Investigation of the role of UNC-45A in Paclitaxel-mediated microtubule stability

项目摘要 微管（MT）是紫杉醇的靶点，紫杉醇是乳腺癌和卵巢癌最广泛的处方药。 紫杉醇通过稳定微管起作用，从而导致细胞死亡。无法预测和影响 癌症患者对紫杉醇的反应是一个主要的临床问题。A45 A是UCS的成员 肌球蛋白II辅伴侣蛋白家族。最近的证据，包括我们最近公布的数据表明， 除了调节肌动球蛋白的收缩性外，α-45 A还是MT去稳定蛋白。在人类癌症中 β-45A过表达与不良结局相关。导致患者结局不佳的一个重要因素是 紫杉醇耐药性在这里，我们想研究是否以及如何将α-45 A赋予癌细胞“免疫”。 紫杉醇治疗的细胞存活优势。目标1。确定在2010年12月25日至26日期间 紫杉醇耐药的人类癌症。我们将（a）确定β-45A过表达是否是一个预测因子， 卵巢癌和乳腺癌临床样本中的紫杉醇耐药性，和（B）确定癌细胞中是否存在紫杉醇耐药性， 从卵巢癌和乳腺癌患者中提取的β-45 A调节细胞对紫杉醇的敏感性。目标2. 确定MT调节的生物物理学和细胞生物学参数由MAP-45 A。我们将（a） 确定在存在以下物质的情况下，Mt-45 A对MT的结合、定位特性和MT去稳定化作用： （B）进行计算建模，以评估紫杉醇和紫杉醇之间的机制联系， 图10示出了用于测定MT-45 A定位和MT稳定性的方法，以及（c）测定MT-45 A对活细胞中MT动力学的影响。 目标3.确定β-内酰胺酶-45A对MT作用的生化基础。我们将确定是否有必要- 45 A在体外寡聚化并作为寡聚体与MT结合（B），和（c）确定与MT结合的β-45 A的结构域， 参与MT结合、MT去稳定化和寡聚化。 相关性：我们的研究将为紫杉醇耐药提供新的生物标志物和分子靶点。 人类癌症，深入了解这种新的ATP非依赖性 蛋白质和MT，以及用于开发包括小分子抑制剂在内的新型治疗靶点的平台 蛋白质-蛋白质（寡聚体）相互作用。

PROJECT ABSTRACT Microtubules (MT) are the target of paclitaxel, the most widely prescribed drug for the breast and ovarian cancers. Paclitaxel acts by stabilizing microtubules which results with cell death. The inability to predict and influence how cancer patients will respond to paclitaxel represents a major clinical problem. UNC-45A is a member of the UCS protein family of myosin II co-chaperones. Recent evidences including our recently published data show that, in addition to regulating actomyosin contractility, UNC-45A is also a MT destabilizing protein. In human cancers UNC-45A overexpression correlates with poor outcome. A significant contributor to poor patient outcomes is chemoresistance to paclitaxel. Here we want to investigate whether and how UNC-45A confers cancer cells’ a cellular survival advantage upon paclitaxel treatment. Aim 1. Determine the contribution of UNC-45A in paclitaxel resistant human cancers. We will (a) Determine whether UNC-45A overexpression is a predictor of paclitaxel resistance in ovarian and breast cancer clinical specimens, and (b) Determine whether in cancer cells derived from ovarian and breast cancer patients, UNC-45A regulates cells’ sensitivity to paclitaxel. Aim 2. Determine the biophysical and cell biological parameters of MT regulation by UNC-45A. We will (a) Determine the binding, localization properties and MT destabilizing effects of UNC-45A on MTs in the presence and absence of paclitaxel, (b) Perform computational modeling to evaluate a mechanistic connection between UNC-45A localization and MT stability and, (c) Determine the effects of UNC-45A on MT dynamics in living cells. Aim 3. Determine the biochemical basis of UNC-45A’s effect on MTs. We will (a) Determine whether UNC- 45A oligomerizes in vitro and binds to MTs as an oligomer (b), and (c) Determine the domain(s) of UNC-45A that are involved in MT-binding, MT-destabilization and oligomerization. RELEVANCE: Our studies will provide us with a new biomarker and molecular target for paclitaxel-resistant human cancers, a deep understanding of the mechanistic interaction between this novel ATP-independent protein and MTs, and a platform for development of novel therapeutic targets including small molecule inhibitors of protein-protein (oligomer) interactions.