P53 Fibril Formation and Disease

描述(申请人提供)：我们在2002年发现，肿瘤抑制因子P53的四聚化(TET)结构域可以从其原始构象转换为具有淀粉样纤维特征的替代构象。此外，我们观察到该结构域的癌症相关突变形式，随着Arg 337突变为His，与野生型结构域相比，显示出显著增强的形成纤维的倾向，P53是一种结构模块化的蛋白质，并且TET结构域的折叠是肿瘤抑制所必需的。此前，我们发现R337H突变以一种pH依赖的方式破坏Tet结构域的稳定性，并为与该突变相关的癌症(儿童肾上腺皮质癌)的组织特异性提供了分子解释。我们认为原纤维的形成与突变型p53肿瘤生物学之间存在联系。与其他癌症相关突变形式的P53的发现一致，带有R337H突变的P53(P53-R337H)在ACC肿瘤细胞的细胞核中聚集在高水平。基于我们对突变的tet结构域的发现，我们假设这些核堆积具有纤维状结构。此外，我们认为(一些)其他形式的突变型P53在纤维状结构中积累。我们试图通过对野生型和突变型p53的多结构域形式的结构研究来检验这些假说。各种生物物理、生化和细胞生物学方法将被应用于研究体外、肿瘤细胞和肿瘤细胞来源的培养细胞中的p53分子。我们的目标是：1)确定野生型和突变型P53在体外是否从天然状态转化为淀粉样纤维；2)确定肿瘤细胞中聚集的野生型和突变型P53是否以纤维状态存在。P53是一种多功能蛋白，受转录、翻译后修饰和细胞定位等多个水平的调控。为了理解与p53突变相关的肿瘤发生，我们必须了解突变是如何影响这些不同的调控过程的。虽然已有多个领域的高分辨率结构和广泛的生物物理数据可用，但全长p53的结构和生物物理性质在很大程度上仍然是一个谜。证明突变的P53分子以纤维形式存在，将解释已报道的P53积累及其核定位的高度稳定性，将为功能丧失提供分子解释，并将为开发新的P53导向疗法提供洞察力。

DESCRIPTION (provided by applicant): We discovered in 2002 that the tetramerization (tet) domain of the tumor suppressor p53 can be converted from its native conformation to an alternative conformation with the characteristics of amyloid fibrils. Further, we observed that a cancer-associated mutant form of this domain, with Arg 337 mutated to His, exhibits a significantly heightened propensity to form fibrils compared to the wild-type domain, p53 is a structurally modular protein, and folding of the tet domain is required for tumor suppression. Previously, we showed that the R337H mutation destabilizes the tet domain in a pH-dependent manner and developed a molecular explanation for the tissue specificity of cancer associated with this mutation (adrenal cortical carcinoma (ACC) in children). We feel that there is a link between fibril formation and mutant p53 tumor biology. Consistent with findings for other cancer-associated mutant forms of p53, p53 with the R337H mutation (p53-R337H) accumulates at high levels in the nuclei of ACC tumor cells. Based on our findings for the mutant tet domain, we hypothesize that these nuclear accumulations have fibrilar structure. Further, we suggest that (some) other mutant forms of p53 accumulate in fibrilar structures. We seek to test these hypotheses through structural studies of multi-domain forms of wild-type and mutant p53. A variety of biophysical, biochemical and cell biology methods will be applied to study p53 molecules in vitro, in tumor cells, and in cultured cells derived from tumors. Our aims are: 1) To determine whether wild-type and mutant p53 convert from the native state to amyloid fibrils in vitro, and 2) To determine whether aggregated forms of wild-type and mutant p53 in tumor cells exist in a fibrilar state. P53 is a multifunctional protein that is regulated at many levels, including transcription, post-translational modification and cellular localization. To understand tumorigenesis associated with mutations to p53, we must understand how these different regulatory processes are affected by mutations. While high-resolution structures and extensive biophysical data are available for several domains, the structural and biophysical properties of full-length p53 remain largely a mystery. Demonstrating that mutant p53 molecules exist as fibrils would explain the high stability reported for p53 accumulations and their nuclear localization, would provide a molecular explanation for loss of function, and would offer insights into the development of novel p53-directed therapeutics.