Designing cell-penetrating peptides for subcellular applications

Abstract

Cellular delivery of functionalised peptides has been a significant challenge in the peptide-design and cell-biology fields. To address this, recently, the Woolfson lab and collaborators developed a two-component system comprising complementary de novo designed acidic and basic coiled-coil peptides. The basic peptide can reliably enter cells and deliver attached peptide and small-molecule cargoes. With the acidic peptide introduced as gene fusions to intracellular proteins of interest (PoI), the basic peptide can be used as an exogenous reagent to target these PoIs directly in cells. This has been demonstrated by targeting engineered and endogenous kinesin motors in HeLa cells. This thesis explores this system further, showing that truncating the basic peptide partner allows the interaction with and allosteric activation of the engineered kinesin to be modulated. Taking insights gained from this system, peptides were designed to target endogenous intracellular coiled-coil-containing proteins more generally. The intermediate filament vimentin is a promising target. It contains large coiled-coil domains and is implicated in cancer cell transformation and embryonic development. Computational, rational, and experimental methods have been combined to design de novo cell-penetrating peptides that bind vimentin-derived peptides in vitro and filaments in live cells. More specifically, the interlock-1A domain of vimentin, which is vital for the association of vimentin proto-fibrils, has been targeted. Thus, the de novo peptides have the potential to disrupt the formation of vimentin filaments and probe their function in cell biology.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Dek N Woolfson (Supervisor) & Paul Verkade (Supervisor)