Evidence That α-Synuclein Does Not Inhibit Phospholipase D

Abstract

Abstractα-Synuclein (αSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding αSyn cause early-onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of αSyn. Several studies have reported evidence that αSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of αSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of αSyn, we sought to extend existing reports on this interaction.Using purified proteins, we tested the ability of αSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether αSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between αSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by αSyn in any of these systems. We propose that the previously reported effects of αSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with αSyn overexpression in cells, but are not likely due to a specific and direct interaction between αSyn and PLD.Parkinson's disease (PD 1 ) is a debilitating neurodegenerative disorder that affects over one million people in the United States (for review, see (1)). Although more than 90% of PD cases are believed to occur sporadically, several genes are known to cause familial forms of PD. Among these, point mutations or increased dosage of the α-synuclein (αSyn) gene are known to cause rare cases of early-onset familial PD (2-7). The αSyn protein is also the major component of Lewy bodies and Lewy neurites, the insoluble aggregates that are neuropathological hallmarks of both familial and sporadic PD (8). Thus, dysregulation of αSyn at either the genetic or protein level can contribute to PD-type neurodegeneration.

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…In these studies, PLD2 was purified by an immunoaffinity technique and no gels of purity are shown. A subsequent study with highly purified PLD2 was unable to replicate the original findings (Rappley et al, 2009), suggesting the presence of an intermediate protein in the original article. Regardless of the interaction between a-synuclein and PLD2, no associations have been made in vivo to support a role for PLD2 in the pathophysiology of a-synuclein (Ahn et al, 2002).…”

Phospholipase D: Enzymology, Functionality, and Chemical Modulation

“…Other proteins originally thought to directly interact with PLD and inhibit activity include α-synuclein, which has subsequently been shown to not inhibit PLD activity in vitro or in cells overexpressing this protein. 388 …”

“…507 By contrast, a collaborative investigation by the Selkoe and Brown laboratories found that under numerous experimental conditions α-synuclein does not inhibit PLD in cells or in vitro . 388 …”

Folding and misfolding of alpha-synuclein on membranes

“…The observation that aS interacts with and inhibits phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (63) also suggests that placing synuclein at the site of docked vesicles could serve to influence the membrane remodeling processes involved in vesicle fusion or budding (although PLD regulation by aS has been contested in recent work (64)). PLD is implicated in the regulation of secretory vesicle budding or fusion through its generation of phosphatidic acid (65, 66), which can play a role in modulating membrane curvature and also regulates phosphatidylinositol-4-phosphate 5-kinase activity (67).…”