Microtubule regulation by small molecules.

小分子的微管调节。

• 批准号: 8941494
• 负责人: Dan L Sackett
• 金额: $ 35.35万
• 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/8941494
• 关键词: Biological; Biological Factors; Blood Cells; Blood Platelets; Cell Differentiation process; Cells; Cellular Morphology; Cellular biology; Chemicals; Chickens; Colchicine; Combretastatin; Cytoplasm; Cytoskeleton; Development; Disease; Epithelial Cells; Error Sources; Erythrocytes; Exposure to; Fluorescence; Genes; Goals; Human; In Vitro; Knowledge; Leukocytes; Mass Spectrum Analysis; Methods; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modification; Movement; Optical Methods; Optics; Paclitaxel; Parasites; Pattern; Pharmaceutical Preparations; Pharmacotherapy; Polymers; Process; Property; Protein Subunits; Regulation; Reporting; Research; Source; Staging; System; Tissues; Tubulin; Vincristine; alpha Tubulin; base; beta Tubulin; brain cell; cell behavior; cell type; chemotherapy; density; novel; physical property; polymerization; pre-clinical research; screening; small molecule; trafficking

Tubulin is a heterodimer composed of one alpha- and one beta-tubulin molecules. There are multiple genes for slightly different forms, or isotypes, of both alpha- and beta-tubulin. These have different tissue- and developmental stage- expression patterns, and all are subject to numerous posttranlational modifications (PTM). Blood cells express a particular isotype of beta-tubulin restricted to these cells, and an isotype of alpha tubulin that is shared by other types of cells as well as blood cells. We have pursued a mass-spectrometry-based study of these tubulins, in order to look for changes during development or following exposure to MT-targeted chemotherapy drugs. We have used readily available chicken red blood cells for this study, but the tubulin isotypes are the same as those expressed in human platelets and white blood cells. We have found that the alpha tubulin contains a PTM that has not been reported previously. This PTM appears to be restricted to blood cells, since we do not find it in tubulin from brain or epithelial cells. We do not yet understand the biological significance of this PTM, its chemical or enzymatic origin, nor how it changes during blood cell differentiation or following exposure to chemotherapy drugs. These are all subjects of ongoing research. Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. The in vitro studies with purified tubulin are a critical way in which compounds are evaluated, and indeed this is often used as a screening method to discover new active compounds as well as to quantitatively evaluate established ones. To further this, we have reviewed approaches to quantitating MT assembly by optical methods, with the goal of maximizing the information that may be obtained and minimizing the errors that the system can produce. We used both optical density-based and fluorescence-based approaches, and evaluated sources of error as well as underappreciated sources of extra information on the polymerization process.

微管蛋白是由一个α-微管蛋白分子和一个β-微管蛋白分子组成的异源二聚体。α-微管蛋白和β-微管蛋白的形式或同种型略有不同，但有多种基因。这些有不同的组织和发育阶段的表达模式，都受到许多翻译后修饰（PTM）。血细胞表达限于这些细胞的β-微管蛋白的特定同种型，以及由其他类型的细胞以及血细胞共享的α微管蛋白的同种型。我们对这些微管蛋白进行了基于质谱的研究，以寻找发育过程中或暴露于MT靶向化疗药物后的变化。本研究中我们使用了容易获得的鸡红细胞，但微管蛋白同种型与人血小板和白色血细胞中表达的微管蛋白同种型相同。我们已经发现，α微管蛋白包含一个PTM，以前没有报道过。这种PTM似乎仅限于血细胞，因为我们在脑或上皮细胞的微管蛋白中没有发现它。我们还不了解这种PTM的生物学意义，它的化学或酶起源，也不知道它在血细胞分化过程中或暴露于化疗药物后如何变化。这些都是正在进行的研究课题。 天然产物历来是大多数微管（MT）靶向小分子的来源，这些小分子的特性使它们成为有用的药物。这仍然适用于我们在本研究中使用的大多数但不是所有化合物。这些包括临床上确定的MT活性药物秋水仙碱、考布他汀、长春新碱、紫杉醇等。几乎所有这些试剂首先在临床前研究中开发，所述临床前研究包括化合物对微管蛋白聚合成微管的影响以及这些化合物对细胞行为的影响的体外研究，特别是检查化合物通过对包含有丝分裂纺锤体的MT阵列的影响破坏有丝分裂的能力。用纯化的微管蛋白进行的体外研究是评价化合物的关键方法，实际上，这通常用作筛选方法来发现新的活性化合物以及定量评价已建立的化合物。为了进一步这一点，我们已经审查的方法来量化MT组装的光学方法，最大限度地提高信息，可以获得和最大限度地减少系统可以产生的错误的目标。我们使用了基于光学密度和基于荧光的方法，并评估了误差来源以及聚合过程中未得到充分重视的额外信息来源。