Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen.

Amy E Rabideau,Xiaoli Liao,Bradley L Pentelute,Gizem Akçay

doi:10.1038/srep11944

Amy E Rabideau, Xiaoli Liao + Show 2 more

Open Access

https://doi.org/10.1038/srep11944

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Journal: Scientific reports	Publication Date: Jul 16, 2015
Citations: 23	License type: CC BY 4.0

Affiliation: Massachusetts Institute of Technology

Abstract

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LFN) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.

Highlights

Nature has evolved several types of translocation systems for delivery of toxic proteins into host cells[1]
We investigated the ability of protective antigen (PA) to translocate complex, cytotoxic small molecules that are frequently used in antibody-drug conjugates: doxorubicin, docetaxel, and monomethyl auristatin F (MMAF)[32]
We investigated whether PA pore could efficiently transport non-canonical polypeptides into the cell cytosol

Summary

Introduction

Nature has evolved several types of translocation systems for delivery of toxic proteins into host cells[1] Certain pathogenic bacteria such as Vibrio cholerae, Corynebacterium diphtheria, and Bacillis anthracis infect host cells with cholera, diphtheria, and anthrax toxin, respectively[2]. The pore-forming protein, PA83 binds to an anthrax toxin receptor on the host cell surface and is proteolytically activated by furin protease to give PA634, triggering oligomerization of PA63 into a heptameric[5] or octameric[6] pre-pore. The entire complex is endocytosed and the low pH triggers a conformational rearrangement of the pre-pore to form a pore that spans through the endosomal membrane[9], initiating translocation of LF into the cytosol[10,11]. Protein cargo partially unfolds in the acidic endosome prior to translocation15,16. Cargo passes through the PA conduit of approximately 12 Å diameter[13,17]

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