Apostolos Koutsokeras1, Panagiotis S Kabouridis. 1. William Harvey Research Institute, Queen Mary's School of Medicine and Dentistry, University of London, Charterhouse Square, London EC1M 6BQ, UK.
Abstract
BACKGROUND: Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes. METHODS: Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy. RESULTS: Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi. CONCLUSIONS: Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved. GENERAL SIGNIFICANCE: These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.
BACKGROUND: Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes. METHODS: Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy. RESULTS: Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi. CONCLUSIONS: Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved. GENERAL SIGNIFICANCE: These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.
Authors: Jean Philippe Richard; Kamran Melikov; Eric Vives; Corinne Ramos; Birgit Verbeure; Mike J Gait; Leonid V Chernomordik; Bernard Lebleu Journal: J Biol Chem Date: 2002-10-30 Impact factor: 5.157