• Intracellular delivery of bioactive peptides to RBL-2H3 cells induces beta-hexosaminidase secretion and phospholipase D activation.

      Howl, John D.; Jones, Sarah; Farquhar, Michelle (Wiley InterScience, 2003)
      This investigation compared the secretory efficacies of a series of peptides delivered to the cytoplasm of RBL-2H3 mast cells. Mimetic peptides, designed to target intracellular proteins that regulate cell signalling and membrane fusion, were synthesised as transportan 10 (TP10) chimeras for efficient plasma membrane translocation. Exocytosis of beta-hexosaminidase, a secretory lysosomal marker, indicated that peptides presenting sequences derived from protein kinase C (PKC; C1 H-CRRLSVEIWDWDL-NH(2)) and the CB(1) cannabinoid receptor (C3 H-RSKDLRHAFRSMFPSCE-NH(2)) induced beta-hexosaminidase secretion. Other peptide cargoes, including a Rab3A-derived sequence and a homologue of C3, were inactive in similar assays. Translocated C1 also activated phospholipase D (PLD), an enzyme intimately involved in the regulated secretory response of RBL-2H3 cells, but C1-induced secretion was not dependent upon phosphatidate synthesis. Neither down-regulation of Ca(2+)-sensitive isoforms of PKC nor the application of a selective PKC inhibitor attenuated the secretory efficacy of C1. These observations indicate that the molecular target of C1 is a protein involved in the regulated secretory pathway that is upstream of PLD but is not a PKC isoform. This study also confirmed that TP10 is a relatively inert cell-penetrating vector and is, therefore, widely suitable for studies in cells that are sensitive to peptidyl secretagogues.
    • Protein Mimicry and the Design of Bioactive Cell-Penetrating Peptides

      Howl, John; Jones, Sarah (2015)
      The multi-domain architecture of many human proteins provides a structural basis for the physical maintenance of interactomes, or networks of protein-protein interactions (PPIs), that are so obviously crucial to cellular functions. Moreover, the structural and electrostatic complementarity provided by PPI interfaces, predominantly located on protein surfaces, is a fundamental component of signal transduction events that are known to be compromised in human diseases including many cancers.The pharmacokinetic advantages provided by cell-penetrating peptides (CPPs) are entirely compatible with the development of intrinsically permeable agents capable of modulating intracellular PPIs. Thus, the term bioportide is a useful descriptor of numerous bioactive CPPs that are distinct from the more usual inert CPP vectors. Herein we illustrate a generic strategy, predominantly centered upon the identification of cationic peptides derived from helical protein domains, which offers a reliable platform to identify bioportides capable of modulating intracellular signal transduction events. In addition, we describe robust methodologies to determine the precise intracellular distribution of fluorescent bioportides and present assays routinely employed to screen for the detrimental pharmacodynamic properties often exhibited by both CPPs and bioportides; namely adverse cytotoxicity and the receptor-independent stimulation of mast cell secretion.