Browsing by Subject "adaptor proteins"

Now showing 1 - 3 of 3
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    Structural Characterization of the ACCH Domain of Angiomotin Family Members
    (Office of the Vice Chancellor for Research, 2016-04-08) Virtanen, Piiamaria; Petrache, Horia; Kimble-Hill, Ann C.
    The 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.
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    Targeting the Role of Tyrosine in Amot Protein-Lipid Binding Events
    (Office of the Vice Chancellor for Research, 2016-04-08) Abufares, Nawara A.; Gebre, Haben; Ray, Bruce D.; Kimble-Hill, Ann C.
    Angiomotins (Amots) are a family of adaptor proteins that have been shown to control cell proliferation and differentiation. Amots can selectively bind with high affinity to phosphoinositol containing membranes through the Amot coiled-coil homology (ACCH) domain. This binding event is linked to endocytosis, changes in cellular polarity, and apical membrane sequestration of nuclear transcription factors associated with development of cancerous phenotypes. Although the lipid selectivity of the protein has been well characterized, the residues involved in the ACCH domain binding these membranes have not been fully described. Understanding the structure-function relationship may provide pathways to modulate protein sorting and downstream signaling events inducing cellular differentiation, cancer cell proliferation, and migration. The fluorescent properties of the ACCH domain were previously used to characterize the binding event. However, the relative proximity of the five native tyrosines to the membrane may have led to differences in perceived lipid binding affinities based on fluorescence resonance energy transfer 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. This was done to determine if each tyrosine had the ability to bury into the hydrophobic region of the membrane mimicked by the carbon chain lengths (alcohol study), or simply interacted with the hydrophilic head groups of the lipid (liposome study). In addition, the full length Amot80 ACCH domains (wild-type and tyrosine-to-phenylalanine mutants) were screened for trends in the varying environments. Interactions were characterized by shifts in maximum wavelengths for absorbance, excitation and emission peaks. A characterization of these shifts with respect to what is seen with the various tyrosine and phenalanine mutants may further our understanding of whether each tyrosine is buried within the protein or interacts with the head groups of the membrane.
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    Toward understanding the structure of Amot’s ACCH Domain
    (Office of the Vice Chancellor for Research, 2016-04-08) Peck, Cameron; Hurley, Thomas D.; Wells, Clark D.; Kimble-Hill, Ann C.
    Amots 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.