Brandis Katrina A, Holmes Isaac F, England Samantha J, Sharma Nijee, Kukreja Lokesh, DebBurman Shubhik K
Biology Department, Lake Forest College, Lake Forest, IL 60045, USA.
J Mol Neurosci. 2006;28(2):179-91. doi: 10.1385/jmn:28:2:179.
Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.
尽管裂殖酵母在模拟重要细胞过程方面有着悠久的历史，但它尚未被用于模拟与人类神经退行性疾病相关的蛋白质错误折叠。由于α-突触核蛋白的错误折叠和聚集与帕金森病（PD）有关，在此，我们报告了一种裂殖酵母（粟酒裂殖酵母）模型，该模型可评估α-突触核蛋白的错误折叠、聚集和毒性，并将这些特性与最近在芽殖酵母（酿酒酵母）中所描述的特性进行比较。野生型α-突触核蛋白和三个突变体（A30P、A53T和A30P/A53T）通过硫胺素可抑制启动子进行表达（使用启动子强度递增的载体：pNMT81、pNMT41和pNMT1），以在活细胞中直接测试α-突触核蛋白错误折叠和聚集的成核聚合假说。为支持该假说，野生型和A53Tα-突触核蛋白在裂殖酵母细胞内以浓度和时间依赖性方式形成了突出的细胞内细胞质包涵体，而A30P和A30P/A53T在整个细胞质中仍呈弥散状态。A53Tα-突触核蛋白比野生型α-突触核蛋白更快地形成聚集体，且所需的α-突触核蛋白浓度更低。出乎意料的是，与芽殖酵母不同，野生型和A53Tα-突触核蛋白在裂殖酵母中并不靶向质膜，即使在低α-突触核蛋白浓度下或作为形成聚集体的前体步骤时也不靶向。尽管α-突触核蛋白广泛聚集，但令人惊讶的是它对裂殖酵母无毒。未来的基因剖析可能会在分子层面深入了解这种对毒性的保护机制。我们推测α-突触核蛋白的毒性可能与其膜结合能力有关。总之，粟酒裂殖酵母和酿酒酵母模拟了α-突触核蛋白生物学中相似但又不同的方面，这两种生物都有助于深入了解α-突触核蛋白在帕金森病发病机制中的作用。
