Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin ␤ is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin ␤ and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin ␤. Processing of APP by meprin ␤ was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin ␤ ؊/؊ mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin ␤ is a physiologically relevant enzyme in APP processing.

Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via β1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Yersinia pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Yersinia pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to nonfunctionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments. *Graphical AbstractBacterial invas on into a human cellvia outer ..•

No abstract

No abstract

Intracellular infections caused by invasive pathogens continue to prove difficult to combat, due in part to the commonly poor membrane permeability of anti-infective drugs. The aim of this study was to improve the intracellular delivery of one such poorly permeable (but broad-spectrum) anti-infective, gentamicin. Gentamicin was encapsulated into liposomal nanocarriers which were then surface functionalized with InvA497, a bacteria-derived invasion protein. Treatment of HEp-2 cells infected with the enteroinvasive bacteria Yersinia pseudotuberculosis or Salmonella enterica with gentamicincontaining, InvA497-functionalized liposomes resulted in a significantly greater reduction in infection load than treatment with non-functionalized liposomes, indicating that such a bacteriomimetic nanocarrier was not only able to promote successful cellular uptake of gentamicin but was also able to mediate anti-infective drug delivery to both cell cytoplasm and intracellular compartments. The developed InvA497-functionalized liposomal nanocarrier therefore holds great promise as a strategy for improving the therapy of intracellular infections.