Examination of Protein-Protein Interaction of GRASP and its Impact on Cell Motility

Open Access
Author:
Reilly, Gregory Peter
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wendy Hanna-Rose, Honors Advisor
  • Lorraine Santy , Thesis Supervisor
Keywords:
  • GRASP
  • HGF
  • HGF Pathway
  • Dock 180
  • Epithelial cells
Abstract:
Epithelial cells typically exist within many parts of the body including lining most of the body’s organs, lining most of the major body cavities and can even specialize into sensory receptors. These cells typically tend to be immobile, but when faced with pathological stress or physiological stress within the body, these cells become motile. One major signaling factor that cues for this motility is Hepatocyte Growth Factor (HGF). HGF signals for various proteins, mainly small GTPases in the Rho family, to produce actin protrusions on the cell. In order to activate these small GTPases, many intermediate steps involving cell trafficking are involved. This cell trafficking pathway has been a primary focus of this lab in the past, which lead to the discovery of the role of a protein in the HGF pathway, GRASP. GRASP acts as a scaffolding protein, allowing the interaction of Cytohesin-2 and Dock-180 to occur, which eventually leads to cell motility. Recently within this lab, a mutant of GRASP was isolated, called GRASP- Double Mutant (GRASP DM), that lacks two putative SH3 binding domains; these domains are known to mediate interactions with other proteins. How these mutations affect the normal protein-protein interactions is currently not fully understood. The aim of my research is to identify proteins that interact with GRASP but not the GRASP DM and then to test the impact of knocking down one or several of these proteins on cell motility. While this goal was not reached, the process of growing and maintaining the GRASP protein was optimized via using exon optimized plasmids, and room temperature to grow the bacteria. Furthermore, the Mira-prep protocol was tested and confirmed to work, and the shelf life of GRASP was discovered to be around 5 weeks old.