Beyond Small Molecules: Orchestrating Cell Fate with Engineered Water-Soluble Membrane Proteins

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Water-soluble membrane proteins (wsMPs) have unrealized potential due to challenges in their application.
• Existing methods for manipulating membrane protein function are limited.

Purpose of the Study:

• To develop a method for creating functional water-soluble membrane proteins (wsMPs) that can be deployed in vivo.
• To demonstrate the ability of these wsMPs to incorporate into cell plasma membranes and alter cellular fate.
• To compare signaling pathway activation by wsMPs versus conventional small molecules.

Main Methods:

• Developed the in vivo deployment of recombinant viable membrane proteins (iDRIVE) strategy.
• Engineered water-soluble pore-forming proteins (MthK) and constitutively active G protein-coupled receptors (GPCRs), including frizzled receptors.
• Validated functional properties in vitro and in vivo using cellular fate assays, signaling pathway analysis, and epigenetic studies.

Main Results:

• Demonstrated successful unidirectional insertion of wsMPs into the plasma membrane via iDRIVE.
• Showcased functional activity of wsMPs, including K+ channels and active GPCRs, in altering cellular fate.
• Induced differential methylation via Wnt signaling activation using wsFrizzled receptors (iDRIVE-FZD), showing more biologically relevant epigenetic changes than small molecule agonists.

Conclusions:

• The iDRIVE method enables the creation and in vivo deployment of functional wsMPs.
• wsMPs can effectively modulate cellular signaling and fate, offering an alternative to small molecule agonists.
• iDRIVE holds promise for future applications in stem cell differentiation and organoid development.