Reversible Polymerization of Low Complexity Polypeptide Sequences as a Framework

低复杂度多肽序列的可逆聚合作为框架

• 批准号: 8899608
• 负责人: STEVEN L MCKNIGHT
• 金额: $ 49.09万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899608
• 关键词: Accidents; Acetylation; Adopted; Applications Grants; Binding; Biogenesis; Biological Assay; C-terminal; Cell Extracts; Cell Nucleus; Cells; Chemicals; Complex; Cytoplasmic Granules; DNA Binding Domain; Dimensions; Disease; Eukaryotic Cell; Fiber; Filament; Gel; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Health; Homeobox; Hydrogels; Ice; In Vitro; Incubated; Isoxazoles; Laboratories; Laboratory Chemicals; Lateral; Leucine Zippers; Life; Link; Liquid substance; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Messenger RNA; Methods; Microscopic; Molecular; Monitor; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Neurons; Nuclear; Nuclear Extract; Nuclear Proteins; Outcome Study; Pattern; Phase; Phase Transition; Physics; Polymers; Precipitation; Procedures; Process; Proteins; RNA; RNA Polymerase II; RNA polymerase II largest subunit; RNA-Binding Proteins; Reaction; Reagent; Relative (related person); Sampling; Science; Serine; Stress; Structure; Surface; TATA-Box Binding Protein; Testing; Tissue Extracts; Transcriptional Activation; Transcriptional Regulation; Trinucleotide Repeats; Tyrosine; Work; X ray diffraction analysis; X-Ray Diffraction; Zinc Fingers; bHLH Domain; base; genetic regulatory protein; in vivo; medical schools; member; polyglutamine; polymerization; polypeptide; red fluorescent protein; research study; sarcoma; solid state nuclear magnetic resonance; transcription factor

DESCRIPTION (provided by applicant): For upwards of 30 years it has been known that regulatory proteins involved in the control of gene expression and mRNA biogenesis contain low complexity (LC) polypeptide sequences that are intrinsically disordered. In the case of gene specific transcription factors, these LC sequences, often typified by long, homopolymeric segments of polyglutamine, have been implicated in the illusive process of transcriptional activation. It is well-understood how the DNA binding domains of transcription factors, including zinc fingers, leucine zippers, bHLH domains, and homeobox domains, facilitate direct and specific interaction between transcription factors and their target genes. By contrast, the field remains largely ignorant of the mechanisms by which the LC sequences typifying "activation domains" facilitate gene expression. In the case of proteins that regulate RNA biogenesis, similarly perplexing LC sequences are often linked to well-folded domains that mediate direct interaction with RNA substrates (including RRM, KH and pumlio domains). By serendipity, members of the McKnight laboratory discovered that a biotinylated isoxazole (b-isox) chemical is capable of reversibly co-precipitating hundreds of RNA binding proteins from cytosolic extracts. This unexpected discovery formed the starting point for experiments showing that LC sequences can reversibly polymerize into cross-beta filaments that phase transition to a hydrogel-like state. In turn, microscopic versions of these hydrogel droplets were evolved into a simplified, quantitative assay for gel retention of homotypic and heterotypic test proteins. By discovering that repeats of the sequence [G/S]Y[G/S] serve as a nucleating substrate for fiber polymerization, it was possible to test the correlative effects of mutational impediments to fiber polymerization in vitro with the ability of RNA binding proteins to move in and out of RNA granules in living cells. As a result of this work, it has been hypothesized that reversible polymerization of LC domains may represent the organizational basis for the formation of RNA granules including P-granules, stress granules, P- bodies and neuronal granules (Kato et al., 2012; Han et al., 2012). As an extension of this work nuclear extracts have been exposed to the b-isox chemical. These experiments led to the reversible precipitation of many nuclear proteins containing LC sequences, including TATA binding protein, TAF15, the largest subunit of RNA polymerase II, and components of the mediator complex. A major objective of the experiments proposed in this grant application will be the test of whether similar processes are employed to control the organization of nuclear puncta, including Cajal bodies, nuclear speckles and "transcription factories".

描述（由申请人提供）： 30 多年来，人们已知参与基因表达和 mRNA 生物合成控制的调节蛋白含有本质上无序的低复杂性 (LC) 多肽序列。就基因特异性转录因子而言，这些 LC 序列通常以聚谷氨酰胺的长均聚物片段为代表，与转录激活的虚假过程有关。转录因子的 DNA 结合域（包括锌指、亮氨酸拉链、bHLH 域和同源盒域）如何促进转录因子与其靶基因之间直接且特异性的相互作用，这一点已众所周知。相比之下，该领域对于代表“激活结构域”的LC序列促进基因表达的机制仍然一无所知。对于调节 RNA 生物合成的蛋白质，类似地令人困惑的 LC 序列通常与介导与 RNA 底物（包括 RRM、KH 和 pumlio 结构域）直接相互作用的良好折叠结构域连接。麦克奈特实验室的成员偶然发现，生物素化异恶唑 (b-isox) 化学物质能够可逆地共沉淀细胞质提取物中的数百种 RNA 结合蛋白。这一意想不到的发现构成了实验的起点，表明LC序列可以可逆地聚合成交叉β丝，并相转变为水凝胶状状态。反过来，这些水凝胶液滴的微观版本演变成一种简化的定量测定，用于同型和异型测试蛋白的凝胶保留。通过发现重复序列 [G/S]Y[G/S] 作为纤维聚合的成核底物，可以测试突变障碍对体外纤维聚合的相关影响与 RNA 结合蛋白的能力进出活细胞中的 RNA 颗粒。这项工作的结果是，人们假设 LC 结构域的可逆聚合可能代表了 RNA 颗粒形成的组织基础，包括 P 颗粒、应激颗粒、P 体和神经元颗粒（Kato 等，2012；韩等人，2012）。作为这项工作的延伸，核提取物已暴露于 b-isox 化学品中。这些实验导致许多含有 LC 序列的核蛋白发生可逆沉淀，包括 TATA 结合蛋白、TAF15、RNA 聚合酶 II 的最大亚基以及介体复合物的成分。本次拨款申请中提出的实验的一个主要目标是测试是否采用类似的过程来控制核斑点的组织，包括卡哈尔体、核斑点和“转录工厂”。