Microtubule regulation by isotype expression and by small molecules.

通过同种型表达和小分子调节微管。

• 批准号: 10266495
• 负责人: Danny Sackett
• 金额: $ 52.12万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10266495
• 关键词: Accounting; Address; Affect; Amino Acids; Anti-Inflammatory Agents; Binding; Biological; Biological Assay; Cell Line; Cells; Cellular Morphology; Cellular biology; Chemistry; Clinical; Code; Coenzymes; Cytoplasm; Cytoskeleton; Development; Disease; Drug Targeting; Elements; Enzymes; Epothilones; Fluorescence; Fluorescence Resonance Energy Transfer; GTP-Binding Protein alpha Subunits, Gs; Generations; Human; Human body; Knowledge; Maps; Measurement; Measures; Mediating; Metabolic; Methods; Microscope; Microscopy; Microtubule Stabilization; Microtubules; Mitochondria; Mitosis; Mitotic spindle; Modification; Monitor; Movement; Myoglobin; Natural Product Drug; Natural Products; Oxidation-Reduction; Oxygen; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Polymers; Post-Translational Protein Processing; Property; Protein Subunits; Proteins; Publishing; Reading; Regulation; Reporting; Research; Role; Science; Site; Structure; Surface; Tail; Taurine; Therapeutic; Time; Tissues; Transfer RNA; Tubulin; Variant; Work; alpha Tubulin; analog; base; beta Tubulin; chemotherapy; combinatorial; dictyostatin; dimer; extracellular; fluorescence lifetime imaging; in vitro Assay; monomer; novel therapeutics; physical property; sensor; small molecule; targeted agent; tool; trafficking; tumor; two-photon

In pursuit of newer microtubule-targeting agents with more favorable spectra of actions as well as more facile chemistry, we have previously developed new methods for synthesizing variants of MT targeting agents. In the report of 2018, we applied our methods to the microtubule-targeting natural product drug dictyostatin, demonstrating that this method allowed synthetic extension of particular sites on precursor molecules to produce new variants of dictyostatin that demonstrated significantly different biological activity. In the report for 2017, we described the development of new analogs of epothilone, a microtubule-stabilizing drug that is already in clinical use. In the current reporting year, we additionally focused on the small molecule PTMs of tubulin, having uncovered a PTM that is new to protein science. The group of PTMs occur at the carboxyl terminal end of both alpha and beta tubulin monomers. One of the earliest discovered PTMs happens on the carboxyl terminus of alpha tubulin. This normally ends with a Tyr residue, but this can be removed enzymatically when the tubulin is polymerized to MT, uncovering the penultimate Glu residue. The Tyr is restored when the MT disassembles. This PTM alters the binding properties of the MT, acting to regulate binding of other proteins to the MT surface. We discovered that there is a previously unrecognized additional regulation step involving addition of a new residue to the Glu instead of the Tyr residue. Surprisingly this residue is taurine. Taurine is a beta-amino acid, known to science for more than 200 years and known to be the most abundant free amino acid in the human body, accounting for about 100 g of a 100 kg human. It has never previous been found covalently incorporated into a protein, even as a PTM. It has been found as a modification of a mitochondrial tRNA, and its function in that role is sufficient to cause clinical disease if absent. The role of the PTM of alpha tubulin with taurine is not known yet, but is under study. Also the tissue and developmental distribution of the taurine-tubulin PTM has not been completely uncovered, though we have preliminary evidence showing that this is not at all uniform through different cell lines and tissue types. Further research will reveal the distribution and function of this unique form of taurine and the significance to MT function of this singular PTM. Often study of the regulation of microtubules and microtubule arrays and the effects of small molecules on those arrays depends on tools of microscopy, both for in vitro assays with purified proteins and especially for assays that address microtubule arrays inside cells, and the effects of small molecules on the biology of cells mediated by effects on the MT arrays. For this reason we have continued our work on new methods that use fluorescence methods and advanced microscope applications to pursue these aims. We have previously published methods to use genetically encoded, FRET-based oxygen sensors based on a myoglobin-mCherry construct to map the intracellular oxygen levels and show how they are affected by varying subatmospheric extracellular oxygen levels, as occur in body tissues. In this reporting period, we extend this approach, by combining mapping of intracellular oxygen levels via the Myoglobin-mCherry sensor with measurement of the cytoplasmic redox level. The redox level is measured noninvasively by using two-photon excitation fluorescence lifetime imaging (FLIM) of free and enzyme-bound NAD(P)H and FAD. This allows a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular oxygen level and coenzyme status redox monitoring in living cells. Our next application will be to perturb these cells using the small molecules that are known to alter MT arrays and also known to be therapeutically significant by inducing variation in reactive oxygen generation within the cytoplasm of the exposed cells.

为了寻求具有更有利的作用光谱和更容易的化学作用的新型微管靶向剂，我们以前已经开发了新的方法来合成不同的MT靶向剂。在2018年的报告中，我们将我们的方法应用于微管靶向天然产品药物dictyostatin，表明这种方法允许前体分子上的特定位点的合成延伸，以产生显示出显著不同生物活性的dictyostatin的新变体。在2017年的报告中，我们描述了已经在临床使用的微管稳定药物埃博西隆的新类似物的开发情况。 在本报告年度，我们还关注了微管蛋白的小分子PTM，发现了一种对蛋白质科学来说是新的PTM。PTM基团位于α和β微管蛋白单体的羧基末端。最早发现的PTMS之一发生在α-微管蛋白的羧基末端。这通常以Tyr残基结束，但当微管蛋白聚合成MT时，这可以通过酶作用去除，揭示出倒数第二个Glu残基。当MT拆卸时，轮胎被恢复。这种PTM改变MT的结合特性，调节其他蛋白质与MT表面的结合。我们发现，有一个以前未被认识到的额外调节步骤，涉及向Glu中添加新的残基，而不是Tyr残基。令人惊讶的是，这种残留物是牛磺酸。牛磺酸是一种β-氨基酸，被科学发现已有200多年的历史，是人体内含量最丰富的游离氨基酸，约占100公斤人体中的100克。它以前从未被发现共价结合到蛋白质中，即使作为PTM也是如此。它已经被发现是线粒体tRNA的修饰，如果没有它的作用，它的功能足以引起临床疾病。α微管蛋白与牛磺酸的PTM的作用尚不清楚，但正在研究中。此外，牛磺酸-微管蛋白PTM的组织和发育分布还没有完全被发现，尽管我们有初步证据表明，在不同的细胞系和组织类型中，这一分布并不完全一致。进一步的研究将揭示这种独特形式的牛磺酸的分布和功能，以及这种单一的PTM对MT功能的意义。 通常，微管和微管阵列的调节以及小分子对这些阵列的影响的研究依赖于显微镜工具，无论是对纯化蛋白质的体外分析，还是对细胞内微管阵列的分析，以及通过对MT阵列的影响而介导的小分子对细胞生物学的影响。出于这个原因，我们继续研究使用荧光方法和先进显微镜应用来实现这些目标的新方法。我们之前已经发表了一些方法，使用基于肌红蛋白-mCherry结构的遗传编码的、基于FRET的氧传感器来绘制细胞内的氧水平，并显示它们如何受到不同的亚大气细胞外氧水平的影响，就像在身体组织中发生的那样。在本报告期间，我们扩展了这一方法，通过肌红蛋白-mCherry传感器绘制细胞内氧水平图与测量细胞质氧化还原水平相结合。利用自由和酶结合的NAD(P)H和FAD的双光子激发荧光寿命成像(FLIM)无创地测量了氧化还原水平。这使得可以通过实时、非侵入性的、逐个细胞的细胞内氧水平和活细胞中辅酶状态的氧化还原监测来同时读取代谢活动。我们的下一个应用将是使用小分子扰乱这些细胞，这些小分子已知可以改变MT阵列，也已知通过在暴露细胞的细胞质内诱导活性氧产生的变化而具有治疗意义。