MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Proc Natl Acad Sci U S A. 2012 Aug 21;109(34):13590-5. doi: 10.1073/pnas.1211557109. Epub 2012 Aug 6.
Aggregation of p53 is initiated by first-order processes that generate an aggregation-prone state with parallel pathways of major or partial unfolding. Here, we elaborate the mechanism and explore its consequences, beginning with the core domain and extending to the full-length p53 mutant Y220C. Production of large light-scattering particles was slower than formation of the Thioflavin T-binding state and simultaneous depletion of monomer. EDTA removes Zn(2+) to generate apo-p53, which aggregated faster than holo-p53. Apo-Y220C also aggregated by both partial and major unfolding. Apo-p53 was not an obligatory intermediate in the aggregation of holo-p53, but affords a parallel pathway that may be relevant to oncogenic mutants with impaired Zn(2+) binding. Full-length tetrameric Y220C formed the Thioflavin T-binding state with similar rate constants to those of core domain, consistent with a unimolecular initiation that is unaffected by neighboring subunits, but very slowly formed small light-scattering particles. Apo-Y220C and aggregated holo-Y220C had little, if any, seeding effect on the initial polymerization of holo-Y220C (measured by Thioflavin T binding), consistent with initiation being a unimolecular process. But apo-Y220C and aggregated holo-Y220C accelerated somewhat the subsequent formation of light-scattering particles from holo-protein, implying coaggregation. The implications for cancer cells containing wild-type and unstable mutant alleles are that aggregation of wild-type p53 (or homologs) might not be seeded by aggregated mutant, but it could coaggregate with p53 or other cellular proteins that have undergone the first steps of aggregation and speed up the formation of microscopically observable aggregates.
p53 的聚集首先由一级过程引发，这些过程产生具有平行展开主要或部分途径的聚集倾向状态。在这里，我们详细阐述了这种机制，并探讨了其后果，从核心结构域开始，扩展到全长 p53 突变体 Y220C。大的光散射颗粒的产生速度比硫代黄素 T 结合状态的形成和单体的同时耗散慢。EDTA 去除 Zn(2+)以生成 apo-p53，其聚集速度比 holo-p53 快。apo-Y220C 也通过部分和主要展开进行聚集。apo-p53 不是 holo-p53 聚集的必需中间产物，但提供了一个平行途径，可能与 Zn(2+)结合受损的致癌突变体有关。全长四聚体 Y220C 以与核心结构域相似的速率常数形成硫代黄素 T 结合状态，这与不受相邻亚基影响的单分子引发一致，但非常缓慢地形成小的光散射颗粒。apo-Y220C 和聚集的 holo-Y220C 对 holo-Y220C 的初始聚合（通过硫代黄素 T 结合测量）几乎没有，如果有的话，种子效应，这与引发是一个单分子过程一致。但是 apo-Y220C 和聚集的 holo-Y220C 稍微加速了来自 holo-蛋白的光散射颗粒的随后形成，这意味着共聚集。对于含有野生型和不稳定突变体等位基因的癌细胞的影响是，野生型 p53（或同源物）的聚集可能不会被聚集的突变体引发，但它可能与已经经历聚集的第一步的 p53 或其他细胞蛋白共聚集，并加速显微镜可见聚集的形成。
