Abstract
The focus of this thesis is on phospholipases, which specifically cleave phospholipids. These lipids are not only the basic building blocks of our cells’ membranes, but they are also potent cell signaling molecules, modulating pathways that determine a cell’s fate. In Part A of this thesis, I study the phospholipase
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called autotaxin (or ATX), an essential enzyme that cleaves a specific lipid type: lysophospholipids. The most physiologically relevant products of autotaxin is a series of lipids called lysophosphatidic acids (LPAs). LPAs are necessary for many life processes, such as embryonic development and blood vessel growth, but are also the drivers for many diseases, as they can trigger cancer metastasis, inflammation and fibrosis, among others. In 2011, the crystal structure of autotaxin revealed a triple interconnected binding side: the tripartite site. This site encompasses the catalytic active site, the hydrophobic pocket, and the allosteric site known as tunnel. Here, I explore the role of the tunnel to unveil a new regulatory mechanism, where the LPA product accelerates its own synthesis by binding to the tunnel. I also investigate how the tunnel acts as a lipid-binding site that facilitates receptor-specific delivery of LPA to cell-surface receptors. As different inhibitors have been designed to block autotaxin’s activity, we compare two different types in liver disease models to gain new insights into the clinical benefit of blocking the tunnel. We also analyze the binding mode of compounds used for positron emission tomography (PET), in hopes that they can be used for non-invasive detection of autotaxin. Additionally, we collaboratively discover and optimize a new hybrid series of autotaxin inhibitors binding both the tunnel and the active site. This part concludes with a discussion of the current view on the enzymatic and non-enzymatic functions of autotaxin. I also examine how clinical success of drug candidates targeting autotaxin depends on binding the tunnel, thereby inhibiting the non-enzymatic signaling functions that I uncover in my research. In Part B of this thesis, I research another family of phospholipases: the glycerophosphodiester phosphodiesterase (or GDPD) family. GDPDs recognize a complex lipid anchor (called glycosylphosphatidylinositol or GPI) that cells attach to some proteins. Cleavage of this anchor by the GDPDs will shed a specific protein off the cell surface into, for instance, the blood, where it can have a specific function, or be used as a biomarker for cancer. The emerging picture in this relatively new field is that the activity of specific GDPDs relates to a positive prognosis of breast cancer and neuroblastoma, among others. Here, I study the determinants of this behavior by removing different stretches of GDE2’s C-terminal amino acid sequence. We then analyze the impact of altered intracellular trafficking on neuroblastoma cell lines. Given the accumulated evidence of the role of GDE2 and GDE3 in malignant cancers, I also venture to identify new clinically relevant substrates they may recognize and cleave. This part concludes with a discussion of the main findings and implications of my work on the GDPD protein family, emphasizing on future research directions.
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