Single Molecule Biophysics of Intrinsically Disordered Proteins in Disease

摘要：在超过50%的人类蛋白质中发现了固有无序蛋白，在这些蛋白质中发挥着重要的作用。 在广泛的细胞功能中，包括转录调节、DNA修复、细胞信号和细胞凋亡。作为一名 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，含有 国内流离失所者往往与癌症有关。国内流离失所者采用各种构象的能力提出了一些关键问题 对标准生化、生物物理和计算技术的挑战。尽管有这些挑战，我们治疗的能力 许多癌症依赖于对疾病分子基础的了解。这反过来又提出了迫切需要 了解IDP功能和功能障碍的机制基础。这项提议将研究蛋白质-核酸的相互作用。 这是由两种紧迫疾病：新冠肺炎和癌症中固有的紊乱蛋白质驱动的。对于F99阶段(目标1) 获奖后，我将在我的计算和实验生物物理学训练的基础上，继续研究SARS- CoV-2核衣壳蛋白及其包装病毒基因组的能力。新冠肺炎大流行之前， 由SARS和MERS引起的冠状病毒暴发，需要对这些病毒进行研究，以便更好地抗击它们。 冠状病毒包含大的rna基因组，这些基因组被包装成一个相对较小的病毒粒子，由核衣壳介导。 蛋白质，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是如何 将他们的30kb基因组打包成一个相对较小(100 Nm)的病毒粒子。冠状病毒的保守结构基序 基因组被称为包装信号，已被证明具有基因组特异性，但包装与包装之间的关系 信号和基因组压缩是不透明的。我的论文工作将单分子荧光光谱与全分子荧光光谱相结合。 原子和粗粒度模拟，以构建对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编码的多价态在选择性基因组包装中的作用。自.以来 核衣壳蛋白的结构在整个冠状病毒中是保守的，这也将为我们提供新的见解 可广泛用于治疗干预的机制。该提案的K00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贡献。融合癌基因是 一种常见的遗传易位事件，通常涉及DNA结合域与IDP融合。在.期间 博士后阶段I将接受超分辨率显微镜的培训，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IDPs驱动转录因子的形成 基因表达部位的集合体(转录缩合物)。我将测试融合癌蛋白导致 形成长寿的异常转录凝聚体，从而驱动增殖基因的表达。这将是 提供对融合癌基因驱动的癌症的分子基础的直接机制洞察。这些综合训练 计划将使我做好准备，使用定量生物物理和单分子技术在 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.