Inducing Native-like Folding of Intrinsically Disordered Proteins using Stabilized a-Helical Peptides

项目摘要/摘要 内源性无序蛋白(IdP)是一类普遍存在的蛋白质，其结构 可塑性使它们在蛋白质-蛋白质相互作用(PPI)和信号转导中发挥枢纽作用 网络。因此，IDP PPI受到严格监管，异常的IDP PPI与 许多疾病状态。因为IdP的折叠和活性依赖于由 它的天然结合伙伴，诱导天然类IDP构象的小分子高度 适合研究IdP功能。然而，复杂的IDP PPI接口是难以实现的目标 传统的小分子。相反，使用稳定的α-螺旋的非传统方法 多肽，如Arora中发展的氢键替代物(HBS)方法 实验室，有望创造成核的小分子，如天然的IdP折叠 既有高亲和力又有高特异性。因此，我假设HBS多肽可以模拟KEY 部分IdP结合伴侣在体外对IdP折叠成核和竞争性抑制IdP PPI 在活体内。总体目标是创建HBS衍生的人造文件夹，以诱导国内流离失所者 比起其他人，偏爱一倍。 一个经典的IdP ppi例子是一般转录辅活化子p300与 缺氧诱导因子C端转录激活结构域的内在无序 1α(HIF1αCTAD)。这种相互作用对于低氧诱导的转录至关重要，导致 许多参与血管生成、侵袭和转移的癌症相关基因表达上调 扩散。阿罗拉实验室已经证明了对Hif1αCTAD螺旋片段模仿 下调低氧诱导转录，减少小鼠异种移植瘤大小。 然而，靶向p300可能会削弱其与众多结合伙伴相互作用的能力。 在目前的提案中，我建议开发一种补充策略，使用HBS多肽来 诱导HIF1CTAD折叠，竞争性抑制p300-HIF1CTAD与α的相互作用。至 为了实现这一目标，我将首先设计和合成模拟p300螺旋的HBS多肽 与Hif1αCTAD相互作用的片段。我将评估HBS多肽结合亲和力和 HIF1α体外CTAD的特异性。然后我将对Hif1αCTAD-HBS的结构进行表征 评价HBS多肽诱导的HIF1αCTAD折叠的复合体。我还将评估哈佛商学院 多肽通过基于细胞的分析来调节HIF1α介导的转录的能力。HBS多肽 诱导具有高亲和力和特异性类HIF1αCTAD折叠可能是有用的 研究HIF1α功能的工具和抑制HIF1α的潜在癌症治疗线索。这 诱导IdP折叠的策略也可以提供一般方法的具体应用 利用结构多肽调节IDP的结构和活性。

PROJECT SUMMARY/ABSTRACT Intrinsically disordered proteins (IDPs) are a ubiquitous class of proteins whose structural plasticity allows them to function as hubs in protein-protein interaction (PPI) and signaling networks. IDP PPIs are therefore tightly regulated, and aberrant IDP PPIs are associated with many disease states. As IDP folding and activity are dependent on the conformations induced by its native binding partners, small molecules that induce native-like IDP conformations are highly desirable for studying IDP function. However, complex IDP PPI interfaces are difficult targets for traditional small molecules. Instead, non-traditional approaches using stabilized α-helical peptides, such as the hydrogen bond surrogate (HBS) approach developed in the Arora laboratory, are promising for creating small molecules that nucleate native-like IDP folding with both high affinity and high specificity. Thus, I hypothesize that HBS peptides can mimic key portions of IDP binding partners to nucleate IDP folding and competitively inhibit IDP PPIs in vitro and in vivo. The overall goal is to create HBS-derived artificial folders that induce IDPs to favor one fold over others. A classic IDP PPI example is the interaction of the general transcriptional coactivator p300 with the intrinsically disordered C-terminal transcription-activation domain of hypoxia-inducible factor 1 α (HIF1α CTAD). This interaction is critical for hypoxia-inducible transcription, leading to the upregulation of many cancer-associated genes involved in angiogenesis, invasion, and proliferation. The Arora lab has shown that mimicry of a HIF1α CTAD helical fragment downregulates hypoxia-inducible transcription and decreases xenograft tumor size in mice. However, targeting p300 may impair its ability to interact with its numerous binding partners. In the current proposal, I propose to develop a complementary strategy to use HBS peptides to induce HIF1α CTAD folding and competitively inhibit the p300-HIF1α CTAD interaction. To achieve this goal, I will first design and synthesize HBS peptides that mimic a p300 helical fragment that interacts with HIF1α CTAD. I will evaluate HBS peptide binding affinity and specificity for HIF1α CTAD in vitro. I will then characterize the structures of HIF1α CTAD-HBS complexes to evaluate HBS peptide-induced folding of HIF1α CTAD. I will also evaluate HBS peptides’ ability to modulate HIF1α-mediated transcription using cell-based assays. HBS peptides that induce native-like HIF1α CTAD folding with high affinity and specificity may serve as useful tools for studying HIF1α function and potential cancer therapeutic leads for HIF1α inhibition. This strategy for inducing IDP folding may also provide a specific application of a general approach to modulate IDP structures and activities using structured peptides.