 protein has been well documented. However, none of the proposed roles of DPP III have been proven, yet, and there are indications that it might also have other roles in the cell physiology. Its proposed role in Nrf2/Keap1 pathway, taken together with its overexpression in tumor tissues of several different tumor types, indicate that it might be a biomarker of certain types of cancer or even the target for anticancer therapy.

The aim of this project is to elucidate the physiological roles, and disease relevance of DPP III by determining its interactors through the combined use of different molecular biology approaches. We plan to use yeast two-hybrid method to identify novel interacting partners of DPP III, and mass spectrometry interactome analysis to identify protein complexes DPP III takes part in.

We plan to confirm the interactions found by Y2H and MS approach by several low-throughput methods. Finding DPP III interactors would enable us to identify other signaling pathways DPP III might be involved in. We will also test if any of newly identified DPP III interacting proteins are also DPP III substrates. Apart from gaining new insights into physiology of DPP III, this project will also enable transfer of technology and knowledge to the Ruđer Bošković Institute, which will have a positive impact on our future research efforts, and represent the first step towards procuring continuous research funding from EU funds.

Principal Investigator

Koraljka Husnjak, PhD
Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany

Co-Principal Investigator

Associates

• Marija Abramić, PhD, IRB, LBPMM
• Zrinka Karačić, mag. chem., IRB, LBPMM
• Ana Tomašić Paić, PhD, IRB, LBPMM
• Snježana Jurić, PhD, IRB, LBPMM
• Akmaral Kussayeva, PhD, LBPMM, IRB
• Sara Matić, MSc, LBPMM, IRB
• Iva Pavlović, PhD, LBPMM, IRB

Activities on the Project

Mihaela Matovina visited dr. Koraljka Husnjak Laboratory at the Institute of Biochemistry II, Medical School of Goethe University in Frankfurt am Main, from October 2nd to 22nd 2016. During the visit, she performed Y2H experiments aimed at determining DPP III protein interaction partners on the whole proteome scale.

Zrinka Karačić, Ana Tomašić Paić and Mihaela Matovina participated at 3rd DPP III Minisymposium on February 10th, 2017 in Graz, Austria. Mihaela Matovina presented the poster entitled „Methodology of DPP III interactome analysis“.

Koraljka Husnjak visited Ruđer Bošković Institute on April 13, 2017. At that time, we discussed the execution of the work plan, and Koraljka Husnjak prepared mouse embryonic fibroblasts (MEFs) from DPP III wt and knock-out (KO) embryos at developmental stage E13.5. We will use those cell lines for the experiments in collaboration with the Laboratory for for mitochondrial bioenergetics and diabetes from RBI and our collaborators from the Institute of Biochemistry of Graz University of Technology, Graz, Austria.