Single Molecule Biophysics of Intrinsically Disordered Proteins in Disease

疾病中内在无序蛋白质的单分子生物物理学

• 批准号: 10818667
• 负责人: Jhullian Jamille Alston
• 金额: $ 9.39万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10818667
• 关键词: 2019-nCoV; Adopted; Affinity; Antiviral Agents; Apoptosis; Architecture; Award; Behavior; Binding; Binding Sites; Biochemical; Biological Assay; Biological Process; Biophysics; COVID-19; COVID-19 pandemic; Cancer Biology; Cancerous; Cell physiology; Cells; Cessation of life; Charge; Chimeric Proteins; Chromosomal translocation; Collection; Computational Technique; Coronavirus; Coronavirus nucleocapsid protein; DNA Binding Domain; DNA Repair; Development; Dimerization; Disease; Disease Outbreaks; Disease Progression; Elements; Enhancers; Etiology; Event; Family; Fluorescence; Fluorescence Spectroscopy; Functional disorder; Fusion Oncogene Proteins; Gene Expression; Genes; Genetic Transcription; Genome; Grain; Human; In Vitro; Intrinsic drive; Knowledge; Lead; Length; Malignant Neoplasms; Mediating; Microscopy; Middle East Respiratory Syndrome; Modeling; Molecular; Molecular Conformation; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Oncogenes; Oncogenic; Output; Phase; Physical condensation; Play; Postdoctoral Fellow; Process; Proliferating; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Proteins; Regulation; Research; Role; Severe Acute Respiratory Syndrome; Signal Transduction; Site; Specificity; Spectrum Analysis; Structure; Surface; Techniques; Testing; Therapeutic; Therapeutic Intervention; Training; Transcriptional Regulation; Viral Genome; Virion; Virus; Virus Diseases; Work; betacoronavirus; biophysical properties; biophysical techniques; cancer type; career; cell growth; combat; dimer; fluorescence imaging; genomic RNA; improved; in vitro Assay; in vivo; insight; member; molecular imaging; molecular modeling; novel; programs; protein function; rational design; simulation; single molecule; success; superresolution microscopy; targeted treatment; transcription factor

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.

摘要：内在无序蛋白 (IDP) 存在于超过 50% 的人类蛋白质中，它们在其中发挥着重要作用 广泛的细胞功能，包括转录调控、DNA 修复、细胞信号传导和细胞凋亡。作为一个 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，含有蛋白质 国内流离失所者通常与癌症有关。国内流离失所者采用多种构象的能力引发了一​​些关键问题 对标准生物化学、生物物理和计算技术的挑战。尽管存在这些挑战，我们的治疗能力 许多癌症取决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。该提案将研究蛋白质-核酸相互作用 由两种紧迫疾病中本质上无序的蛋白质驱动：COVID-19 和癌症。对于 F99 阶段（目标 1） 获得该奖项后，我将在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。 COVID-19 大流行之前曾发生过 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，这些基因组被包装成相对较小的病毒粒子，由核衣壳介导 蛋白质，一种高度无序的多域RNA结合蛋白。当前的一个悬而未决的问题是SARS-CoV-2如何 将其 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。我的论文工作将单分子荧光光谱与全 原子和粗粒度模拟，以构建对 N 蛋白如何驱动 RNA 包装的机制理解。 该项目的成功将揭示 IDP 编码的多价在选择性基因组包装中的作用。自从 核衣壳蛋白的结构在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。本提案的 K00 阶段（目标 2）将研究 IDP 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 一种常见的基因易位事件，通常涉及 DNA 结合域与 IDP 融合。期间 博士后第一阶段将获得超分辨率显微镜方面的培训，以研究转录活性的影响 融合癌基因。多项研究表明，来自转录因子的 IDP 驱动转录因子的形成。 基因表达位点的组装体（转录缩合物）。我将检验融合癌蛋白导致的假设 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直接的机制见解。这些综合训练 计划将为我使用定量生物物理和单分子技术在以下领域取得成功的研究生涯做好准备 机械癌症生物学领域。