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 蛋白的一个基本特性是新的脂质结合结构域（ACCH 结构域），它表现出脂质选择性和使膜变形的能力，然而，实现脂质特异性和膜变形的生物物理机制。这一基本知识空白对于填补至关重要，因为 Amot 作为顶膜上皮细胞信号转导的衔接蛋白，影响细胞分化、癌细胞增殖和迁移。该提案的核心假设是，不同 Amot 亚型的脂质结合和支架功能能够对细胞极性事件进行特异性调节。最终影响导管细胞增生进展为乳腺癌。该假设是基于 Amot 的各种同工型在上皮-间质细胞转变（导管中的关键步骤）中发挥不同作用的基础上提出的。据统计，增生的个体患乳腺癌的可能性高出 3-5 倍，而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能是控制分化的治疗靶点。和癌细胞的增殖。 该假设将通过追求三个具体目标进行检验：1) 确定 Amot ACCH 结构域与膜表面结合的分子基础；2) 确定 Amot 作为促进不对称细胞膜组织的极性蛋白的作用；3) 确认Amot 定向膜组织（细胞极性）细胞增殖和迁移之间的联系旨在使用定向定点诱变作为测量 ACCH 酪氨酸与合成膜环境的直接接触和插入的手段。 F??目标 2 旨在使用含有 PIP 和模拟筏混合物的合成膜来研究 Amot 结构在细胞极性中的作用，目标 3 将利用对调节 ACCH 脂质结合特异性的理解。体外细胞测定来描绘影响能力 该方法是创新的，因为它结合了生物物理和细胞生物学技术来解决癌症生物学背景下 Amot 脂质结合活性的结构-功能关系。 ACCH 结构域作为一种分子开关，应针对与导管癌相关的细胞分化和增殖进行特异性控制。该提案在癌症生物学背景下采用生物物理技术，是候选者成为独立癌症的目标的逻辑进展。研究员在课程期间进行的研究。该奖项将成为追求学术终身教职的平台，为实现这一目标，候选人将通过教学和体验途径获得培训，以表征化学工程师和生物物理化学家的功能。影响进展的关键蛋白质 然而，需要增强她对癌症生物学以及分子生物学、生物显微镜和生物统计学应用学科的了解，并为她成为一名独立的癌症研究员做好准备。将正式课程工作与导师实验室的实践工作相结合。这些课程与她的具体目标、生物统计学以及她的资助能力的进一步发展有关，将在获奖过程中与另一项教学一起讨论。课程导师 Hurley 博士和 Wells 博士将根据各自的研究专业领域、统计相关分析开发、出版物/资助写作和研究道德，每周为她提供非正式的投入。机构资源，例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，该提案的所有要素（例如研究、培训等）将用于准备和申请第四个 NIH R01。获奖年份以进一步提高她的独立研究地位。