Control of Guanine Exchange Factor Activity Using De Novo Designed Protein Inhibitors

Area of Science:

• Molecular Biology
• Cell Signaling
• Protein Engineering

Background:

• Guanine Exchange Factors (GEFs) activate RhoA GTPases, crucial for cellular processes.
• Understanding GEF function requires studying their activity in real-time within living cells.
• Existing tools lack the speed and reversibility needed for dynamic GEF modulation.

Purpose of the Study:

• To develop a versatile platform for engineering specific inhibitors against Dbl family GEFs.
• To create tools for rapid and reversible modulation of endogenous GEF activity in live cells.
• To demonstrate the utility of generative protein design for creating intracellular signaling modulators.

Main Methods:

• Utilized generative protein design to engineer high-affinity, specific inhibitors for Dbl family GEFs.
• Validated inhibitor efficacy both in vitro and in cellular environments.
• Implemented optogenetic control by coupling a GEF inhibitor to a light-activated module.

Main Results:

• Engineered proteins demonstrated high affinity and specificity for target GEFs.
• Modulation of GEF activity was confirmed both in vitro and within living cells.
• Successful optogenetic control of GEF activity was achieved using a light-activated inhibitor.

Conclusions:

• Generative protein design is a powerful approach for creating specific modulators of intracellular signaling pathways.
• The developed platform expands the toolkit for studying GEF and GTPase biology in real-time.
• This work enables new avenues for investigating cellular signaling dynamics and developing targeted therapeutics.