Biophysical studies of oncogenic Cdc42Hs constructs

描述(申请人提供)：CDC42Hs是RAS超家族信号转导蛋白的成员，与鸟嘌呤核苷酸结合，在多个信号转导途径中发挥分子计时开关的作用。这一提议的核心是研究与其致癌潜力相关的Cdc42Hs形式的结构和动力学，并将为调节这一活动的机制提供原子水平的信息。将要研究的CDC42Hs结构是：A)CDC42Hs(无核苷酸)和B)Cdc42Hs(F28L_？L8)。确定核苷酸缺失形式的Cdc42Hs的结构和动力学将有助于了解cdc42Hs的构象(S)，它促进蛋白质效应物的结合，如DBL，刺激致癌活性。对Cdc42Hs(F28L_？L8)的结构和动力学的研究应该揭示出Cdc42Hs中的相互作用可以阻断单个突变的Cdc42Hs(F28L)引起的致癌活性。 这项研究的近期目标是使用核磁共振和荧光作为结构技术，以更好地了解CDC42H的变化及其与调节因子的相互作用影响导致癌症的各种重要信号转导途径的过程。这项拟议研究的长期目标是更好地了解蛋白质结构和动力学与异常细胞功能的关系，这将为未来开发合理的抗癌药物设计方案奠定基础。CDC42Hs是RAS超家族蛋白质中的一员，与结肠癌、乳腺癌和肺癌有关。这些研究将提供特定细胞信号传递过程的特定方面的信息，这些信息可能随后用于开发新的抗癌治疗方法，这些治疗方法比传统的化疗方法更具特异性，毒性可能更小。我有在生物分子研究中使用荧光光谱学的背景，并曾使用核磁共振光谱学研究小肽在溶液中的结构。这项建议的指导阶段将使我有机会在尚未掌握的这些技术的几种用途上获得经验，这对我向独立调查的过渡至关重要。康奈尔大学分子医学系在将物理方法应用于生物过程，特别是信号转导方面拥有丰富的经验。对于这个项目来说，这是一个很好的环境，也是为了促进我的职业发展。该提案的研究生涯发展计划分两个阶段进行。第一阶段(指导)将允许研究无核苷酸的Cdc42Hs结构。这一阶段在结构工具、数据分析程序和科学合作方面的经验将为成功过渡到第二阶段(独立)培养必要的准备，在此期间我将研究Cdc42Hs(F28L_？L8)的结构和动力学，从而导致与癌症相关的生物分子结构生物学的进一步研究。

DESCRIPTION (provided by applicant): Cdc42Hs, a member of the Ras superfamily signal transduction proteins, binds guanine nucleotides and acts as a molecular timing switch in multiple signal transduction pathways. This proposal is centered around studying the structure and dynamics of Cdc42Hs forms associated with its oncogenic potential and will provide atomic level information into mechanisms that regulate this activity. The Cdc42Hs constructs that will be studied are, A) Cdc42Hs(nucleotide-free) and, B) Cdc42Hs(F28L_?L8). Defining the structure and dynamics of the nucleotide-depleted form of Cdc42Hs will shed light on the conformation(s) of Cdc42Hs that facilitate the binding of protein effectors, such as Dbl, that stimulate oncogenic activity. The study of the structure and dynamics of Cdc42Hs(F28L_?L8) should reveal information on interactions in Cdc42Hs that block oncogenic activity caused by the single mutant Cdc42Hs(F28L). The immediate goals of this research are to use NMR and fluorescence as structural techniques to gain a better understanding of the processes by which changes in Cdc42Hs and its interaction with regulators affect various important signal transduction pathways that lead to cancer. The long-term goals of this proposed research are to gain a better understanding of how protein structure and dynamics correlate with aberrant cell function, which will lay the foundation for future research into the development of a rational anti-cancer drug design program. Cdc42Hs is a member of the Ras superfamily of proteins, which have been implicated in cancers in the colon, breasts, and lungs. These studies will provide information on particular aspects of specific cell-signaling processes that may be subsequently used in the development of new anti-cancer treatments that are specific and may be less toxic than traditional chemotherapy approaches. I have a background in the use of fluorescence spectroscopy in the study of biological molecules and have used NMR spectroscopy to study structure of small peptides in solution. The mentored phase of this proposal will provide me with the opportunity to gain experience in several uses of these techniques that have not yet been mastered that will be crucial to my transition to independent investigation. The Department of Molecular Medicine at Cornell University has great experience in the area of applying physical methods to biological processes, and in particular, signal transduction. It is an excellent setting for this project, and also to foster the development of my career. The research career development plan of this proposal is two-phased. Phase I (Mentored) will allow for the study of the nucleotide-free construct of Cdc42Hs. The experience with the structural tools, data analysis procedures, and scientific collaboration in this phase will foster the necessary preparation for a successful transition into Phase II (Independent), during which time I will study the structure and dynamics of Cdc42Hs(F28L_?L8), leading to further research in the structural biology of biomolecules related to cancer.