Undergraduate Honors Thesis

 

Quantifying the Binding Affinity of Disease Linked Ras Mutants to the Ras Binding Domain of PI3Kγ by Microscale Thermophoresis Public Deposited

https://scholar.colorado.edu/concern/undergraduate_honors_theses/3484zj18b
Abstract
  • The Ras family of GTPases (H/K/N-Ras) mediate numerous signaling pathways involved in cell proliferation, metabolism and cell motility through effectors such as Raf and phosphatidylinositol-3-kinase (PI3K). Ras bound to GTP adopts an active conformation that is able to bind to its effectors. Its intrinsic GTP hydrolysis activity yields an inactive, GDP-bound conformation, regulating the activation of its effectors as a molecular “on-off” switch. Ras mutations are present in around 30% of human cancers, predominantly in positions 12, 13, and 61. Mutations at these positions are thought to inhibit downregulation by GTPase Activating Proteins (GAPs) that normally increase its intrinsic rate of GTP hydrolysis by several orders of magnitude, leaving Ras in a constitutively activated form that hyperactivates effector pathways. However, a significant number of disease-linked Ras mutations occur at other positions. Notably, over 30 occur at interfacial positions that contact the Ras Binding Domain (RBD) of PI3K. We hypothesize that some of these mutations may increase (or decrease) the binding affinity of Ras to PI3K, which in turn would be predicted to recruit more (or less) PI3K to the plasma membrane and increase (or decrease) production of the signaling lipid phosphatidylinositol-3,4,5-triphosphate (PIP3). An increase in PIP3 production is observed in many human cancers and is thought to hyperactive downstream effectors of the PI3K/Akt/mTOR pathway involved in the regulation of cellular growth.  A decrease in PIP3 production, by contrast, may yield growth defects that underly other human diseases linked to growth deficiencies.

    The work presented in this thesis builds off of the significant progress of previous lab members, who designed the protein constructs used in this project and the methods for purification. My work includes optimizing the existing protein purification methodology and improving the microscale thermophoresis (MST) assay used to quantify the binding affinity of Ras to the RBD of PI3Kγ in solution.  I also helped design a robust method to standardize the analysis MST data. Perhaps most significantly, I collected MST data extensively, in conjunction with my undergraduate colleague Ian Fleming, that characterize the binding affinity of standard Ras, GDP-loaded standard Ras, and the Ras variants Q25L, D38E, Y40C, and R41Q to the PI3Kγ RBD. Our initial work has been published in Analytical Biochemistry (2022), in a paper reporting the first Ras mutation, Q25L, found to trigger increased affinity for Ras binding to the PI3Kγ RBD.  More recently, new findings first presented herein show that the Ras mutation R41Q triggers an even larger increase in Ras affinity for Ras binding to the PI3Kγ RBD.  Future work by the single molecule subgroup in our laboratory will test our predictions that (i) the Ras Q25L and R41Q mutations will each generate increased binding of full length PI3K to membrane-anchored Ras on a target membrane, and (ii) the resulting larger population of active, membrane-bound PI3K lipid kinase molecules will in turn exhibit increased net PIP3 production under physiological conditions.

     

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  • 2023-04-12
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  • 2023-04-20
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