WIRE Collection: null

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

Mon, 01 Jan 2007 00:00:00 GMT

Title: The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells Authors: Holton, Marylouisa; Yang, Di; Wang, Weiguang; Mohamed, Tamer M. A.; Neyses, Ludwig; Armesilla, Angel Luis Abstract: 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.

Mon, 01 Jan 2007 00:00:00 GMT

Title: Mitoparans: mitochondriotoxic cell penetrating peptides and novel inducers of apoptosis. Authors: Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. Abstract: 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. Description: Acknowledgments The authors would like to thank Keith Holding at the University of Wolverhampton for his outstanding technical support. This work was supported in part by Samantha Dickson Brain Tumour Trust.

Identification and biological applications of rhegnylogically-organized cell penetrating peptides.

Mon, 01 Jan 2007 00:00:00 GMT

Title: Identification and biological applications of rhegnylogically-organized cell penetrating peptides. Authors: Howl, John D.; Jones, Sarah Abstract: 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. Description: Acknowledgments: We are extremely grateful to Ülo Langel (Stockholm) who provided access to the CPP prediction algorithm. This work also benefited from collaborations with Shant Kumar (Manchester) and Matjaž Zorko (Ljubljana). These investigations were financially supported in part by the Samantha Dickson Brain Tumour Trust.

Chimeric peptides as tumour-selective delivery systems.

Sat, 01 Jan 2005 00:00:00 GMT

Title: Chimeric peptides as tumour-selective delivery systems. Authors: Jones, Sarah; Howl, John D. Abstract: 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. Description: Abstracts from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Duke University Press.