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

基本信息

批准号：
10305403
负责人：
Jhullian Jamille Alston
金额：
$ 3.15万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10305403
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 Knowledge Lead Length Malignant Neoplasms Mediating Microscopy Middle East Respiratory Syndrome Modeling Molecular Molecular Conformation Nucleic Acids Nucleocapsid Proteins Oncogenes Oncogenic Output Phase Physical condensation Play Postdoctoral Fellow Process Proteins RNA RNA Binding RNA Recognition Motif RNA-Binding Proteins Regulation Research Resolution 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 design dimer fluorescence imaging genomic RNA improved in vitro Assay in vivo insight member molecular imaging molecular modeling novel programs protein function simulation single molecule success targeted treatment transcription factor

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 Knowledge Lead Length Malignant Neoplasms Mediating Microscopy Middle East Respiratory Syndrome Modeling Molecular Molecular Conformation Nucleic Acids Nucleocapsid Proteins Oncogenes Oncogenic Output Phase Physical condensation Play Postdoctoral Fellow Process Proteins RNA RNA Binding RNA Recognition Motif RNA-Binding Proteins Regulation Research Resolution 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 design dimer fluorescence imaging genomic RNA improved in vitro Assay in vivo insight member molecular imaging molecular modeling novel programs protein function simulation single molecule success targeted treatment transcription factor

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项目摘要

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：在超过50％的人类蛋白质中发现了本质上无序的蛋白质（IDP），它们起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。作为它们在与细胞生长，增殖和死亡相关的关键过程中重要性的结果，含有蛋白质的蛋白质 IDP通常与癌症有关。 IDP采用广泛构象的能力提高了许多关键标准生化，生物物理和计算技术的挑战。尽管面临这些挑战，但我们的治疗能力许多癌症取决于对疾病分子基础的理解。反过来，这迫切需要了解IDP功能和功能障碍的机械基础。该建议将研究蛋白质核酸相互作用在两种压力疾病中的内在无序蛋白质驱动：COVID-19和癌症。对于F99阶段（目标1）该奖项，我将基于我的计算和实验性生物物理学培训，以继续研究SARS- COV-2 Nucleocapsid蛋白及其包装病毒基因组的能力。互联19大流行，之前 SARS和MERS引起的冠状病毒爆发需要研究这些病毒，以便更好地对抗它们。冠状病毒含有大的RNA基因组，这些基因组被包装到相对较小的病毒体中，由Nucleocapsid介导蛋白质，一种高度无序的多域RNA结合蛋白。当前的一个杰出问题是SARS-COV-2如何将他们的30 kb基因组打包成一个相对较小的（<100 nm）的病毒粒子。冠状病毒中保守的结构基序已证明称为包装信号的基因组赋予基因组特异性，但包装之间的关系信号和基因组压实是不透明的。我的论文工作结合了单分子荧光光谱和原子和粗粒模拟，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编码的多价性在选择性基因组包装中的作用。自从 Nucleocapsid蛋白的结构在整个冠状病毒中都保存下来可以广泛针对治疗干预的机制。该提案的K00阶段（AIM 2）将研究 IDP在转录调节，基因组组织和癌症发展中的贡献。融合融合基因是一个常见的遗传易位事件，通常涉及DNA结合结构域与IDP融合。在博士后I将获得超分辨率显微镜的训练，以研究转录活性的影响融合融合。几项研究表明，来自转录因子的IDP驱动转录的形成基因表达部位的组件（转录冷凝）。我将测试融合 - 共蛋白铅的假设形成驱动增殖基因表达的长寿命异常转录缩合物。这会提供直接的机械洞察力，以了解融合 - 癌基因驱动的癌症的分子基础。这些结合培训计划将使用定量的生物物理和单分子技术为成功的研究职业做好准备机械癌症生物学领域。