Purification of 伪-Synuclein from Human Brain Reveals an Instability of Endogenous Multimers as the Protein Approaches Purity
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- 作者：Eric S. Luth ; Tim Bartels ; Ulf Dettmer ; Nora C. Kim ; Dennis J. Selkoe
- 刊名：Biochemistry
- 出版年：2015
- 出版时间：January 20, 2015
- 年：2015
- 卷：54
- 期：2
- 页码：279-292
- 全文大小：551K
- ISSN：1520-4995
文摘
Despite two decades of research, the structure鈥揻unction relationships of endogenous, physiological forms of 伪-synuclein (伪Syn) are not well understood. Most in vitro studies of this Parkinson鈥檚 disease-related protein have focused on recombinant 伪Syn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that 伪Syn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant 伪Syn, physiological tetramers purified from human erythrocytes have substantial 伪-helical content and resist pathological aggregation into 尾-sheet rich fibers. Here, we report the first method to fully purify soluble 伪Syn from the most relevant source, human brain. We describe protocols that purify 伪Syn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant 伪Syn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue 尾-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived 伪Syn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying 伪Syn to homogeneity destabilizes native, 伪-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification.