Poster Presentation 50th Lorne Proteins Conference 2025

Decoding PLD3: Structural and functional discoveries with therapeutic potential for Alzheimer’s Disease (#153)

Maria Eleni Georgopoulou 1 2 3 , Kenta Ishii 1 3 4 , Stefan J Hermans 1 3 , Tracy L Nero 1 5 , Nancy C Hancock 1 3 , Gabriela AN Crespi 1 3 , Michael A Gorman 1 , Jonathan H Gooi 1 3 6 , Michael W Parker 1 2 3 5 7
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
  3. Structural Biology Laboratory, St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
  4. Present address: Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia
  5. Australian Cancer Research Foundation Facility for Innovative Cancer Drug Discovery, Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
  6. Present address: Research, Innovation and Commercialisation, University of Melbourne, Parkville, Victoria, Australia
  7. Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia

Approximately 421,000 Australians are affected by dementia; without any medical advancements by 2054 this number will double 1. One emerging target in Alzheimer’s disease (AD) pathology, particularly in late-onset AD which is the most common dementia form, is human phospholipase D3 (PLD3, UniProt ID: Q8IV08) 2. PLD3 variants are linked with increased vulnerability to neurodegeneration and amyloid pathology 3, with recent studies establishing a direct association between the enzyme and AD 4,5. PLD3 is one of the six members of the Phospholipase D (PLD) family. It is a type II transmembrane protein with a short cytosolic N-terminus, an α-helical single transmembrane domain and a luminal C-terminal domain. When the protein reaches acidic compartments, a cleavage action takes place releasing the soluble luminal domain 6 and there is no evidence so far of any membrane signal transduction abilities 2.

Our research aims to shed light on PLD3 biology and provide the groundwork for the development of novel AD therapeutics. We designed a construct, and then expressed and purified the soluble (luminal) domain of human PLD3 2. Extensive characterisation, including dynamic light scattering and mass photometry, revealed a dynamic equilibrium between monomers and dimers with the latter favoured at lower pH. Enzymatic assays confirmed PLD3 had 5’ exonuclease activity whereas it had no measurable phospholipase activity 2. Our PLD3 structural studies resulted in crystals diffracting at 2.3 Å resolution and the structure was solved (PDB ID: 8V5T) by molecular replacement using an AlphaFold2 model. We mapped the location of reported PLD3 AD risk variants onto our structure to derive the molecular basis for their effect on PLD3 function 2. We also compared our structure to those of other PLD family members to identify features unique to PLD3. I will report insights on the functional and structural aspects arising from our experiments, and describe ongoing efforts using structure-based drug design to discover small molecule modulators of PLD3 function that might be developed into new therapies to treat AD.

  1. Dementia Australia, 2023, Dementia Prevalence Data 2024-2054
  2. Ishii K, et al. Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions. The FEBS Journal, (2024).
  3. Cruchaga C, et al. Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer’s disease. Nature 505, 550-554 (2014).
  4. Yuan P, et al. PLD3 affects axonal spheroids and network defects in Alzheimer’s disease. Nature 612, 328-337 (2022).
  5. Van Acker ZP, et al. Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism. Nature Communications 14, 2847 (2023).
  6. Gonzalez AC, et al. Unconventional trafficking of mammalian phospholipase D3 to lysosomes. Cell Reports 22, 1040-1053 (2018).