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

使用稳定的α-螺旋肽诱导本质无序蛋白质的类似天然折叠

基本信息

批准号：
9190844
负责人：
Nicholas Alan Sawyer
金额：
$ 5.25万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9190844
关键词：
Affinity Binding Binding Proteins Biological Assay C-terminal Cells Circular Dichroism Complement Complex Computer Simulation Disease EP300 gene Evaluation Fluorescence Fluorescence Polarization Genes Genetic Transcription Goals HIF1A gene Hydrogen Bonding Hypoxia In Vitro Laboratories Luciferases Malignant Neoplasms Measurement Measures Mediating Molecular Conformation Mus NMR Spectroscopy Peptides Play Protein Conformation Protein Family Proteins Research Role STAT2 gene Side Signal Transduction Specificity Structure TP53 gene Therapeutic Therapeutic Intervention Transcription Coactivator Transcriptional Activation Transcriptional Regulation Tumor Volume Up-Regulation Xenograft procedure alpha helix angiogenesis base biological systems covalent bond design flexibility functional group genome-wide hypoxia inducible factor 1 improved in vitro activity in vivo interest mimetics mimicry novel peptide structure peptidomimetics protein aminoacid sequence protein folding protein function protein protein interaction protein structure research study small molecule tool tumor microenvironment tumor xenograft

项目摘要

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.

项目摘要/摘要本质上无序的蛋白质（IDP）是一类无处不在的蛋白质可塑性使它们可以在蛋白质 - 蛋白质相互作用（PPI）和信号传导中充当轮毂网络。因此，IDP PPI受到严格调节，异常IDP PPI与许多疾病状态。由于IDP折叠和活动取决于诱导的构象它的本地结合伙伴，影响本机样IDP构象的小分子高度理想研究IDP功能。但是，复杂的IDP PPI接口是困难的目标传统的小分子。相反，使用稳定的α-螺旋的非传统方法肽，例如在Arora中开发的氢键替代（HBS）方法实验室有望创建小分子，使核本机样IDP折叠与高亲和力和高特异性。那我假设HBS肽可以模仿钥匙 IDP结合伙伴与核IDP折叠的一部分，并在体外竞争抑制IDP PPI 和体内。总体目标是创建HBS衍生的人工文件夹，以影响IDP 偏爱另一折。经典的IDP PPI示例是一般转录共激活器P300与缺氧诱导因子的本质上无序的C末端转录激活结构域 1α（HIF1αCTAD）。这种相互作用对于低氧诱导转录至关重要，导致与血管生成，侵袭和增殖。 Arora实验室表明，模仿HIF1αCTAD螺旋片段的模仿下调缺氧诱导的转录，并减少小鼠的Xenographic肿瘤大小。但是，针对P300可能会损害其与众多绑定伙伴互动的能力。在当前的建议中，我建议制定完整的策略，以使用HBS肽诱导HIF1αCTAD折叠并有竞争力抑制p300-HIF1αCTAD相互作用。到实现此目标，我将首先设计并合成模仿P300螺旋的HBS肽与HIF1αCTAD相互作用的片段。我将评估HBS胡椒结合亲和力和体外HIF1αctad的特异性。然后，我将表征HIF1αCTAD-HBS的结构复合物评估HBS胡椒诱导的HIF1αCTAD折叠。我还将评估HBS 肽使用基于细胞的测定法调节HIF1α介导的转录的能力。 HBS肽这会影响像天然一样的HIF1αCTAD折叠，具有高亲和力和特异性可能是有用的研究HIF1α功能和潜在的癌症治疗铅的工具用于HIF1α抑制作用。这引起IDP折叠的策略也可能提供一般方法的特定应用使用结构化肽调节IDP结构和活动。