Defining the oligomerization state of γ-synuclein in solution and in cells.

γ-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member α-synuclein, γ-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of γ-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase Cß (PLCß), altering its interaction with G proteins. As a first step in determining its role, we have characterized its oligomerization using fluorescence homotransfer, photon-counting histogram analysis, and native gel electrophoresis. We found that when its expressed in Escherichia coli and purified, γ-synuclein appears monomeric on chromatographs under denaturing conditions, but under native conditions, it appears as oligomers of varying sizes. We followed the monomer-to-tetramer association by labeling the protein with fluorescein and following the concentration-dependent loss in fluorescence anisotropy resulting from fluorescence homotransfer. We also performed photon-counting histogram analysis at increasing concentrations of fluorescein-labeled γ-synuclein and found concentration-dependent oligomerization. Addition of PLCß2, a strong γ-synuclein binding partner whose cellular expression is correlated with γ-synuclein, results in disruption of γ-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express γ-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified γ-synuclein form oligomers, when binding partners are present, as in cells, it dissociates to a monomer to bind these partners, which in turn may modify protein function and integrity.

γ-Synuclein interacts with phospholipase Cß2 to modulate G protein activation.

Phospholipase Cß2 (PLC ß2) is activated by G proteins and generates calcium signals in cells. PLCß2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCß2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCß2 are associated. In solution, purified γ-synuclein binds to PLCß2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCß2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gßγ). Binding of γ-synuclein reduces the catalytic activity of PLCß2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCß2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCß2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gßγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCß2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCß2.