Development of a Generalizable Method to Activate Small GTPase Signaling in Living Cells

Small GTPases comprise a superfamily of over 167 proteins in the human genome and are critical regulators of a variety of pathways including cell migration and proliferation. The activity of these proteins is regulated by a molecular switch that cycles between two distinct states: a GTP-bound active state or a GDP-bound inactive state. The active GTP-bound conformation of the protein has a significantly higher binding affinity towards downstream effectors leading to diverse phenotypic changes. Dysregulation of small GTPase activity has been implicated in numerous human diseases, particularly cancers.
The critical role of these enzymes in disease pathology has spurred numerous efforts to modulate their activity. Traditional challenges include the inherent difficulties of targeting small GTPases with small molecules and navigating the complex cellular feedback mechanism involved. While innovative protein engineering strategies have been developed to manipulate specific small GTPases within cellular contexts, these approaches require considerable target-specific customization and are not universally applicable.
Addressing this gap, my dissertation introduces a generalized strategy to modulate small GTPase activity, crucial for dissecting disease-associated signaling pathways. Chapter 1 provides an overview of current biochemical techniques for regulating small GTPase activity. In Chapter 2, we present an innovative split-protein based method to control the activity of Cdc42 within mammalian cells, demonstrating its ability to induce filopodia formation in response to a specific molecular input. Building on this foundation, Chapter 3 extends the application of this fragmentation site to other small GTPases, including Rac1, RhoA and KRas. Specifically, we developed a novel and rapid assay for detecting new split-GTPase fragment reassembly and subsequent pathway reprogramming in living cells. Chapter 4 further explores the versatility of this system, showing that these fragmentation sites can be used with customizable inputs for protein reassembly, offering a “plug and play” solution for the direct activation of small GTPases tailored to specific research needs. Finally, Chapter 5 envisages the potential of our split-GTPase technology in tackling significant health-related biological problems, highlighting the potential to illuminate the mechanisms underlying disease processes.
This thesis work paves the way for a deeper understanding of small GTPase function and their implications in human health, offering a novel toolset for the scientific community to explore and manipulate cellular signaling pathways.