Browsing by Author "Abufares, Nawara A."

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    Targeting the Role of Tyrosine in Amot Protein-lipid Binding Events
    (Office of the Vice Chancellor for Research, 2015-04-17) Abufares, Nawara A.; Kimble-Hill, Ann C.
    Amot 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 Medicine
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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.