• The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells

      Holton, Marylouisa; Yang, Di; Wang, Weiguang; Mohamed, Tamer M. A.; Neyses, Ludwig; Armesilla, Angel Luis (Elsevier, 2007)
      Plasma membrane calcium/calmodulin-dependent ATPases (PMCAs) are high affinity calcium pumps that extrude calcium from the cell. Emerging evidence suggests a novel role for PMCAs as regulators of calcium/calmodulin-dependent signal transduction pathways via interaction with specific partner proteins. In this work, we demonstrate that endogenous human PMCA2 and -4 both interact with the signal transduction phosphatase, calcineurin, whereas, no interaction was detected with PMCA1. The strongest interaction was observed between PMCA2 and calcineurin. The domain of PMCA2 involved in the interaction is equivalent to that reported for PMCA4b. PMCA2-calcineurin interaction results in inhibition of the calcineurin/nuclear factor of activated T-cells signalling pathway.
    • Mitoparans: mitochondriotoxic cell penetrating peptides and novel inducers of apoptosis.

      Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. (Australian Peptide Association, 2007)
      Introduction: The amphipathic helical peptide mastoparan (MP; H-INLKALAALAKKIL-NH2) inserts into biological membranes to modulate the activity of heterotrimeric G proteins and other targets. Moreover, whilst cell free models of apoptosis demonstrate MP to facilitate mitochondrial permeability transition and release of apoptogenic cytochrome c, MP-induced death of intact cells has been attributed to its non-specific membrane destabilising properties (necrotic mechanisms). However, MP and related peptides are known to activate other signalling systems, including p42/p44 MAP kinases and could therefore, also modulate cell fate and specific apoptotic events. The ability of MP to facilitate mitochondrial permeability in cell free systems has lead to proposals that MP could be of utility in tumour therapeutics provided that it conferred features of cellular penetration and mitochondrial localization. We have recently reported that our highly potent amphipathic MP analogue mitoparan (mitP; [Lys5,8Aib10]MP; Aib = -aminoisobutyric acid) specifically promotes apoptosis of human cancer cells, as was confirmed by in situ TUNEL staining and activation of caspase-3. Moreover, we have also demonstrated that mitP penetrates plasma membranes and redistributes to co-localize with mitochondria. Complementary studies, using isolated mitochondria, further demonstrated that mitP, through co-operation with a protein of the permeability transition pore complex voltage-dependent anion channel (VDAC), induced swelling and permeabilization of mitochondria, leading to the release of the apoptogenic factor cytochrome c. An expanding field of peptide and cell penetrating peptide (CPP) research has focussed on the selective targeting of tumours by engineering constructs that incorporate cell-specific or tissue–specific address motifs. Peptidyl address motifs could enhance the selectivity of drug delivery whilst the improved cellular uptake offered by CPP enhances bioavailability. Thus and as a potential therapeutic strategy, we extended our findings to design target-specific mitP analogues. The integrin-specific address motif RGD and a Fas ligand mimetic WEWT were incorporated by N-terminal acylation of mitP to produce novel tandem-linked chimeric peptides.
    • Identification and biological applications of rhegnylogically-organized cell penetrating peptides.

      Howl, John D.; Jones, Sarah (Australian Peptide Association, 2007)
      Introduction: Many different cell penetrating peptides (CPPs) have been utilized as vectors to affect the highly efficient intracellular delivery of bioactive moieties. A majority of such studies employ sychnologically-organized tandem combinations of a cargo (message) and a CPP (address). To date, bioactive cargoes have included peptides, proteins and a range of oligonucleotides attached either by direct chemical conjugation or as a component of a larger macromolecular complex. Moreover, a majority of CPPs, including the commonly used sequences Tat and penetratin, are designed to be both biologically and toxicologically inert. More recently, a QSAR-based algorithm has been developed to predict cryptic polycationic CPP motifs within the primary sequences of proteins. As described here, this novel technology has enabled the study of rhegnylogic CPPs in which multiple pharmacophores for cellular penetration and desirable biological activities are discontinuously organized within the primary sequence of single peptide. This organization differs from the more commonly utilized sychnologic strategy which joins functionally discrete and continous address and messages together in a tandem construct.
    • Chimeric peptides as tumour-selective delivery systems.

      Jones, Sarah; Howl, John D. (Society for Neuro-oncology and Duke University Press, 2005)
      The cell-type-specific targeting of cytotoxic agents and other functional moieties can be achieved by using peptidyl address motifs that selectively bind protein targets expressed at high density at the cell membrane. Indeed, numerous studies have confirmed the utility of ligands for G protein–coupled receptors as components of heterofunctional peptide chimeras that are selective biological probes. Our current efforts are directed toward the further development of chimeric peptidyl constructs that employ sequences derived from GPCR ligands or cell penetrant motifs to affect the selective delivery of cytotoxins and signal transduction modulators to tumor cells. We have designed and synthesized a range of hybrid constructs consisting of cytotoxins (peptide and non-peptide) covalently linked to an address peptide derived from the C-terminal of gastrin (G7; H-AYGWMDF-NH2). The G7 homing motif targets a novel binding site expressed by U373MG astrocytic tumor cells that is distinct from classical CCK1/CCK2 receptors. Moreover, biological responses following activation of this novel membrane-bound protein may offer additional therapeutic advantages. For example, G7 receptor activation is reported to inhibit the motility of malignant astrocytoma in vivo while avoiding the growth-promoting effects of gastrin (Pannequin et al., J. Pharmacol. Exp. Ther. 302, 274, 2002). We evaluated the cytotoxicity of our chimeric peptides by comparing changes in cellular viability using MTT conversion assays. Our data indicate that chimeric peptides dose-dependently and rapidly (<8 h) reduced the viability of U373MG cells. Moreover, as a chimeric amino-terminal extension, the G7 address motif enhanced the cytotoxicity of both mastoparan (H-INLKALAALAKKIL-NH2) and D(KLAKLAK)2 peptides reported to stimulate necrosis and/or apoptosis of eukarytoic cells. In conclusion, hybrid G7 chimeras enhance the efficacy of cytotoxic agents and may be valuable probes to investigate and manipulate additional aspects of astrocytoma cell biology. This work was supported by The Wellcome Trust.
    • Applications of cell-penetrating peptides as signal transduction modulators.

      Jones, Sarah; Howl, John D. (Washington, D.C.: CRC Press, 2006)
      THIS BOOK: Since the first Handbook of Cell-Penetrating Peptides was prepared in 2001, the wealth of new information on the use of these peptides as transport systems has in fact served to confound the field. The constant internal change in the field of cell-penetrating peptides (CPPs) is due to recent research uncovering apparent ambiguities in cellular uptake. There is still neither a common terminology nor a uniform explanation for the penetrative mechanism of cell-penetrating peptides. In this second edition of the Handbook of Cell-Penetrating Peptides, the authors summarize the current state of the field including recent reevaluations of earlier studies of CPP mechanisms. Beginning with an overview of the classes of peptides and their individual uptake mechanisms, from the earlier lipid models to the more recent endocytotic pathways, the book demonstrates the diversity and the opportunity for these biologically active proteins to serve as future drug leads. The text then covers the use of CPPs in gene modulation, addressing the application of antisense and decoy oligonucleotides, as well as the new avenue of research targeting specific tumors and other tissues-questions that had barely been asked when the first edition was published. (CRC Press)
    • A rhegnylogic strategy for the synthesis of signal transduction modulatory, cell penetrating peptides

      Jones, Sarah; Ostlund, Pernilla; Langel, Ulo; Zorko, Matjaz; Nicholl, Iain D.; Howl, John D. (Wiley InterScience, 2006)
      INTRODUCTION: Many cell-penetrating peptides (CPP) have been utilised as biologically inert vectors. A majority of these studies employ sychnologically organised constructs in which a bioactive cargo (message) is chemically conjugated to the CPP (address). Previously, we have adopted a sychnologic strategy to modulate intracellular signal transduction. Using chimeric constructs composed of the CPP transportan 10, conjugated to partial sequences that correspond to functional domains of signal transduction proteins, we have selectively modulated a variety of cellular activities including secretion and activation of p42/p44 mitogen-activated protein kinases [1, 2]. However, a QSAR-based algorithm can now be used to predict CPP that reside within the primary sequences of proteins [3]. We have adapted this strategy to identify CPP within signal transducing proteins including functional domains that govern protein-protein interactions. Data presented herein indicate that it is now feasible to identify rhegnylogic sequences, containing vectoral-independent discontinuously organised pharmacophores, that are cell penetrant modulators of signal transduction pathways.
    • Mitoparan and target-selective chimeric analogues: membrane translocation and intracellular redistribution induces mitochondrial apoptosis.

      Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. (Amsterdam: Elsevier, 2008)
      Mastoparan, and structurally-related amphipathic peptides, may induce cell death by augmentation of necrotic and/or apoptotic pathways. To more precisely delineate cytotoxic mechanisms, we determined that [Lys(5,8)Aib(10)]mastoparan (mitoparan) specifically induces apoptosis of U373MG and ECV304 cells, as demonstrated by endonuclease and caspase-3 activation and phosphatidylserine translocation. Live cell imaging confirmed that, following translocation of the plasma membrane, mitoparan specifically co-localizes with mitochondria. Complementary studies indicated that mitoparan induces swelling and permeabilization of isolated mitochondria, through cooperation with a protein of the permeability transition pore complex VDAC, leading to the release of the apoptogenic factor, cytochrome c. N-terminal acylation of mitoparan facilitated the synthesis of chimeric peptides that incorporated target-specific address motifs including an integrin-specific RGD sequence and a Fas ligand mimetic. Significantly, these sychnologically-organised peptides demonstrated further enhanced cytotoxic potencies. We conclude that the cell penetrant, mitochondriotoxic and apoptogenic properties of mitoparan, and its chimeric analogues, offer new insights to the study and therapeutic induction of apoptosis.
    • Biofilms, dormancy and resistance

      Smith, Anthony W.; Brown, Michael R. W. (Cambridge University Press, 2003)
      All cellular life-forms can exist in replicating and non-replicating states. Organisms replicate only when the conditions are beneficial, and when not replicating they concentrate on survival of these environmental stresses. Many bacteria, harmful to humans, survive the period of infection in a low growth state. This book addresses the basic science of microbial dormancy and low growth states, putting this in the context of human medicine. Such fundamental topics as bacterial growth and non-growth, culturability and viability are covered, as well as survival of the host’s immune response, and inter-bacterial signalling. Following this introduction, more medically-focused topics are discussed, namely antibiotic resistance arising during stationary phase, biofilms, the bacteria which cause gastric ulcers and tuberculosis as the classic persistent bacterial infection. This book will be of interest to graduate students and researchers in medical microbiology, immunology and infectious disease medicine who are interested in bacterial dormancy in relation to disease. (Cambridge University Press)
    • Antimicrobial agents and biofilms

      Brown, Michael R. W.; Smith, Anthony W. (Cambridge University Press, 2003)
      Interest in biofilms has increased dramatically in recent years. New microscopic and molecular techniques have revolutionized our understanding of biofilm structure, composition, organization, and activities. This book brings advances in the prevention and treatment of biofilm-related diseases to the attention of clinicians and medical researchers. Human tissues often support complex microbial communities growing as biofilms that can cause infections. As microbes in biofilms exhibit increased tolerance toward anti-microbial agents and decreased susceptibility to host defense systems, biofilm-associated diseases have become increasingly difficult to treat. (Cambridge University Press)
    • Biofilms and protozoa: a ubiquitous health hazard

      Smith, Anthony W.; Brown, Michael R. W. (IWA Publishing, 2003)
      This timely book will introduce its readers to the structure and function of biofilms at a fundamental level as determined during the past decade of research, including: Extracellular polymers as the biofilm matrix; Biofilm phenotype (differential gene expression, interspecies signalling); Biofilm ecology; Biofilm monitoring; Resistance of biofilms to antimicrobial agents and Biofilm abatement. (IWA Publishing)
    • E. coli O157 persistence in the environment.

      Brown, Michael R. W.; Smith, Anthony W.; Barker, John; Humphrey, Thomas J.; Dixon, Bernard (Society for General Microbiology, 2002)
    • Resistance of medical biofilms

      Brown, Michael R. W.; Smith, Anthony W. (IWA Publishing, 2003)
      This timely book will introduce its readers to the structure and function of biofilms at a fundamental level as determined during the past decade of research, including: Extracellular polymers as the biofilm matrix; Biofilm phenotype (differential gene expression, interspecies signalling); Biofilm ecology; Biofilm monitoring; Resistance of biofilms to antimicrobial agents and Biofilm abatement. (IWA Publishing)
    • Cell penetrating peptides as signal transduction modulators

      Jones, Sarah; Howl, John D. (CRC Press (Taylor & Francis), 2007)
      THIS BOOK: Since the first Handbook of Cell-Penetrating Peptides was prepared in 2001, the wealth of new information on the use of these peptides as transport systems has in fact served to confound the field. The constant internal change in the field of cell-penetrating peptides (CPPs) is due to recent research uncovering apparent ambiguities in cellular uptake. There is still neither a common terminology nor a uniform explanation for the penetrative mechanism of cell-penetrating peptides. In this second edition of the Handbook of Cell-Penetrating Peptides, the authors summarize the current state of the field including recent reevaluations of earlier studies of CPP mechanisms. Beginning with an overview of the classes of peptides and their individual uptake mechanisms, from the earlier lipid models to the more recent endocytotic pathways, the book demonstrates the diversity and the opportunity for these biologically active proteins to serve as future drug leads. The text then covers the use of CPPs in gene modulation, addressing the application of antisense and decoy oligonucleotides, as well as the new avenue of research targeting specific tumors and other tissues-questions that had barely been asked when the first edition was published. By summarizing the diffuse information regarding CPPs, including the ambiguities and variety of mechanisms, the Handbook of Cell-Penetrating Peptides provides the most solid foundation available from which to expand the potential of this rapidly growing field of medicine. (CRC Press)
    • Peptidyl-based delivery systems as a strategy for the therapeutic intervention of human astrocytoma and medulloblastoma

      Jones, Sarah; Howl, John D. (Springer Verlag, 2006)
      THIS BOOK: Understanding Biology Using Peptides: Proceedings of the 19th American Peptide Symposium highlights many of the recent developments in peptide science, with a particular emphasis on how these advances are being applied to basic problems in biology and medicine. Specific topics covered include novel synthetic strategies, peptides in biological signaling, post-translational modifications of peptides and proteins, peptide quaternary structure in material science and disease, and peptides as tools in drug discovery. (Springer Verlag)
    • Design, synthesis and applications of cell-penetrant peptides as signal transduction modulators

      Howl, John D.; Farquhar, Michelle; Jones, Sarah (Springer Verlag, 2006)
      THIS BOOK: Understanding Biology Using Peptides: Proceedings of the 19th American Peptide Symposium highlights many of the recent developments in peptide science, with a particular emphasis on how these advances are being applied to basic problems in biology and medicine. Specific topics covered include novel synthetic strategies, peptides in biological signaling, post-translational modifications of peptides and proteins, peptide quaternary structure in material science and disease, and peptides as tools in drug discovery. (Springer Verlag)
    • Chimerism: a strategy to expand the utility and applications of peptides

      Howl, John D. (Clifton, N.J.: Humana Press, 2005)
      The modular nature of peptides can be exploited in the synthesis of chimeric sequences that combine diverse motifs in a single molecule. A theoretical consideration of the classification of peptides further expounds the multigeneric nature of peptide chimeras. Strategies for chimeric peptide syntheses include the chemical cross-linking of monomers and tandem combination by conventional SPPS. Additional details of chimeric peptide synthesis are also provided elsewhere in this volume. This chapter also explores some of the more common applications of chimeric peptides with particular emphasis on the molecular pharmacology of sequences that include address motifs for G protein-coupled receptors. Specific details of the biological properties of chimeras containing mastoparan, an amphiphilic tetradecapeptide component of wasp venom, further illustrate the novel and often unpredictable biological actions of chimeric constructs. These and numerous additional studies confirm that chimerism is an established strategy for the synthesis of molecular probes and bioactive agents.
    • 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.