Single Molecule Biophysics of Intrinsically Disordered Proteins in Disease

翻译后摘要：在超过50%的人类蛋白质中，它们发挥着重要的作用 在广泛的细胞功能中，包括转录调节、DNA修复、细胞信号传导和凋亡。作为 由于它们在与细胞生长、增殖和死亡相关的关键过程中的重要性， 国内流离失所者往往与癌症有关。国内流离失所者能够采取各种各样的形态，这提出了一些关键问题。 对标准生物化学、生物物理学和计算技术的挑战。尽管存在这些挑战，我们治疗 许多癌症取决于对疾病分子基础的理解。这反过来又迫切需要 了解IDP功能和功能障碍的机制基础。这项提案将研究蛋白质-核酸相互作用 由两种紧迫疾病中的内在无序蛋白质驱动：COVID-19和癌症。F99阶段（目标1） 获奖后，我将在我的计算和实验生物物理学培训的基础上继续研究SARS- CoV-2核衣壳蛋白及其包装其病毒基因组的能力。2019冠状病毒病大流行之前， SARS和MERS引起的冠状病毒爆发，需要对这些病毒进行研究，以便更好地对抗它们。 冠状病毒含有大的RNA基因组，这些RNA基因组被包装成相对较小的病毒体，由核衣壳介导 蛋白，一种高度无序的多结构域RNA结合蛋白。目前一个悬而未决的问题是SARS-CoV-2如何 将它们的30 kb基因组包装成相对较小（<100 nm）的病毒体。冠状病毒的保守结构基序 被称为包装信号的基因组已被证明赋予基因组特异性，但包装之间的关系 信号和基因组压缩是不透明的。我的论文工作结合单分子荧光光谱与所有- 原子和粗粒度的模拟，以构建一个N蛋白如何驱动RNA包装的机械理解。 该项目的成功将揭示IDP编码的多价性在选择性基因组包装中的作用。以来 核衣壳蛋白的结构在整个冠状病毒中是保守的，它也将提出新的见解， 这些机制可以广泛用于治疗干预。本提案的K 00阶段（目标2）将研究 IDPs在转录调控、基因组组织和癌症发展中的作用。融合癌基因是 一种常见的遗传易位事件，其通常涉及DNA结合结构域与IDP融合。期间 博士后阶段I将获得超分辨率显微镜培训，以调查转录活性的影响。 融合癌基因一些研究表明，来自转录因子的IDP驱动转录因子的形成。 在基因表达位点的转录浓缩物。我将检验融合癌蛋白导致 形成长寿命的异常转录缩合物，驱动增殖基因的表达。这将 为融合癌基因驱动的癌症的分子基础提供了直接的机制性见解。这些综合训练 计划将准备我成功的研究生涯中使用定量生物物理和单分子技术， 机械癌症生物学领域。

Abstract: Intrinsically disordered proteins (IDPs) are found in over 50% of human proteins where they play essential roles in a wide range of cellular functions including transcriptional regulation, DNA repair, cell signaling, and apoptosis. As a result of their importance in key processes associated with cellular growth, proliferation, and death, proteins containing IDPs are often associated with cancer. The ability of IDPs to adopt a wide range of conformations raises a number of key challenges to standard biochemical, biophysical, and computational techniques. Despite these challenges, our ability to treat many cancers depends on an understanding of the molecular basis for diseases. This, in turn, presents a pressing need to understand the mechanistic basis of IDP function and dysfunction. This proposal will study protein-nucleic acid interactions driven by intrinsically disordered proteins in two pressing diseases: COVID-19 and cancer. For the F99 phase (Aim 1) of the award, I will build upon my computational and experimental biophysics training to continue investigating the SARS- CoV-2 nucleocapsid protein and its ability to package its viral genome. The COVID-19 pandemic, preceded by previous coronavirus outbreaks caused by SARS and MERS, necessitates study of these viruses in order to better combat them. Coronaviruses contain large RNA genomes that are packaged into a relatively small virion, mediated by the nucleocapsid protein, a highly disordered multidomain RNA binding protein. A current outstanding question is how SARS-CoV-2 package their 30 kb genomes into a relatively small (<100 nm) virion. The conserved structural motifs in coronavirus genomes known as packaging signals has been shown to confer genome specificity, yet the relationship between packaging signals and genome compaction are opaque. My thesis work combines single-molecule fluorescence spectroscopy with all- atom and coarse-grained simulations to construct a mechanistic understanding of how N protein drives RNA packaging. Success of this project will reveal the role of IDP-encoded multivalency in selective genome packaging. Since the architecture of the nucleocapsid protein is conserved throughout coronaviruses it will also present new insight into mechanisms that can be broadly targeted for therapeutic intervention. The K00 phase (Aim 2) of this proposal will study the contribution of IDPs in transcriptional regulation, genome organization and cancer development. Fusion-oncogenes are a common genetic translocation event which often involve a DNA binding domain becoming fused to an IDP. During the post-doctoral phase I will obtain training in super-resolution microscopy to investigate the effects of transcriptionally active fusion-oncogenes. Several studies have shown that IDPs from transcription factors drive the formation of transcriptional assemblies (transcriptional condensates) at sites of gene expression. I will test the hypothesis that fusion-oncoproteins lead to the formation of long-lived aberrant transcriptional condensates that drive the expression of proliferative genes. This will provide direct mechanistic insight into the molecular basis of fusion-oncogene driven cancers. These combined training plans will prepare me for a successful research career using quantitative biophysical and single-molecule techniques in the field of mechanistic cancer biology.