title: Evidence of protein transduction but not intercellular transport by proteins fused to HIV tat in retinal cell culture and in vivo.
abstract: The human immunodeficiency virus type-1 Tat protein is known to exit virally infected cells and enter the nucleus of adjacent uninfected cells. This property has been mapped to an 11-amino-acid protein transduction domain (PTD). When the PTD of Tat is fused to heterologous proteins and added exogenously to cells, the fusion peptide is able to demonstrate protein transduction across plasma membranes. Recent reports indicate that endogenously expressed Tat fusion peptides can demonstrate intercellular transport and improve biodistribution of therapeutic protein in the context of adenovirus vectors. Intercellular transport and protein transduction have not been observed in some studies and in the former have been attributed to an artifact of fixation. We have attempted to resolve these studies using an approach that unambiguously distinguishes cells that express Tat fusion protein from those that receive it from their environment. We find no evidence of intercellular transport in the context of an adenovirus vector in cell culture or in vivo. Instead, we find that Tat fusion peptides are down regulated in terms of expression not only in the context of adenovirus vectors, but also when expressed from transfected plasmid DNA. However, when Tat fusion peptides are released from cells by degradation of the plasma membrane, the fusion peptides demonstrate protein transduction without the need for cell fixation, indicating a unidirectional transport of Tat fusion proteins across the plasma membrane. Our data are consistent with previously reported studies and help to explain the apparently different results obtained from several different laboratories.
authors.affiliation: Department of Ophthalmology and Visual Sciences and Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112-5330, USA.
authors.forename: Siobhan M
authors.initials: SM
authors.lastname: Cashman
authors.forename: David J
authors.initials: DJ
authors.lastname: Morris
authors.forename: Rajendra
authors.initials: R
authors.lastname: Kumar-Singh
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002130:Calcium Phosphates / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D015536:Down-Regulation; D005434:Flow Cytometry; D015696:Gene Products, tat / Q000235:genetics*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012160:Retina / Q000166:cytology*; D014162:Transfection
chemical_list: D002130:Calcium Phosphates; D015696:Gene Products, tat; D008164:Luminescent Proteins; D010455:Peptides; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous