IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation

Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation

The intracellular pharmacokinetics of terminally capped peptides.

Item does not contain fulltextWith significant progress in delivery technologies, peptides and peptidomimetics are receiving increasing attention as potential therapeutics also for intracellular applications. However, analyses of the intracellular behavior of peptides are a challenge; therefore, knowledge on the intracellular pharmacokinetics of peptides is limited. So far, most research has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Here, we studied the structure-activity relationship of peptides with respect to intracellular residence time and proteolytic breakdown. The peptides comprised a collection of interaction motifs of SH2 and SH3 domains with different charge but that were of similar size and carried an N-terminal fluorescein moiety. First, we show that electroporation is a highly powerful technique to introduce peptides with different charge and hydrophobicity in uniform yields. Remarkably, the peptides differed strongly in retention of intracellular fluorescence with half-lives ranging from only 1 to more than 10 h. Residence times were greatly increased for retro-inverso peptides, demonstrating that rapid loss of fluorescence is a function of peptide degradation rather than the physicochemical characteristics of the peptide. Differences in proteolytic sensitivity were further confirmed using fluorescence correlation spectroscopy as a separation-free analytical technique to follow degradation in crude cell lysates and also in intact cells. The results provide a straightforward analytical access to a better understanding of the principles of peptide stability inside cells and will therefore greatly assist the development of bioactive peptides

Black Tea Theaflavins Inhibit Formation of Toxic Amyloid-β and α-Synuclein Fibrils

Causal therapeutic approaches for amyloid diseases such as Alzheimer's and Parkinson's disease targeting toxic amyloid oligomers or fibrils are still emerging. Polyphenols from green tea, especially (-)-epigallocatechin gallate (EGCG), have recently been found to redirect amyloid formation pathways leading to the assembly of small, non-toxic aggregate structures. Here, we show that theaflavins (TF1, TF2a, TF2b, TF3), the main polyphenolic components found in fermented black tea, are potent inhibitors of amyloid-beta (A{beta}) and alpha-synuclein ({alpha}S) fibrillogenesis. Their mechanism of inhibiting amyloid formation was compared to that of two established inhibitors of amyloid formation, EGCG and congo red. All three compounds reduce the fluorescence of the amyloid indicator dye thioflavin T. Mapping the binding regions of TF3 and revealed that all three bind to two regions of the peptide, aa 12-23 and aa 24-36, albeit with different specificities. However, their mechanisms of amayloid inhibition differ. Like EGCG but unlike congo red, theaflavins stimulate the assembly of A{beta} and {alpha}S into non-toxic, spherical aggregates that are incompetent to seed amyloid formation and remodel A{beta} fibrils into non-toxic, spherical aggregates. These findings suggest that theaflavins might be useful to remove toxic amyloid deposits in Alzheimer's and Parkinson's disease brains