<b><i>Pseudomonas</i> exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine‐rich loop of diphtheria toxin</b>

Chiron, Marielle; Ogata, Masato; FitzGerald, David J.

doi:10.1046/j.1365-2958.1996.d01-1721.x

Cited by 18 publications

(28 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several studies showed that there is no clear correlation between processing and toxicity. Indeed, mutants more efficiently processed by furin intracellularly were found to be consistently less toxic than PE (33,36). Our mutants also showed a lack of correlation between processing and toxicity because, within cells, they were cleaved by furin with the same efficiency as native PE (data not shown) although their toxicity varied over A, mutant toxicity is expressed as the IC 50 that is the toxin concentration resulting in 50% protein synthesis inhibition.…”

Section: Discussionmentioning

confidence: 79%

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Méré

Morlon-Guyot

Bonhoure

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 ␣-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. Exotoxin A (PE)1 is one of the major virulence factors secreted by Pseudomonas aeruginosa. This toxin is able to kill a large range of mammalian cell lines by inhibiting their protein synthesis (1), and it is one of the favorite toxins to prepare immunotoxins that are promising agents for the treatment of cancers (2). PE is a single chain 66-kDa protein organized in three structural (3) and functional (4) domains successively involved in the intoxication process. First, domain I binds to the ␣ 2 -macroglobulin/low density lipoprotein receptor-related protein (5), enabling internalization via receptor-mediated endocytosis. Domain II will then mediate translocation into the cytosol of the entire toxin (6) or of a carboxyl-terminal fragment generated by furin proteolysis and encompassing domain III and most of domain II (1). Finally, domain III will catalyze the

show abstract

Section: Discussionmentioning

confidence: 79%

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Méré

Morlon-Guyot

Bonhoure

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…However, all these substrates have the sequence Arg-X-Lys/Arg-Arg or Lys-X-X-Arg2 at their cleavage site. In contrast, LoVo cells act very inefficiently on Pseudomonas exotoxin and Shiga toxin that have the cleavage sequence Arg-X-X-Arg2, which is present in UCE (28,29). Despite this, we cannot exclude the possibility that one of the proconvertases expressed in the LoVo cells is activating a small proportion of UCE.…”

Section: Discussionmentioning

confidence: 92%

Human Mannose 6-Phosphate-uncovering Enzyme Is Synthesized as a Proenzyme That Is Activated by the Endoprotease Furin

Do¹,

Lee

Ghosh

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

N-Acetylglucosamine-1-phosphodiester ␣-N-acetylglucosaminidase, also known as "uncovering" enzyme (UCE), is localized in the trans-Golgi network, where it removes a covering N-acetylglucosamine from the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Here we show that UCE is synthesized as an inactive proenzyme that is activated by the endoprotease furin, which cleaves an RARLPR2D sequence to release a 24-amino acid propiece. As furin is localized in the trans-Golgi network, newly synthesized UCE is inactive until it reaches this terminal Golgi compartment. LoVo cells (derived from a human colon adenocarcinoma) lack furin activity and have extremely low UCE activity. Addition of furin to LoVo cell extracts restores UCE activity to normal levels, demonstrating that the UCE proenzyme is stable in this cell type. LoVo cells secrete acid hydrolases with phosphomannose diesters as a consequence of the deficient UCE activity. This demonstrates for the first time that UCE is the only enzyme in these cells capable of efficiently uncovering phosphomannose diesters. UCE also hydrolyzes UDPGlcNAc, a sugar donor for Golgi N-acetylglucosaminyltransferases. The fact that UCE is not activated until it reaches the trans-Golgi network may ensure that the pool of UDP-GlcNAc in the Golgi stack is not depleted, thereby maintaining proper oligosaccharide assembly.

show abstract

“…In the first construct, we added additional amino acid residues to the PEA275-280 sequence (T 273 RHRQPRGWE 282 , Fpe) to enhance the rate of furin-mediated cleavage (23,24). In the second construct, we used the furin site from diphtheria toxin (A 187 GNRVRRSVG 196 , Fdt), which has been reported to facilitate furin-mediated cleavage (27). In the third construct, we used the arginine-rich sequence (RRRRRRRRR, R 9 ), a furin cleavage sequence with multiple cleavage sites.…”

Section: Resultsmentioning

confidence: 99%

“…Furin-mediated cleavage of PEA and PEA-derived immunotoxins is both an obligatory and a rate-limiting step for their cytotoxic activity (25,26). In fact, only 5% to 10% of cell-associated PEA can be cleaved by furin within cells (27). In this study, we report the generation of a new class of conjugates, called novel immunoproapoptotic proteins, which contain furin-sensitive sequences.…”

Section: Introductionmentioning

confidence: 90%

Recombinant Immunoproapoptotic Proteins with Furin Site Can Translocate and Kill HER2-Positive Cancer Cells

Wang¹,

Zhao²,

Ren

et al. 2007

Cancer Research

View full text Add to dashboard Cite

We previously reported the selective killing of HER2-positive tumor cells by a class of immunoproapoptotic proteins containing single-chain antibody, translocation domain of Pseudomonas exotoxin A (domain II; PEA II), and constitutively active human apoptotic molecules. In this study, a novel class of antitumor immunoproapoptotic proteins was explored to mediate tumor-specific apoptosis both in vitro and in vivo. Three furin cleavage sequences, including a synthetic polyarginine tract, and two furin cleavable sequences from PEA and diphtheria toxin were respectively used to replace PEA II in the previously constructed immunoproapoptotic protein. When produced and secreted by the genetically modified Jurkat cells, the novel targeted proapoptotic proteins selectively bound to HER2, which is often overexpressed on tumor cell surface. Followed by receptor-mediated endocytosis and furin cleavage in the endosome, the recombinant proteins could translocate into the cytosol, leading to irreversible cell death. Moreover, delivery of these proteins by either i.m. plasmid injection or i.v. injection of plasmidexpressing Jurkat cells led to tumor regression and prolonged animal survival in a nude mouse xenograft tumor model, indicating in vivo antitumor activity of the recombinant proteins. We conclude that the new class of immunoproapoptotic proteins show comparable activity with PEA II-containing counterpart and provide an attractive therapeutic alternative as they contain much less exogenous fragments.

show abstract

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine‐rich loop of diphtheria toxin

Cited by 18 publications

References 16 publications

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Human Mannose 6-Phosphate-uncovering Enzyme Is Synthesized as a Proenzyme That Is Activated by the Endoprotease Furin

Recombinant Immunoproapoptotic Proteins with Furin Site Can Translocate and Kill HER2-Positive Cancer Cells

Contact Info

Product

Resources

About