Dataset Information

Cytosolic delivery of proteins by cholesterol tagging.

ABSTRACT: Protein-based imaging agents and therapeutics are superior in structural and functional diversity compared to small molecules and are much easier to design or screen. Antibodies or antibody fragments can be easily raised against virtually any target. Despite these fundamental advantages, the power and impact of protein-based agents are substantially undermined, only acting on a limited number of extracellular targets because macrobiomolecules cannot spontaneously cross the cell membrane. Conventional protein delivery techniques fail to address this fundamental problem in that protein cargos are predominantly delivered inside cells via endocytosis, a remarkably effective cell defense mechanism developed by Mother Nature to prevent intact biomolecules from entering the cytoplasm. Here, we report a unique concept, noncovalent cholesterol tagging, enabling virtually any compact proteins to permeate through the cell membrane, completely bypassing endocytosis. This simple plug-and-play platform greatly expands the biological target space and has the potential to transform basic biology studies and drug discovery.

SUBMITTER: Tai W

PROVIDER: S-EPMC7304968 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cytosolic delivery of proteins by cholesterol tagging.

Tai Wanyi W Zhao Pengfei P Gao Xiaohu X

Science advances 20200619 25

Protein-based imaging agents and therapeutics are superior in structural and functional diversity compared to small molecules and are much easier to design or screen. Antibodies or antibody fragments can be easily raised against virtually any target. Despite these fundamental advantages, the power and impact of protein-based agents are substantially undermined, only acting on a limited number of extracellular targets because macrobiomolecules cannot spontaneously cross the cell membrane. Convent ...[more]

PMID: 32596467

Similar Datasets

Project description:The delivery of functional proteins remains a major challenge in advancing biological and pharmaceutical sciences. Herein, we describe a powerful, simple, and highly effective strategy for the intracellular delivery of functional cargoes. Previously, we demonstrated that cell-penetrating peptide (CPP) additives equipped with electrophilic thiol-reactive moieties temporarily attach to the cellular membrane, thereby facilitating the cellular uptake of protein- and antibody-CPP cargoes through direct membrane transduction at low concentrations. Now, we hypothesize that CPP-additives with an increased retention on the cellular membrane will further enhance intracellular uptake. We discovered that adding a small hydrophobic peptide sequence to an arginine-rich electrophilic CPP-additive further improved the uptake of protein-CPP conjugates, whereas larger hydrophobic anchors showed increased cytotoxicity. Cell viability and membrane integrity measurements, structure-activity relationship studies, and quantitative evaluation of protein-CPP uptake revealed important design principles for cell-surface-retained CPP-additives. These investigations allowed us to identify a nontoxic, thiol-reactive CPP-additive containing the hydrophobic ILFF sequence, which can deliver fluorescent model proteins at low micromolar concentrations. This hydrophobic CPP-additive allowed the addition of protein cargoes for intracellular delivery after initial additive incubation. Time-lapse fluorescence microscopy and membrane tension analysis of cells treated with fluorescent ILFF-CPP-additives supported the claim of increased cell surface retention and suggested that the protein-CPP cargoes enter the cell through a mechanism involving lowered cell membrane tension. Finally, we demonstrated that our newly engineered hydrophobic CPP-additive enabled the uptake of a functional macrocyclic peptidic MDM2-inhibitor and a recombinant genome editing protein. This indicates that the developed hydrophobic CPP-additive holds promise as a tool to enhance the intracellular delivery of peptide and protein cargoes.

Dataset Information

Cytosolic delivery of proteins by cholesterol tagging.

Publications

Cytosolic delivery of proteins by cholesterol tagging.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets