Systematic Survey of the Role of IGF in the Link Between Diabetes and Cancer

Epidemiological studies have proposed a link between type II diabetes and cancer via the IGF/insulin signaling pathway, which includes insulin-like peptides (IGF1, IGF2, and insulin), insulin receptors (IR-A, IR-B, IGF1R, and hybrids), and insulin substrate proteins (IRS1-6). In this study, up- and down-regulation of various components in the IGF/insulin signaling pathway are compared to clinical outcomes for cancer patients; the components include diagnosis age, overall survival, tumor invasion and vascularization, and body mass index. It was found that the up-regulation of insulin growth Factor (IGF)/insulin components was associated with overall survival and tumor invasion and vascularization, while the down-regulation of equivalent components was not associated with clinical outcomes assessed in this study. Particularly, the up-regulation of DOK5, IGF2, and IRS2 in colorectal cancer and IGF1R in liver cancer is associated with significantly decreased overall survival. Functional aberrations in either of the two proteins in co-expression pairs were identified for each cancer and correlated with overall survival and diagnosis age. Specific biomarkers proposed in this study will be further analyzed to fine-tune consistent associations that can be translated to reliable prognostic standards for the roles of IGF/insulin signaling pathway modulations that promote cancer

A review of factors affecting the success of membrane protein crystallization using bicelles

Author's manuscript made available in accordance with the publisher's policy.Several reports have been published detailing various platforms for obtaining crystals of membrane proteins to determine their structure including those that use disk shaped bilayers called bicelles. While these crystals have been readily grown and used for x-ray diffraction, the general understanding as to why bicelles are adequate for such a procedure or how to rationally choose conditions remains unknown. This review intends to discuss issues of protein stabilization and precipitation in the presence of lipids that may influence crystal formation

Incorporating Identity Safety into the Laboratory Safety Culture

Chemistry practitioners, particularly in educational settings, often associate building strong safety cultures with compliance or regulatory requirements around laboratory glass-ware, equipment, flammable and incompatible materials, signage, container labels, and safety data sheets. Other fields of science also emphasize biohazardous materials, animal handling, human subject, and ergonomics. However, little attention in the literature has gone toward describing the interpersonal interactions and behaviors affecting the physical and emotional safety and wellbeing of laboratory trainees and personnel from marginalized backgrounds. This work unifies known approaches of building strong safety cultures and principles for preventing identity cues that threaten safety within a laboratory environment. Specifically, this work uses the four principles of chemical safety RAMP model as a conceptual framework for integrating identity safety within the laboratory safety culture

Structural Characterization of the ACCH Domain of Angiomotin Family Members

poster abstractThe Angiomotin (Amot) family of adaptor proteins directly coordinates signaling events during cellular and neural differentiation and proliferation. A critical feature of all Amot proteins is a novel lipid binding domain, the Amot coiled-coil homology (ACCH) domain, which confers its association with membranes and affects membrane curvature and deformation. Specifically, this domain has the unique ability to selectively bind monophosphorylated phosphatidylinositols (PIs) and cholesterol. Furthermore, Amot family members bind core polarity proteins that control the organization of the apical domain of epithelial cells as well as Yap, a transcriptional coactivator that appears to be the key regulator of cell growth. Amots have been shown to have a critical role in endothelial and epithelial cell migration, invasion, and tubule formation, and they are believed to regulate angiogenesis, which promotes tumor growth and metastasis. Amot overexpression and mutations have been linked to neuroepithelial tumors, such as glioblastomas, brain hemangioendotheliomas, neurofibromatosis, and many other cancers, such as breast cancer. The role of Amots in epithelial and endothelial cancer growth and metastasis have been linked to poor prognosis and unfavorable clinical outcomes. Understanding the structure-function relationship of the ACCH domain may provide pathways to modulate protein sorting and downstream signaling events inducing cellular differentiation, cancer cell proliferation, and cell migration. The goal of this project is to generate a solution structure of the Amot80/130 and AmotL2 (Mascot) ACCH domains using SAXS and WAXS data as well as various protein modeling software, thereby suggesting possible routes to modulate their activity associated with various tumors. Additionally, this structure will be compared against theoretical models to determine the statistical accuracy of the theoretical models. Furthermore, we hypothesize that generating these models will allow us to determine the structure of another analogue of A80/130, the Angiomotin-like 1 (JEAP) ACCH domain

Using the Predicted Structure of the Amot Coiled Coil Homology Domain to Understand Lipid Binding

Angiomotins (Amots) are a family of adapter proteins that modulate cellular polarity, differentiation, proliferation, and migration. Amot family members have a characteristic lipid-binding domain, the coiled coil homology (ACCH) domain that selectively targets the protein to membranes, which has been directly linked to its regulatory role in the cell. Several spot blot assays were used to validate the regions of the domain that participate in its membrane association, deformation, and vesicle fusion activity, which indicated the need for a structure to define the mechanism. Therefore, we sought to understand the structure-function relationship of this domain in order to find ways to modulate these signaling pathways. After many failed attempts to crystallize the ACCH domain of each Amot family member for structural analysis, we decided to pursue homologous models that could be refined using small angle x-ray scattering data. Theoretical models were produced using the homology software SWISS-MODEL and threading software I-TASSER and LOMETS, followed by comparison to SAXS data for model selection and refinement. We present a theoretical model of the domain that is driven by alpha helices and short random coil regions. These alpha helical regions form a classic dimer interface followed by two wide spread legs that we predict to be the lipid binding interface

Using Phosphatidylinositol Phosphorylation as Markers for Hyperglycemic Related Breast Cancer

Studies have suggested that type 2 diabetes (T2D) is associated with a higher incidence of breast cancer and related mortality rates. T2D postmenopausal women have an ~20% increased chance of developing breast cancer, and women with T2D and breast cancer have a 50% increase in mortality compared to breast cancer patients without diabetes. This correlation has been attributed to the general activation of insulin receptor signaling, glucose metabolism, phosphatidylinositol (PI) kinases, and growth pathways. Furthermore, the presence of breast cancer specific PI kinase and/or phosphatase mutations enhance metastatic breast cancer phenotypes. We hypothesized that each of the breast cancer subtypes may have characteristic PI phosphorylation profiles that are changed in T2D conditions. Therefore, we sought to characterize the PI phosphorylation when equilibrated in normal glycemic versus hyperglycemic serum conditions. Our results suggest that hyperglycemia leads to: 1) A reduction in PI3P and PIP3, with increased PI4P that is later converted to PI(3,4)P2 at the cell surface in hormone receptor positive breast cancer; 2) a reduction in PI3P and PI4P with increased PIP3 surface expression in human epidermal growth factor receptor 2-positive (HER2+) breast cancer; and 3) an increase in di- and tri-phosphorylated PIs due to turnover of PI3P in triple negative breast cancer. This study begins to describe some of the crucial changes in PIs that play a role in T2D related breast cancer incidence and metastasis

Targeting the Role of Tyrosine in Amot Protein-lipid Binding Events

poster abstractAmot proteins have been shown to control cell proliferation and differentiation and can selectively bind with high affinity to phosphoinositol containing membranes. This binding event is linked to endocytosis, changes in cellular polarity, and apical membrane sequestration of nuclear transcription factors associated with development of cancer phenotypes. Although the lipid selectivity of the protein has been well characterized, the mechanisms involved in the Amot coiled-coil homology domain (ACCH) binding these membranes are not yet known. The fluorescence properties of the ACCH domain were used to characterize the binding event, however it became clear each of the five native tyrosines proximity to membrane might differ based on fluorescence resonance energy transfer experiments with fluorescently tagged lipids. A variety of short peptides correlating to the amino acid sequence of Amot surrounding these tyrosines were assayed and observed in different membrane mimicking environments to determine if each tyrosine had the ability to bury into the hydrophobic region of the membrane (alcohol study), or simply interacted with the hydrophilic head groups (liposome study). Interactions were characterized by shifts in absorbance, excitation and emission scans peaks. A characterization of these shifts with respect to what is seen with the various tyrosine-phenalanine mutants will further our understanding of whether each tyrosine is buried within the protein or interacts with the membrane. Mentor: Ann Kimble-Hill, Department of Biochemistry, IU School of Medicin

Defining the Roles of Various Lysines and Arginines in Amot Lipid Binding

poster abstractOne of the defining traits of cancerous cells is proliferation. The focus of this study is on the proliferation of mammary cells. As an adaptor protein, the Amot membrane binding event is key to the localization and sorting of proteins responsible for cellular differentiation, proliferation, and migration. The Amot coiled-coil homology domain (ACCH) is a lipid-binding domain responsible for cholesterol affinity and binding to endothelial membranes. Our working hypothesis is that the ability to modulate Amot lipid-binding will lead to means to prevent ductal cell hyperplasia progression into breast cancer tumors. We will determine which residues are responsible for lipid-binding by changing positively charged lysine and arginine into uncharged or negatively charged amino acids. Approximately 40 of these mutations have been screened using a liposome binding assay which mimics how the protein binds with the cell membrane by using an in vitro mixture of lipids similar to that seen in endothelial cells. Forster resonance energy transfer (FRET) was used to confirm significant decreases in lipid binding of ACCH mutants selected from the liposome binding assay, as energy transfer only occurs when the tyrosines in the protein and the Dansylated liposome are in close proximity to each other. In order to saturate the binding affinity of the mutants, the liposomes will be combined with cholesterol in increasing amounts. It has been found that Amot protein is concentrated in areas of PI with higher levels of cholesterol. This will provide a target for the ACCH domain to associate with in the membrane. Mutants deemed important from this study will then be transformed into human cells to study their effects on cell polarity, signal transduction, cell shape, and cellular proliferation

Reorganization of Ternary Lipid Mixtures of Non-Phosphorylated Phosphatidylinositol Interacting with Angiomotin

Phosphatidylinositol (PI) lipids are necessary for many cellular signaling pathways of membrane associated proteins, such as Angiomotin (Amot). The Amot family regulates cellular polarity, growth, and migration. Given the low concentration of PI lipids in these membranes, it is likely that such protein-membrane interactions are stabilized by lipid domains or small lipid clusters. By small-angle x-ray scattering, we show that non-phosphorylated PI lipids induce lipid de-mixing in ternary mixtures of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), likely due to preferential interactions between the head groups of PE and PI. These results were obtained in the presence of buffer containing concentrations of Tris, HEPES, NaCl, EDTA, DTT, and Benzamidine at pH 8.0 that in previous work showed an ability to cause PC to phase separate but are necessary to stabilize Amot for in vitro experimentation. Collectively, this provided a framework for determining the effect of Amot on lipid organization. Using fluorescence spectroscopy, we were able to show that the association of Amot with this lipid platform causes significant reorganization of the lipid into a more homogenous organization. This reorganization mechanism could be the basis for Amot membrane association and fusigenic activity previously described in the literature and should be taken into consideration in future protein-membrane interaction studies

Toward understanding the structure of Amot’s ACCH Domain

poster abstractAmots are a family of adaptor proteins widely involved in cell signaling and lipid binding. Amot80 has been linked to cellular proliferation in breast cancer via the VEGF and MAPK signaling pathways, while Amot130 and AmotL1 have been linked to cellular inhibition via the HIPPO signaling pathway. Amot family members also have a characteristic lipid-binding domain – named the ACCH Domain for its predicted coil-coil structure – that has the ability to selectively target phosphoinositols followed by deformation of the membrane. Understanding the structure-function relationship of this domain may provide options to modulate these signaling pathways, directly affecting cellular differentiation, proliferation, and migration. Extensive crystallization attempts for this domain have failed, leading to a bioinformatics and biophysics-combined approach. Using SAXS, data for the globular structure of Amot80 has been generated and analyzed. Additionally, the threading programs ITASSER and LOMETS were used to develop 20 computational theoretical models. By fitting the computational models to the SAXS data, potential ACCH domain models were generated, and then scored based on accuracy of fit via C-score, TMScore, and RMSD values. This 3D model can then be used to discover how Amot interacts with lipids and further the understanding of Amot’s role in the cancer-signaling cascade