Large glycosylating toxins are major virulence factors of various species of pathogenic Clostridia. Prototypes are Clostridium difficile toxins A and B, which cause antibiotics-associated diarrhea and pseudomembranous colitis. The current model of the toxins' action suggests that receptor binding is mediated by a C-terminal domain of combined repetitive oligopeptides (CROP). This model is challenged by the glycosylating Clostridium perfringens large cytotoxin (TpeL toxin) that is devoid of the CROP domain but still intoxicates cells. Using a haploid genetic screen, we identified LDL receptor-related protein 1 (LRP1) as a host cell receptor for the TpeL toxin. LRP1-deficient cells are not able to take up TpeL and are not intoxicated. Expression of cluster IV of LRP1 is sufficient to rescue toxin uptake in these cells. By plasmon resonance spectroscopy, a K D value of 23 nM was determined for binding of TpeL to LRP1 cluster IV. The C terminus of TpeL (residues 1335-1779) represents the receptor-binding domain (RBD) of the toxin. RBD-like regions are conserved in all other clostridial glycosylating toxins preceding their CROP domain. CROP-deficient C. difficile toxin B is toxic to cells, depending on the RBD-like region (residues 1349-1811) but does not interact with LRP1. Our data indicate the presence of a second, CROP-independent receptor-binding domain in clostridial glycosylating toxins and suggest a two-receptor model for the cellular uptake of clostridial glycosylating toxins.toxin receptor | receptor-mediated endocytosis

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Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf AVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf AVKA allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf AVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf AVKA . Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.

Background: Clostridium difficile binary toxin CDT enters host cells via its receptor LSR. Results: Regions responsible for the CDT-LSR interaction were identified. Conclusion: An immunoglobulin-like domain of LSR and amino acids 757-866 of the binding component of CDT determine the toxin-receptor interaction. Significance: Knowledge of the toxin-receptor interaction is key to the development of anti-toxin strategies.

Metallic Zn alloys have recently gained interest as potential candidates for developing platforms of bioresorbable vascular stents (BVS). Previous studies revealed that Mg alloys used for BVS can degrade too early, whereas PLLA materials may fail to provide effective scaffolding properties. Here we report on results of a new bioresorbable, metallic stent made from a Zn-Ag alloy studied in a porcine animal model of thrombosis and restenosis. While the tensile strength (MPa) of Zn-3Ag was higher than that of PLLA and resembled Mg’s (WE43), fracture elongation (%) of Zn-3Ag was much greater (18-fold) than the PLLA’s or Mg alloy’s (WE43). Zn-3Ag exposed to HAoSMC culture medium for 30 days revealed degradation elements consisting of Zn, O, N, C, P, and Na at a 6 nm surface depth. Platelet adhesion rates and blood biocompatibility did not differ between Zn-3Ag, PLLA, Mg (WE43), and non-resorbable Nitinol (NiTi) stent materials. Balloon-expandable Zn-3Ag alloy BVS implanted into iliofemoral arteries of 15 juvenile domestic pigs were easily visible fluoroscopically at implantation, and their bioresorption was readily detectable via X-ray over time. Histologically, arteries with Zn-3Ag BVS were completely endothelialized, covered with neointima, and were patent at 1, 3, and 6 months follow-up with no signs of stent thrombosis. Zn-3Ag alloy appears to be a promising material platform for the fabrication of a new generation of bioresorbable vascular stents.