Defining the Oligomerization State of γ-Synuclein in Solution and in Cells
| Autor: | Suzanne Scarlata, Cassandra Zurawsky, Urszula Golebiewska |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
G protein
Cells animal diseases Phospholipase C beta Biology Fibril Biochemistry Article chemistry.chemical_compound gamma-Synuclein Cell Line Tumor Humans chemistry.chemical_classification Gamma-synuclein HEK 293 cells nervous system diseases Solutions HEK293 Cells Enzyme Monomer nervous system chemistry Cell culture Fluorescence anisotropy HeLa Cells |
| Zdroj: | Biochemistry. 53:293-299 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi401479a |
| Popis: | 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 Cβ (PLCβ), 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 PLCβ2, 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|