Defining the Oligomerization State of 纬-Synuclein in Solution and in Cells

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• 作者：Urszula Golebiewska ; Cassandra Zurawsky ; Suzanne Scarlata
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：January 21, 2014
• 年：2014
• 卷：53
• 期：2
• 页码：293-299
• 全文大小：322K
• ISSN：1520-4995

文摘

纬-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member 伪-synuclein, 纬-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of 纬-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase C尾 (PLC尾), 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, 纬-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 纬-synuclein and found concentration-dependent oligomerization. Addition of PLC尾2, a strong 纬-synuclein binding partner whose cellular expression is correlated with 纬-synuclein, results in disruption of 纬-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express 纬-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified 纬-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.