Characterization of Phospholipase D3’s Activity, Structure, and Regulation in Lysosomal Metabolism and Disease

The phospholipase D3, along with the closely related phospholipase D4, has recently been described as lysosomal 5’-exonuclease specifically degrading single-stranded nucleic acids. Not only does their deficiency lead to a severe deregulation of the innate immune system and to a massive autoinflammatory phenotype, but variants in the PLD3 gene have been linked to the development of late-onset Alzheimer’s Disease by genome-wide association studies. These studies however have been disputed among different authors using different data sets to confirm or refute the association. To clarify this association, as well as to refine its role in the innate immune system, more data on PLD3’s cellular and molecular function is necessary. This thesis therefor aimed to provide methods and cellular data for the further characterization of PLD3’s function.

Using a stable mammalian expression system, the soluble domain of PLD3 was produced with high purity. With an adaptation concerning the cell lines used for expression, this protocol was also applicable to produce high amounts of PLD3 with a homogenous glycosylation pattern that could be used in a collaboration project to resolve the crystal structure of PLD3 binding a single-stranded oligonucleotide. Furthermore, the recombinant enzyme was used to develop a fluorescence-based assay for the specific determination of PLD3’s 5’-exonuclease activity, which also proved to be applicable for whole cell lysates. This enabled the functional characterization of sequence variations and mutations, including those linked to the development of neurodegenerative diseases. Another focus of this thesis was to provide insights into PLD3’s regulation inside the cell. This included a more thorough description of PLD3’s proteolytic processing inside the lysosome and contributed to the discovery of homodimerization being crucial for its correct folding and trafficking.

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