Increased cytotoxicity of interleukin 2-Pseudomonas exotoxin (IL2-PE) chimeric proteins containing a targeting signal for lysosomal membranes

Fishman, Ala; Barkana, Yaniv; Steinberger, Ida; Lorberboum-Galski, Haya

doi:10.1021/bi00186a025

Cited by 8 publications

(7 citation statements)

References 31 publications

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“…In moderately PE‐sensitive cells such as lymphocytes which are unable to process PE, the toxin intracellular pathway is essentially restricted through its interaction with the LRP (Kounnas et al ., 1992) to endocytic elements involved in the transferrin cycle (Alami et al ., 1998). In agreement with this localization, we observed cell‐free PE translocation from endosomes (Alami et al ., 1998) and signals targeting PE chimeras to the lymphocyte endocytic pathway enhanced toxicity to this cell type (Fishman et al ., 1994).…”

Section: Discussionmentioning

confidence: 99%

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A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

Taupiac

Bébien

Alami

et al. 1999

Molecular Microbiology

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SummaryPseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains. Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death. To investigate the role of the six a-helices (A±F) that form the translocation domain, we deleted them successively one at a time. All mutants showed native cell-binding and catalytic activities, indicating that deletions speci®cally affected translocation activity. This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectly by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication. Deletion of B, C and D helices resulted in non-toxic and non-translocating molecules, whereas mutants lacking the A or E helix displayed signi®cant cytotoxicity albeit 500-fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three-to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtained until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule.

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Section: Discussionmentioning

confidence: 99%

“…Fusion of the alkaline‐phosphatase lysosomal targeting signal to the amino terminus of a chimeric protein obtained by coupling interleukin 2 to a truncated PE lacking its cell recognition domain enhanced cytotoxicity to lymphocytes two‐ to threefold (Fishman et al ., 1994).…”

Section: Introductionmentioning

confidence: 99%

A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

Taupiac

Bébien

Alami

et al. 1999

Molecular Microbiology

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“…Construction of a plasmid encoding the IL2-Bax protein; expression of the protein and partial puri¢cation pHL906 [20], which carries the fusion gene IL2-PE40, was cut with HindIII and PpuMI, removing the PE sequence, and the vector fragment was eluted.…”

Section: Methodsmentioning

confidence: 99%

Interleukin 2‐Bax: a novel prototype of human chimeric proteins for targeted therapy

Aqeilan¹,

Yarkoni²,

Lorberboum-Galski³

1999

FEBS Letters

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During the past few years many chimeric proteins have been developed to target and kill cells expressing specific surface molecules. Generally, these molecules carry a bacterial or plant toxin that destroys the unwanted cells. The major obstacle in the clinical application of such chimeras is their immunogenicity and non-specific toxicity. We have developed a new generation of chimeric proteins, taking advantage of apoptosis-inducing proteins, such as the human Bax protein, as novel killing components. The first prototype chimeric protein, IL2-Bax, directed toward IL2R-expressing cells, was constructed, expressed in Escherichia coli and partially purified. IL2-Bax increased the population of apoptotic cells in a variety of target T cell lines, as well as in human fresh PHA-activated lymphocytes, in a dose-dependent manner and had no effect on cells lacking IL2R expression. The IL2-Bax chimera represents an innovative approach for constructing chimeric proteins comprising a molecule that binds a specific cell type and an apoptosis-inducing protein. Such new chimeric proteins could be used for targeted treatment of human diseases.z 1999 Federation of European Biochemical Societies.

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“…However, the apparent recognition by native CTX of a number of different targets, particularly cardiac cells, potentially limits their therapeutic utility. Nevertheless, other toxins, such as pseudomonas exotoxin, have been demonstrated to possess a cell recognition site which is distinct from its lytic domain [13]. Before one could determine whether the cytotoxins similarly possess distinct binding and lytic domains, information about their binding and lytic parameters needs to be obtained.…”

Section: Introductionmentioning

confidence: 99%

Heart and T-Lymphocyte Cell Surfaces Both Exhibit Positive Cooperativity in Binding a Membrane-Lytic Toxin

Stevens‐Truss

Messer

Hinman

1996

Journal of Membrane Biology

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Cobra venom cytotoxins (CTX) have been shown to disrupt cells as different as immunocytes, skeletal myocytes, erythrocytes and tumor cells. Nevertheless, even subpopulations of tumor cells are differentially susceptible to CTX by an order of magnitude. In the present study, our objective was to compare CTX-specific binding with cytolytic potency for two disparate cell types in vitro. We investigated the lytic activity of cytotoxin-III from Naja naja atra (NNA, fraction D) using heart cells and human leukemic T-cells (CEM cells). For both cell types, 50% cytolysis, assessed by tetrazolium dye conversion, occurred with microM concentrations of toxin (EC50 = 2.2 microM). We examined the binding of radiolabeled CTX III to both heart cells and CEM cells and found the apparent dissociation constant (KDapp) to be 0.69 microM and 0.75 microM, for CEM and heart cells respectively. The Bmax for the CEM cells was 1.0 fmoles/cell and that for heart cells was 5.2 fmoles/cell, both exhibiting positive cooperativity between the sites (Hill coefficients 1.4, T-cells; 1.6, heart). Relatively modest dissociation constants plus high numbers of binding sites per cell are consistent with a model of CTX binding to plasma membranes by interaction with phospholipids in the bilayer. Our results suggest that the lytic activity of this cytotoxin follows its binding to a population of sites on the cells in a cooperative fashion.

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Increased cytotoxicity of interleukin 2-Pseudomonas exotoxin (IL2-PE) chimeric proteins containing a targeting signal for lysosomal membranes

Cited by 8 publications

References 31 publications

A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

Interleukin 2‐Bax: a novel prototype of human chimeric proteins for targeted therapy

Heart and T-Lymphocyte Cell Surfaces Both Exhibit Positive Cooperativity in Binding a Membrane-Lytic Toxin

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