Kinetic Variability in Seeded Formation of ALS-Linked SOD1 Fibrils Across Multiple Generations
Kinetic Variability in Seeded Formation of ALS-Linked SOD1 Fibrils Across Multiple Generations
复制标题
DOI：
10.1021/acschemneuro.9b00464
复制
发表时间：
2020-02-05
影响因子：
5
通讯作者：
Shaw, Bryan F.
中科院分区：
文献类型：
--
作者：
Baumer, Katelyn M.;Koone, Jordan C.;Shaw, Bryan F.
超氧化物歧化酶-1（SOD 1）在体外和体内随机聚集成淀粉样原纤维，即分离的SOD 1蛋白群体（在微孔板威尔斯孔或活细胞内）以跨越概率分布的速率自组装成淀粉样蛋白。这种随机性已被归因于不同程度的单体消耗无定形和纤维状聚集（阿利亚其他外）的竞争途径。在此，以高迭代（类似于300）进行基于微孔板的硫磺素-T（ThT）荧光测定，以确定当使用祖（“亲本”）SOD 1原纤维接种多代子代原纤维（子代、孙女和曾孙女原纤维）的形成时，这种观察到的随机性是否持续。祖原纤维的群体以不同的速率和荧光强度形成随机，然而，后代原纤维以更相似的速率形成，而不管祖原纤维的形成速率如何。例如，以类似于30小时或类似于15小时的滞后时间形成的祖原纤维群体都产生具有类似于8小时的滞后时间的子代原纤维。同样，具有高或低最大荧光的祖原纤维群体（例如，450或类似于75 A.U.）两者都产生了具有更相似的最大荧光（类似于125 A.U.）的子代原纤维。繁殖率被认为是更依赖于单体浓度比种子浓度。这些结果可以合理的初级成核和单体依赖的二次成核的经典速率定律。我们还发现，在体外生长的原纤维的一些“家庭”的播种倾向表现出有限的寿命（类似于小分子晶体和胶体的播种中观察到的）。本研究的唯一生物学结论是，细胞中天然SODI的浓度对接种聚集速率的影响可能比感染细胞的朊病毒样种子的浓度更强。
The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically in vitro and in vivo, that is, isolated populations of SOD1 proteins (within microplate wells or living cells) self-assemble into amyloid at rates that span a probability distribution. This stochasticity has been attributed to variable degrees of monomer depletion by competing pathways of amorphous and fibrillar aggregation (inter alia). Here, microplate-based thioflavin-T (ThT) fluorescence assays were performed at high iteration (similar to 300) to establish whether this observed stochasticity persists when progenitor ("parent") SOD1 fibrils are used to seed the formation of multiple generations of progeny fibrils (daughter, granddaughter, and great-granddaughter fibrils). Populations of progenitor fibrils formed stochastically at different rates and fluorescence intensity, however, progeny fibrils formed at more similar rates regardless of the formation rate of the progenitor fibril. For example, populations of progenitor fibrils that formed with a lag time of similar to 30 h or similar to 15 h both produced progeny fibrils with lag times of similar to 8 h. Likewise, populations of progenitor fibrils with high or low maximum fluorescence (e.g., 450 or similar to 75 A.U.) both produced progeny fibrils with more similar maximum fluorescence (similar to 125 A.U.). The rate of propagation was found to be more dependent on monomer concentration than seed concentration. These results can be rationalized by classical rate laws for primary nucleation and monomer-dependent secondary nucleation. We also find that the seeding propensity of some "families" of in vitro grown fibrils exhibit a finite lifetime (similar to that observed in the seeding of small molecule crystals and colloids). The single biological takeaway of this study is that the concentration of native SODI in a cell can have a stronger effect on rates of seeded aggregation than the concentration of prion-like seed that infected the cell.