Dataset Information

Structural complementation of the catalytic domain of pseudomonas exotoxin A.

ABSTRACT: The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions-supplied exogenously as genetic elements or purified protein fragments-had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.

SUBMITTER: Boland EL

PROVIDER: S-EPMC3997303 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structural complementation of the catalytic domain of pseudomonas exotoxin A.

Boland Erin L EL Van Dyken Crystal M CM Duckett Rachel M RM McCluskey Andrew J AJ Poon Gregory M K GM

Journal of molecular biology 20131108 3

The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and ...[more]

PMID: 24211469

Similar Datasets

Project description:The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT6, can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, ZHER2:2891, or the dual-HER2-binding hybrid ZHER2:2891-ADAPT6 were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC50 values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT6 as targeting domain, ZHER2:2891 gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, ZHER2:2891 has the most favorable characteristics as targeting domain for PE25.

Dataset Information

Structural complementation of the catalytic domain of pseudomonas exotoxin A.

Publications

Structural complementation of the catalytic domain of pseudomonas exotoxin A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets