Defining the Tubulin Cytoskeleton of Drug Resistant Cells By High Resolution MS

项目概述：微管是动态蛋白质聚合物，在细胞分裂、维持细胞形状、囊泡运输和运动中起重要作用。微管已被证明是抗癌药物的有效靶点，如微管稳定剂紫杉醇、艾硫洛酮类药物和迪德莫内酯。然而，对这些微管稳定药物的耐药性是一个反复出现的问题，而肿瘤产生耐药性的过程尚不清楚。耐药的一个可能原因是微管的微管蛋白同型组成的变化。微管由α -微管蛋白和β -微管蛋白异二聚体组成，其中人类有6种α -微管蛋白和7种β -微管蛋白同型体。微管蛋白同型表达的改变会影响微管的稳定性，导致微管稳定药物的疗效降低。对微管蛋白的翻译后修饰（PTMs）进一步促进了微管的多样性。微管改变也可能影响与微管支架结合的蛋白质的能力，这是另一种可能的耐药性机制。本研究的目的是确定微管蛋白同型表达和翻译后修饰以及微管相关蛋白在对微管稳定药物产生耐药性过程中的变化。具体目的：(1)建立一种高分辨率质谱方法来完整表征微管蛋白同型，为了解微管蛋白同型表达与耐药性之间的关系提供基础；(2)检测敏感和耐药细胞系中表达的微管蛋白和PTMs；(3)使用自顶向下和自底向上的蛋白质组学方法定量测定耐药细胞系中与微管相关的蛋白质的表达水平和修饰程度的变化。使用高分辨率质谱法，我们将测量对微管稳定药物敏感和耐药的细胞系中存在的微管蛋白的完整质量，并准确确定与任何PTMs或突变一起存在的微管蛋白同型。此外，我们将鉴定与微管相关的蛋白质，并确定这些蛋白质在耐药细胞系中发生的变化。相关性：这些研究将有助于我们理解细胞如何对紫杉醇和其他微管稳定药物产生耐药性，并可能提供见解，为什么某些肿瘤类型对这些药物的反应比其他药物更好，而有些则完全没有反应。最终，这些信息将帮助我们开发新的癌症治疗方法，并了解如何治疗特定的肿瘤。

DESCRIPTION (provided by applicant): Project Summary: Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, transport of vesicles, and motility. Microtubules are proven to be an effective target for anticancer drugs, such as the microtubule-stabilizing agents Taxol, the epothiolones and discodermolide. However, there is a recurring problem of resistance to these microtubule-stabilizing drugs and the process by which tumors develop resistance is not well understood. One possible cause of resistance is a change in the tubulin isotype composition of the microtubules. Microtubules are made up of alpha- and beta-tubulin heterodimers, of which there are 6 alpha- and 7 beta-tubulin isotypes in humans. Alterations in tubulin isotype expression can affect the stability of the microtubules leading to reduced efficacy of microtubule-stabilizing drugs. Further contributing to the diversity of microtubules are post- translational modifications (PTMs) to tubulin. Microtubule alterations may also affect the ability of proteins that bind to the microtubule scaffold, another possible mechanism of drug resistance. The objective of this research is to ascertain the changes in tubulin isotype expression and post-translational modification and in proteins associated with microtubules in the development of resistance to microtubule-stabilizing drugs. Specific Aims: (1) To establish a high resolution mass spectrometric methodology for complete characterization of tubulin isotypes, providing a basis for understanding the relationship between tubulin isotype expression and resistance; (2) to examine the expressed tubulins and PTMs that occur in sensitive and drug resistant cell lines; (3) to quantitate changes in the level of expression and extent of modification of proteins that are associated with microtubules in resistant cell lines using top down and bottom up proteomic methodologies. Using high resolution mass spectrometry, we will measure the intact mass of the tubulins present in cell lines sensitive and resistant to microtubule-stabilizing drugs and accurately determine the tubulin isotypes present along with any PTMs or mutations. Also, we will identify proteins associated with the microtubules and determine changes that occur with these proteins in resistant cell lines. Relevance: These studies will contribute to our understanding of how cells become resistant to Taxol and other microtubule-stabilizing drugs and may provide insight as to why certain tumor types respond better to these drugs than others and some do not respond at all. Ultimately, this information will help us to develop new cancer therapeutics and also to understand how to treat specific tumors.