Prolonged display or rapid internalization of the IgG-binding protein ZZ anchored to the surface of cells using the diphtheria toxin T domain

Nizard, Philippe; Chenal, Alexandre; Beaumelle, Bruno; Fourcade, A; Gillet, Daniel

doi:10.1093/protein/14.6.439

Cited by 22 publications

(27 citation statements)

References 21 publications

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“…Interestingly, DTox included in different parts of the MRTs caused similar defects in lipid membranes, which suggests a possibility to use the DTox as an endosomolytic module in different polypeptide contexts, which agrees with findings made by Nizard et al (35).…”

Section: Discussionsupporting

confidence: 90%

Targeting Cancer Cells by Novel Engineered Modular Transporters

et al. 2006

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A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed highaffinity interactions with ErbB1 receptors and A/B-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and Á OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications.

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Section: Discussionsupporting

confidence: 90%

Targeting Cancer Cells by Novel Engineered Modular Transporters

et al. 2006

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“…Recombinant Proteins-The recombinant T domain containing mutation C201S (native diphtheria toxin numbering) and referred to as "wild type" (WT) has been described previously (15,16). His to Phe mutations were introduced by sitedirected mutagenesis in plasmid pA/T C201S and checked by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…His to Phe mutations were introduced by sitedirected mutagenesis in plasmid pA/T C201S and checked by DNA sequencing. Protein expression was performed as described previously (15,16) except that yields were increased by growing overnight precultures at 25°C. The T domains were purified from the soluble cytoplasmic fraction as described (9,15).…”

Section: Methodsmentioning

confidence: 99%

Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain

Perier

Chassaing

Raffestin

et al. 2007

Journal of Biological Chemistry

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The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His 223 -His 257 and His 251 are the most sensitive triggers for the formation of the molten globule state in solution, whereas His 322 -His 323 and His 251 are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

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“…Recombinant Proteins-The recombinant T domain has been described previously (39,40). Cys-201 (native diphtheria toxin numbering) has been mutated to Ser.…”

Section: Methodsmentioning

confidence: 99%

Membrane Protein Insertion Regulated by Bringing Electrostatic and Hydrophobic Interactions into Play

Chenal

Savarin

Nizard

et al. 2002

Journal of Biological Chemistry

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153

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The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.Folding and insertion of membrane proteins (1, 2), binding of hormones to membrane receptors (3), action of antibiotic peptides (4, 5), protein translocation, and internalization of toxins (6) are examples of phenomena that require the interactions of polypeptide chains with membranes. Because of the anisotropic nature of membranes, the initial steps of the association and the final structure and localization of polypeptide chains within membranes depend on a combination of hydrophobic and electrostatic interactions (7). Hydrophobic interactions are dominant for the insertion of transmembrane polypeptides. Electrostatic interactions are important for the binding of antibiotic peptides (5,8), the association of proteins with the surface of the membrane (9 -11), and as determinants of the topology of integral membrane proteins after biosynthesis (12). In most cases, electrostatic interactions are the result of the attraction between anionic phospholipid head groups and basic amino acid side chains (13,14). However, there are examples where electrostatic repulsions are involved in the membrane association of peptides, particularly when their structure and localization within the membrane is regulated by the pH (15-19). The interplay of hydrophobicity and electrostatics and their distribution within the polypeptide sequence have only been studied in detail for small peptides (3-5, 7, 13-15, 17-20). In the case of proteins, the role of these effects on the association with and the insertion into membranes are still poorly understood (2,(21)(22)(23)(24).The study of the membrane insertion process of the translocation (T) 1 domain of diphtheria toxin (25) can provide precious insight into the interactions between proteins and membranes and the refolding mechanisms of membrane proteins. During intoxication of cells (25), the toxin reaches the early endosomes through the clathrin-coated pathway (26). Beca...

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Prolonged display or rapid internalization of the IgG-binding protein ZZ anchored to the surface of cells using the diphtheria toxin T domain

Cited by 22 publications

References 21 publications

Targeting Cancer Cells by Novel Engineered Modular Transporters

Targeting Cancer Cells by Novel Engineered Modular Transporters

Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain

Membrane Protein Insertion Regulated by Bringing Electrostatic and Hydrophobic Interactions into Play

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