Lysosomal biology in autophagy: peculiarities of the interaction of DPPII and ADA in lysosomes

Autophagy is a cellular recycling pathway that targets macromolecules for lysosomal degradation. We aim at a better understanding of the role and regulation of two lysosomal enzymes critical for macromolecule turnover: dipeptidyl peptidase II (DPPII, EC 3.4.14.2) and adenosine deaminase (ADA, EC 3.5.4.4).

In lysosomes a wide variety of macromolecules, including proteins, complex carbohydrates, glycolipids and nucleic acids are degraded. The role of lysosomes in supporting cellular activities is especially important under conditions of nutritional deprivation, which leads to the activation of autophagy. Lysosomes are a major recycling center, providing the basic building blocks and energy for synthesis of essential biomolecules necessary for cell survival.

DPPII is a proline-specific dipeptidyl peptidase family enzyme, localized in lysosomes. It releases N-terminal dipeptides from peptides with proline or alanine in the penultimate position at acidic pH. Some neuropeptides were shown to be cleaved by DPPII. DPPII participates in degradation of myofibril proteins, in cell differentiation, pathogenesis of autoimmune diseases, and protection from cell death. Also, DPPII was suggested to play a role in the degradation of collagens.

It was shown that ADA may convert adenosine to inosine in lysosomes. Inosine can be transported to the cytosol and converted by nucleoside phosphorylase into hypoxanthine and ribose 1-phosphate, which can be used to support cellular energy requirements, particularly important under conditions of nutrient deprivation. Lysosomal compartmentalization of ADA may be important for maintaining cellular energy requirements under conditions when cytosolic adenosine levels are low.

Using a resonant mirror biosensor, fluorescence polarization, and differential spectroscopy techniques we have shown that DPPII binds ADA. The goal of the present project is to (a) investigate the physiological regulation of this interaction and the underlying molecular mechanisms; and (b) on a broader scale to study the regulation of protein-protein interactions inside the lysosome of fed control cells and of starving cells actively undergoing autophagy.

To this end, the next tasks are:

(1) To prove the formation of the enzymes complex in vitro, DPPII from bovine kidney cortex and lung and ADA from bovine lung will be isolated and purified up to the electrophoretically homogeneous state at the Laboratory of Metabolism of Adenylic Compounds at the Institute of Biochemistry of the Armenian National Academy of Science (IBANAS) using standard protein chemistry methods.

(2) Quantitative mass spectrometry (MS)-based proteomics study at the Department of Biology, University of Fribourg (UniFr) of protein complexes inside lysosomes and their regulation in starvation conditions. As focus will be set on the DPPII-ADA complex.

Participants:

Prof. Jörn Dengjel, University of Fribourg

Dr. Alvard Antonyan, Armenian National Academy of Science