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.
The proposed work is relevant to public health because over 2.5 million women in the United States are affected by breast cancer, either living with the diagnosis, in active treatment, or in remission. Specifically, the work is directed toward understanding the biophysical mechanisms involved in the arrangement of cancer related proteins mammary cell membrane. Thus the proposed research is relevant to the part of NIH-NCI's mission that pertains to developing knowledge to diagnose and give a prognosis for breast cancer.
|Johnson, Merrell A; Seifert, Soenke; Petrache, Horia I et al. (2014) Phase coexistence in single-lipid membranes induced by buffering agents. Langmuir 30:9880-5|