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

2.50
Hdl Handle:
http://hdl.handle.net/2436/29936
Title:
Intracellular delivery of bioactive peptides to RBL-2H3 cells induces beta-hexosaminidase secretion and phospholipase D activation.
Authors:
Howl, John D.; Jones, Sarah; Farquhar, Michelle
Abstract:
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.
Citation:
ChemBioChem, 4(12): 1312-1316
Publisher:
Wiley InterScience
Journal:
ChemBioChem
Issue Date:
2003
URI:
http://hdl.handle.net/2436/29936
DOI:
10.1002/cbic.200300694
PubMed ID:
14661273
Additional Links:
http://www3.interscience.wiley.com/journal/106567337/abstract; http://direct.bl.uk/bld/PlaceOrder.do?UIN=142527065&ETOC=RN&from=searchengine
Type:
Article
Language:
en
ISSN:
1439-4227
Appears in Collections:
Molecular Pharmacology Research Group

Full metadata record

DC FieldValue Language
dc.contributor.authorHowl, John D.-
dc.contributor.authorJones, Sarah-
dc.contributor.authorFarquhar, Michelle-
dc.date.accessioned2008-06-12T08:32:38Z-
dc.date.available2008-06-12T08:32:38Z-
dc.date.issued2003-
dc.identifier.citationChemBioChem, 4(12): 1312-1316en
dc.identifier.issn1439-4227-
dc.identifier.pmid14661273-
dc.identifier.doi10.1002/cbic.200300694-
dc.identifier.urihttp://hdl.handle.net/2436/29936-
dc.description.abstractThis 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.en
dc.language.isoenen
dc.publisherWiley InterScienceen
dc.relation.urlhttp://www3.interscience.wiley.com/journal/106567337/abstracten
dc.relation.urlhttp://direct.bl.uk/bld/PlaceOrder.do?UIN=142527065&ETOC=RN&from=searchengineen
dc.subjectMolecular Biologyen
dc.subjectChimerismen
dc.subjectsecretionen
dc.subject.meshAmino Acid Sequenceen
dc.subject.meshAnimalsen
dc.subject.meshCell Line, Tumoren
dc.subject.meshDose-Response Relationship, Drugen
dc.subject.meshEnzyme Activationen
dc.subject.meshGlycerophospholipidsen
dc.subject.meshMast Cellsen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshPeptidesen
dc.subject.meshPhospholipase Den
dc.subject.meshProtein Kinase Cen
dc.subject.meshRatsen
dc.subject.meshReceptor, Cannabinoid, CB1en
dc.subject.meshRecombinant Fusion Proteinsen
dc.subject.meshbeta-N-Acetylhexosaminidasesen
dc.subject.meshrab3A GTP-Binding Proteinen
dc.titleIntracellular delivery of bioactive peptides to RBL-2H3 cells induces beta-hexosaminidase secretion and phospholipase D activation.en
dc.typeArticleen
dc.identifier.journalChemBioChemen
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