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: 8539483
• 负责人: Ann Carol Kimble-Hill
• 金额: $ 9.75万
• 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/8539483
• 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（由适用提供）：所有AMOT蛋白的必要特性是一种新型的脂质结合域（ACCH结构域），它证明了脂质选择性和变形膜的能力。然而，实现脂质特异性和膜变形的生物物理机制尚不清楚。这种基本知识的差距对于填充至关重要，因为AMOT充当顶端膜上上皮细胞信号中的衔接蛋白，该项目的远距离目标是将ACCH域的结构与其脂质结合能力相关联，从而驱动蛋白质分类和下游信号事件。该提议的中心假设是，不同AMOT同工型的脂质结合和脚手架功能可以对细胞极性事件进行特定的调节，最终影响导管细胞增生到乳腺癌中的发展。该假设是基于AMOT的各种同工型在上皮 - 间质细胞转变中起着不同作用的基础，这是导管细胞增生肿瘤发生的关键步骤。从统计学上讲，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功能的特定调节可能代表控制取消细胞的分化和扩散的治疗靶标。 该假设将通过追求三个特定目的来检验：1）确定AMOT ACCH结构域与膜表面相关的分子基础； 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指导的膜组织（细胞极性）细胞增殖与迁移之间的联系。 AIM 1旨在使用针对目标的定位诱变作为一种手段，以通过f rster共振能量传递和较小的角度散射来测量与合成膜环境的ACCH酪氨酸直接接触和插入。 AIM 2旨在使用含有PIP和木筏模拟混合物的合成机制来研究AMOT结构在细胞极性中的作用。 AIM 3将使用对体外细胞分析的背景下调节ACCH脂质结合特异性的理解，以描绘影响影响的能力 细胞迁移，分化和增殖。该方法具有创新性，因为它结合了生物物理和细胞生物学技术，以解决癌症生物学背景下AMOT脂质结合活性的结构功能关系。提出的工作很重要，因为它可以将ACCH结构域描述为分子开关，该分子开关应针对特定控制与导管癌相关的细胞分化和增殖。提案，这是候选人成为独立癌症研究者的目标的逻辑发展。在本奖项期间开发的研究将成为追求学术任期教师任命的平台。为了实现这一目标，将通过教学和经验丰富的途径获得培训。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关键蛋白的功能，从而影响进展 乳腺癌。但是，有必要增强她对癌症生物学的了解以及分子生物学，生物学显微镜和生物统计学的应用学科。为了解决这些培训目标，并更好地使她成为一名独立的癌症研究人员，这是一项特定的培训计划，将正式课程工作与指导者实验室的手工一席之地相结合。这些课程与特定目标，生物统计学和赠款的进一步发展有关。在奖励期间，将通过另一个教学课程和讲习班来解决负责任的研究。联合官员，博士。 Hurley and Wells将根据其研究专业知识领域，统计相关的评估开发，出版/赠款写作和研究道德的领域，每周向她的培训提供非正式的意见。专业和科学培训将包括利用机构资源，例如部门和癌症中心中心，程序间合作以及研究核心设施。因此，该提案的所有要素（例如研究，培训等）将在奖励的第四年准备并申请NIH R01，以进一步进一步实现她的独立研究状况。