Engineering of bacterial exotoxins for highly efficient and receptor‐specific intracellular delivery of diverse cargos

Ryou, Jeong-Hyun; Sohn, Youn Soo; Hwang, Dosam; Park, Woo‐Yong; Kim, Nury; Heo, Won Do; Kim, Mi Young; Kim, Kwang Ho

doi:10.1002/bit.25935

Cited by 21 publications

(26 citation statements)

References 30 publications

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“…Prior to modulation of the ERK2‐mediated signaling process using PEA‐15, we attempted the cytosolic delivery of PEA‐15. We previously developed a protein delivery platform comprising a receptor binding domain of Shiga‐like toxin (StxB) and a translocation domain of Pseudomonas exotoxin A (TDP) (Figure b) (Ryou et al, ). The toxin‐based delivery system was shown to efficiently transfer diverse proteins into the cytosol of the StxB‐targeted Globotriaosylceramide (Gb3) positive cell lines.…”

Section: Resultsmentioning

confidence: 99%

“…The proteins were over‐expressed by addition of 0.5 mM of isopropyl‐d‐1‐thiogalactopyranosid (IPTG) when OD 600 reached about 0.6 and further incubation at 18°C for 20 hr. Cells were harvested and disrupted through sonication, and the proteins were purified by Ni‐NTA agarose resins and size exclusion chromatography as previously described (Ryou et al, ).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were incubated with 100 nM of proteins fused with a bacterial‐toxin based delivery system as described in our previous work (Ryou et al, ), followed by washing them twice with DPBS. The cells were fixed using 4% paraformaldehyde for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway

Ryou

Sohn

Kim

et al. 2018

Biotech & Bioengineering

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The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway

Ryou

Sohn

Kim

et al. 2018

Biotech & Bioengineering

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“…Many bacterial toxins have been extensively studied for their promising clinical applications [43][44][45][46][47]. A previous study has shown that human gingival squamous carcinoma cell line Ca9-22 transfected with Actinobacillus actinomycetemcomitans-CdtB expressing plasmid, manifested G2 phase arrest and cell growth inhibition [48].…”

Section: Discussionmentioning

confidence: 99%

Hyaluronic Acid Nanoparticles-encapsulated Bacterial Genotoxin for Targeted Therapy of Radioresistant Prostate Cancer

Chen¹,

Lai²,

Wu³

et al. 2020

Preprint

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Background: Prostate cancer (PCa) is one of the most commonly diagnosed cancers in men and usually becomes refractory because of recurrence and metastasis. CD44, a transmembrane glycoprotein, serves as a receptor for hyaluronic acid (HA) and has been found to be abundantly expressed in cancer stem cells (CSCs) that often exhibit a radioresistant phenotype. Cytolethal distending toxin subunit B (CdtB), produced by Campylobacter jejuni, is a genotoxin acts as a type I deoxyribonuclease (DNase I), which is responsible for creating DNA double-strand breaks (DSBs). Nanoparticles loaded with antitumor drugs and specific ligands that recognize cancerous cell receptors are promising methods to overcome the therapeutic challenges. Results: In this study, PCa cells with DOC-2/DAB2 interactive protein (DAB2IP) knockdown were employed to imitate the CSC with radioresistant phenotype. We further prepared HA-decorated nanoparticles-encapsulated CdtB (HA-CdtB-NPs) and investigated the targeted therapeutic activity in radioresistant PCa cells. Our results showed that HA-CdtB-NPs sensitized radioresistant PCa cells by enhancing DSB and causing G2/M cell-cycle arrest, without affecting the normal prostate epithelial cells. These results demonstrate that HA-CdtB-NPs possess maximum target-specificity and delivery efficiency of CdtB into the nucleus, and enhance the effect of radiation in radioresistant PCa cells. Conclusions: These findings indicate that HA-loaded CdtB nanoparticles exert target-specificity accompanied with radiomimetic activity, which can be developed as an effective agent for overcoming radioresistance in PCa.

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“…The structurally and functionally distinct toxic cargo delivered across the AB 5 toxin group highlights the advantages of the common non-covalent architecture as an extensible platform for delivery of protein cargo. While direct genetic fusions of protein cargo to the B subunits are possible 18 , 19 , assembly and glycolipid binding are often inhibited 20 . The relatively simple architecture of the heat labile enterotoxin (LT) family is particularly attractive for further development of modular non-covalent protein carriers.…”

Section: Introductionmentioning

confidence: 99%

Engineering an AB5 Protein Carrier

Lichtenstein

Höcker

2018

Sci Rep

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The promise of biologic therapeutics is hindered by the challenge to deliver their activity to biochemically relevant sites within diseased cells. The favourable application of the natural protein carriers of the AB5 toxin family to this challenge has been restricted owing to still unresolved requirements for assembling non-native cargo into carrier complexes. Here, we clarify the properties of fusion peptides which allow co-assembly of a selected fluorescent protein cargo with the non-toxic B subunit of a heat-labile enterotoxin. We establish the influence of sequence length, sequence identity and secondary structure of these linking domains on the assembly and disassembly of the complexes. Through our engineering framework we identify several non-native, reduced length fusion sequences that robustly assemble with the native carriers, maintain their ability to deliver protein cargo to cells, and demonstrate substantially refined in vitro properties. Constructs based upon these sequences should prove directly applicable to a variety of protein delivery challenges, and the described design framework should find immediate application to other members of the AB5 protein carrier family.

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Engineering of bacterial exotoxins for highly efficient and receptor‐specific intracellular delivery of diverse cargos

Cited by 21 publications

References 30 publications

Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway

Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway

Hyaluronic Acid Nanoparticles-encapsulated Bacterial Genotoxin for Targeted Therapy of Radioresistant Prostate Cancer

Engineering an AB5 Protein Carrier

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