Intacellular Switches

When scientists hope to deduce the function of a particular gene, the most common approach is to inhibit that gene or its protein product and then examine the cellular aftermath. Whether inhibition is achieved through small molecules, siRNA, or gene editing, proper interpretation of the cause (the gene/the protein it produces) and its effect (cell signaling, trafficking, organ development, disease manifestation, etc.) requires that inhibition is precise enough to inhibit only the gene of interest and rapid enough that the cell cannot compensate for the inhibition. Accordingly, the ideal technology to study gene function can be imagined as a cellular switch that precisely, rapidly, and reversibly inhibits gene activity.

To this end, our lab employs intracellular elastin-like polypeptides (ELPs) to reversibly modulate protein function through subtle changes in temperature. This approach works at the protein level and is achieved by fusing ELPs to a given cellular protein. The resulting ELP fusion protein retains the functionality of the wild type cellular protein but only at temperature ranges permitted by the attached thermally responsive ELP peptide sequence. As temperature is raised above the transition temperature (Tt) of the attached ELP, the ELP fusion proteins are induced to rapidly assemble together. Depending on the cellular protein, this assembly may inhibit or activate the protein of interest and its relevant pathway. Importantly, temperature-mediated assembly is reversed by decreasing temperature below the Tt of the attached ELP. In addition, ELPs are non-toxic, non-immunogenic, and genetically encodable, just like the endogenous cellular proteins to which they are cloned.

In 2014, our lab reported that temperature mediated assembly of ELP-Clathrin light chain (CLC) fusion proteins can be employed to reversibly inhibit Clathrin Mediated Endocytosis (Pastuszka et al., 2014). We are currently expanding on this result and are characterizing fusion proteins composed of ELPs and Caveolin 1 to reversibly inhibit Caveolae Mediated Endocytosis. In addition to cellular trafficking, we are also exploring Epidermal Growth Factor Receptor ELP fusion proteins to reversibly activate EGFR signaling in the absence of EGF ligand. Overall, we envision this ELP based cellular switching as a widely applicable tool for the study of cellular events.

Selected Publications

Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains
Martha K. Pastuszka, Curtis T. Okamoto, Sarah F. Hamm-Alvarez, and J. Andrew MacKay
Adv Funct Mater. 2014 Sep 10; 24(34): 5340–5347.

Triggered Sorting and Co-Assembly of Genetically Engineered Protein Microdomains in the Cytoplasm
Pu Shi, Yi-An Lin, Martha Pastuszka, Honggang Cui, and J. Andrew MacKay
Adv Mater. 2014 Jan 22; 26(3): 449–454.

A Tunable and Reversible Platform for the Intracellular Formation of Genetically Engineered Protein Microdomains
Martha K. Pastuszka, Siti M. Janib, Isaac Weitzhandler, Curtis T. Okamoto, Sarah Hamm-Alvarez, and J. Andrew MacKay
Biomacromolecules. 2012 Nov 12; 13(11): 3439–3444.