Cell migration is characterized by restricted temporal-spatial distribution of intracellular proteins that regulate actin protrusion and trailing edge retraction. Microtubule plus end tracking proteins (+TIPs) are part of the cell migration machinery, targeting the growing plus ends of the microtubules, controlling the cell dynamics during migration. A major class of +TIPs, microtubule end-binding (EB) protein family, are known to target the microtubules independently at the plus end by recognizing the growing structure of the microtubules through their N-terminal domain. The EB1 Cterminal domain is an á-helical coiled coil responsible for interacting with other classes of +TIPs such as SxIP proteins. SxIP proteins are rich in basic amino acids, containing a conserved sequence of Ser-x-Ile-Pro. Thus, they need interaction with EB1 to target the plus end of the microtubules. According to the conserved sequence in SxIP proteins, we designed 9-11 residue peptides to determine the main residues responsible for interacting with EB1. To examine the interaction between the designed peptides and EB1, we applied a solid-base binding assay and determined the Kd values for each peptide. We observed a significant decrease in binding affinity of a scrambled amino acid sequence, but not the other peptides. Therefore, we labeled the peptide with fluorescein to see the fluorescence polarization while binding to EB1 and also inhibiting binding of labeled peptide to EB1 by higher concentration of un-labeled peptide in solution. In addition, we studied the peptide localization by using a carrier reagent and microinjecting the peptide directly into B16F10 mouse melanoma cells, CHO cells and NIH 3T3 cells. We observed intracellular localization consistent with microtubules plus ends. The results of the studies are valuable in determining the minimal recognition sequence for EB1 and designing of small molecule inhibitors to block interaction between SxIP proteins and EB1. An EB1 inhibitor may block cellular processes in disease such as metastasis of malignant cells during cancer progression. However, it is important to examine larger peptide sequences containing other frequent residues identified in other SxIP proteins.
|Advisor:||Schober, Joseph M.|
|Commitee:||Khazeli, Sadegh, Kontoyianni, Maria, Neumann, William L.|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 52/05M(E), Masters Abstracts International|
|Subjects:||Cellular biology, Biochemistry, Pharmacy sciences|
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