Department of Chemistry and Biochemistry , Baylor University , Waco , Texas 76706 , United States.
ACS Chem Neurosci. 2020 Feb 5;11(3):304-313. doi: 10.1021/acschemneuro.9b00464. Epub 2020 Jan 15.
The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically and , 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 (∼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 ∼30 h or ∼15 h both produced progeny fibrils with lag times of ∼8 h. Likewise, populations of progenitor fibrils with high or low maximum fluorescence (e.g., ∼450 or ∼75 A.U.) both produced progeny fibrils with more similar maximum fluorescence (∼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 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 SOD1 in a cell can have a stronger effect on rates of seeded aggregation than the concentration of prion-like seed that infected the cell.
超氧化物歧化酶 1（SOD1）无种子聚集随机形成淀粉样原纤维，也就是说，在微孔板孔或活细胞内，SOD1 蛋白的孤立群体以跨越概率分布的速率自我组装成淀粉样纤维。这种随机性归因于无定形和纤维状聚集（除其他外）竞争途径导致单体耗竭程度不同。在此，进行了基于微孔板的硫黄素 T（ThT）荧光测定法，其迭代次数较高（约 300 次），以确定当使用前体（“亲本”）SOD1 纤维来种子形成多代后代纤维（子、孙和曾孙女纤维）时，是否存在这种观察到的随机性。然而，亲本纤维以不同的速率和荧光强度随机形成，而不管亲本纤维的形成速率如何，后代纤维都以更相似的速率形成。例如，形成滞后时间约为 30 h 或 15 h 的亲本纤维群体都产生滞后时间约为 8 h 的后代纤维。同样，具有高或低最大荧光（例如，约 450 或 75 AU）的亲本纤维群体都产生更相似的最大荧光（约 125 AU）的后代纤维。传播速率发现更依赖于单体浓度而不是种子浓度。这些结果可以用初级成核和单体依赖性二次成核的经典速率定律来合理化。我们还发现，一些“家族”的生长纤维的成核倾向具有有限的寿命（类似于小分子晶体和胶体的成核观察到的寿命）。本研究的唯一生物学结论是，细胞中天然 SOD1 的浓度对成核聚集的速率的影响要强于感染细胞的类朊样种子的浓度。
