Defining the role of Amot lipid binding in cellular proliferation and migration

定义 Amot 脂质结合在细胞增殖和迁移中的作用

• 批准号: 8354099
• 负责人: Ann Carol Kimble-Hill
• 金额: $ 9.62万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8354099
• 关键词: Actins; Adaptor Signaling Protein; Address; Adhesions; Affect; Affinity; Amino Acids; Apical; Appointment; Area; Award; Binding; Biological; Biological Assay; Biometry; Breast Cancer Model; Cancer Biology; Cancer Center; Cancerous; Cell Count; Cell Polarity; Cell Proliferation; Cell membrane; Cells; Cellular Assay; Cellular Membrane; Cellular biology; Characteristics; Chemical Engineering; Cholesterol; Circular Dichroism; Collaborations; Complex; Core Facility; DNA Sequence Rearrangement; Development; Diagnosis; Differentiation and Growth; Dimensions; Discipline; Disease remission; Docking; Ductal Carcinoma; Ductal Epithelial Cell; E-Cadherin; Educational workshop; Elements; Endosomes; Energy Transfer; Environment; Epithelial; Epithelial Cells; Equilibrium; Event; Faculty; Family; Family member; Fluorescence; Goals; Grant; Growth; Hand; Head; Hyperplasia; Image; In Vitro; Individual; Isoenzymes; Knowledge; Laboratories; Life; Link; Lipid Binding; Lipids; Liposomes; MAP Kinase Gene; Malignant Neoplasms; Mammary gland; Measurement; Measures; Membrane; Mentors; Mesenchymal; Microscopy; Mission; Modeling; Molecular; Molecular Biology; Monitor; Morphology; N-Cadherin; Outcome; Phase; Play; Prevention; Proliferating; Property; Protein Dynamics; Protein Isoforms; Proteins; Public Health; Publications; Regulation; Relative (related person); Research; Research Ethics; Research Personnel; Research Training; Resources; Role; Signal Transduction; Site-Directed Mutagenesis; Sorting - Cell Movement; Specificity; Structure; Surface; Techniques; Testing; Time; Training; Tryptophan; Tyrosine; United States; United States National Institutes of Health; Woman; Work; Writing; active method; apical membrane; assay development; base; cell growth; cell motility; density; design; innovation; insight; malignant breast neoplasm; migration; mutant; novel; outcome forecast; prevent; programs; protein transport; responsible research conduct; scaffold; study characteristics; therapeutic target; tissue culture; tumor; tumorigenesis

DESCRIPTION (provided by applicant): An essential property of all Amot proteins is a novel lipid-binding domain (ACCH domain) that demonstrates lipid selectivity and the ability to deform membranes. However, the biophysical mechanisms through which lipid specificity and membrane deformation is achieved remains unclear. This fundamental gap of knowledge is critical to fill, as Amot serves as an adaptor protein in apical membrane epithelial cell signaling which impacts cellular differentiation, cancer cell proliferation, and migration. The long-range goal of this project is to correlate the structure of Amot's ACCH domain with its lipid binding capabilities which drives protein sorting and downstream signaling events. The central hypothesis of this proposal is that the lipid-binding and scaffolding functions of the different Amot isoforms enables specific modulation of cellular polarity events which ultimately impacts the progression of ductal cell hyperplasia into breast cancer. This hypothesis was formulated on the basis that various isoforms of Amot play distinct roles in epithelial-mesenchymal cell transition, a key step in ductal cell hyperplasia tumorigenesis. Statistically, individuals with hyperplasia are 3-5 times more likely to develop breast cancer, and ductal carcinoma makes up approximately 80% of all breast cancer cases. Therefore, specific regulation of Amot function may represent a therapeutic target to control differentiation and proliferation of cancerous cells. This hypothesis will be tested by pursuing three specific aims: 1) Determine the molecular basis through which Amot ACCH domains associate with membrane surfaces; 2) Establish the role of Amot as a polarity protein that promotes asymmetric cellular membrane organization; and 3) Confirm the link between Amot-directed membrane organization (cellular polarity) cellular proliferation and migration. Aim 1 is designed to use targeted site directed mutagenesis as a means to measure direct contact and insertion of ACCH tyrosines with synthetic membrane environments by F¿rster resonance energy transfer and small angle scattering. Aim 2 is designed to use synthetic membranes containing PIP and raft-mimicking mixtures to study the role of Amot structure in cellular polarity. Aim 3 will use an understanding of modulating ACCH lipid binding specificity in the context of in vitro cellular assays to delineate ability to affect cellular migration, differentiation, and proliferation. The approach is innovative as it combines biophysical and cellular biology techniques to address the structure-functional relationship of Amot lipid binding activity in the context of cancer biology. The proposed work is significant, because it may describe the ACCH domain as a molecular switch that should be targeted for specific control of cellular differentiation and proliferation associated with ductal carcinoma. Ths proposal, which employs biophysical techniques in the context of cancer biology, is a logical progression in the candidate's goal of becoming an independent cancer researcher. Research developed during the course of this award will become the platform for pursuing an academic tenure track faculty appointment. To achieve this goal, training will be obtained through didactic and experiential avenues. The candidate will apply her training as a chemical engineer and biophysical chemist to characterize the function of a critical protein that impacts the progression of breast cancer. However, there is a need to enhance her knowledge of cancer biology and the applied disciplines of molecular biology, biological microscopy and biostatistics. To address these training goals and better prepare her to become an independent cancer researcher a specific training plan that integrates formal course work with hand-on work in the laboratory of her mentors. The courses are relevant to the specific aims, biostatistics, and further development of her grantsmanship. Responsible conduct of research will be addressed during the course of the award with another didactic course and workshops. Co- mentors, Drs. Hurley and Wells, will each provide weekly informal input into her training based on their areas of research expertise, statistically relevant assay development, publication/grant writing and research ethics. Professional and scientific training will include utilizing institutional resource such as departmental and Cancer Center seminars, inter-program collaborations, and research core facilities. Hence, all elements of this proposal (e.g. research, training, etc.) will be used o prepare and apply for a NIH R01 in the fourth year of the award to further her independent research status.

描述（由申请人提供）：所有Amot蛋白的基本特性是一种新型脂质结合结构域（ACCH结构域），其表现出脂质选择性和使膜变形的能力。然而，脂质特异性和膜变形的生物物理机制仍然不清楚。这一基本的知识空白是至关重要的，因为Amot作为顶膜上皮细胞信号传导中的衔接蛋白，影响细胞分化，癌细胞增殖和迁移。该项目的长期目标是将Amot的ACCH结构域的结构与其驱动蛋白质分选和下游信号事件的脂质结合能力相关联。该提议的中心假设是，不同Amot同种型的脂质结合和支架功能能够特异性调节细胞极性事件，其最终影响导管细胞增生进展为乳腺癌。这一假设是基于Amot的各种亚型在上皮-间充质细胞转化中发挥不同的作用，这是导管细胞增生肿瘤发生的关键步骤。据统计，患有增生的个体患乳腺癌的可能性高3 - 5倍，导管癌约占所有乳腺癌病例的80%。因此，Amot功能的特异性调节可以代表控制癌细胞分化和增殖的治疗靶点。 该假设将通过追求三个特定目标进行测试：1）确定Amot ACCH结构域与膜表面相关联的分子基础; 2）确定Amot作为促进不对称细胞膜组织的极性蛋白的作用;和3）确认Amot指导的膜组织（细胞极性）细胞增殖和迁移之间的联系。目的1旨在使用靶向定点突变作为一种手段来测量直接接触和插入的ACCH酪氨酸与合成膜环境的福斯特共振能量转移和小角散射。目的2是利用含有PIP和模拟筏的混合物的合成膜来研究Amot结构在细胞极性中的作用。目的3将使用在体外细胞试验的背景下调节ACCH脂质结合特异性的理解来描述影响ACCH的能力。 细胞迁移、分化和增殖。该方法是创新的，因为它结合了生物物理学和细胞生物学技术，以解决Amot脂质结合活性在癌症生物学背景下的结构-功能关系。这项拟议的工作很重要，因为它可能将ACCH结构域描述为一个分子开关，应该靶向特异性控制与导管癌相关的细胞分化和增殖。这个建议，它采用生物物理技术在癌症生物学的背景下，是一个合乎逻辑的进展，在候选人的目标成为一个独立的癌症研究人员。在这个奖项的过程中开发的研究将成为追求学术终身教职的平台。为实现这一目标，将通过教学和经验途径进行培训。候选人将应用她作为化学工程师和生物物理化学家的培训来表征影响进展的关键蛋白质的功能 乳腺癌然而，有必要提高她的癌症生物学和分子生物学，生物显微镜和生物统计学的应用学科的知识。为了实现这些培训目标，并更好地为她成为一名独立的癌症研究人员做好准备，她制定了一个具体的培训计划，将正式课程与导师实验室的实践工作相结合。这些课程与她的专业的具体目标、生物统计学和进一步发展有关。在颁奖过程中，将通过另一个教学课程和讲习班来解决负责任的研究行为。共同导师Hurley博士和威尔斯博士将根据他们的研究专长、统计相关测定开发、出版物/赠款写作和研究道德领域，每周为她的培训提供非正式投入。专业和科学培训将包括利用机构资源，如部门和癌症中心研讨会，项目间合作和研究核心设施。因此，本提案的所有内容（如研究、培训等）将用于准备和申请NIH R01在该奖项的第四年，以进一步她的独立研究地位。