Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide.

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.

Processing of Pseudomonas aeruginosa exotoxin A is dispensable for cell intoxication.

Exotoxin A is a major virulence factor of Pseudomonas aeruginosa. This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of the toxin proteins with different unfolding abilities so that they inhibited or did not inhibit endosomal translocation of the chimera. We then assayed the amount of toxin fragments delivered to the cytosol during cell intoxication. Finally we used furin inhibitors and examined the fate and intracellular localization of the toxin and its receptor. The results showed that exotoxin cytotoxicity results largely from endosomal translocation of the entire toxin. We found that the C-terminal fragment was unstable in the cytosol.

Acid-triggered membrane insertion of Pseudomonas exotoxin A involves an original mechanism based on pH-regulated tryptophan exposure.

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Gelsolin binds to polyphosphoinositide-free lipid vesicles and simultaneously to actin microfilaments.

Gelsolin is a calcium-, pH- and lipid-dependent actin filament severing/capping protein whose main function is to regulate the assembly state of the actin cytoskeleton. Gelsolin is associated with membranes in cells, and it is generally assumed that this interaction is mediated by PPIs (polyphosphoinositides), since an interaction with these lipids has been characterized in vitro. We demonstrate that non-PPI lipids also bind gelsolin, especially at low pH. The data suggest further that gelsolin becomes partially buried in the lipid bilayer under mildly acidic conditions, in a manner that is not dependent of the presence of PPIs. Our data also suggest that lipid binding involves a number of sites that are spread throughout the gelsolin molecule. Linker regions between gelsolin domains have been implicated by other work, notably the linker between G1 and G2 (gelsolin domains 1 and 2 respectively), and we postulate that the linker region between the N-terminal and C-terminal halves of gelsolin (between G3 and G4) is also involved in the interaction with lipids. This region is compatible with other studies in which additional binding sites have been located within G4-6. The lipid-gelsolin interactions reported in the present paper are not calcium-dependent, and are likely to involve significant conformational changes to the gelsolin molecule, as the chymotryptic digest pattern is altered by the presence of lipids under our conditions. We also report that vesicle-bound gelsolin is capable of binding to actin filaments, presumably through barbed end capping. Gelsolin bound to vesicles can nucleate actin assembly, but is less active in severing microfilaments.