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The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 A-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target. A structural study of one of two human phospholipase D enzymes, hPLD1, helps define the determinants of substrate recognition, the highly regulated enzymatic activity, as well as activation by PIP2 and RhoA.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.1668F is a founder variant among Ashkenazi Jews (allele frequency of -.2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

Mammalian phospholipase D (PLD) generates phosphatidic acid, a dynamic lipid secondary messenger involved with a broad spectrum of cellular functions including but not limited to metabolism, migration, and exocytosis. As a promising pharmaceutical target, the biochemical properties of PLD have been well characterized. This has led to the recent crystal structures of human PLD1 and PLD2, the development of PLD specific pharmacological inhibitors, and the identification of cellular regulators of PLD. In this review, we discuss the PLD1 and PLD2 structures, PLD inhibition by small molecules, and the regulation of PLD activity by effector proteins and lipids.