Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration

Nagahara, Hikaru; Vocero-Akbani, Adamina; Snyder, Eric L.; Ho, Alan L.; Latham, Dawn G.; Lissy, Natalie A.; Becker-Hapak, Michelle; Ezhevsky, Sergei A.; Dowdy, Steven F.

doi:10.1038/4042

Cited by 893 publications

(813 citation statements)

References 19 publications

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“…pGEM-H2a, was digested with NcoI and EcoRI to have the insert H2a which was then fused into the restriction sites NcoI and EcoRI of pTat-HA vector (a generous gift from Dr Steve F Dowdy at University of California, San Diego School of Medicine) which contains a 6 Â His tag. Tat-H2a was expressed in Escherichia coli as described previously (Nagahara et al, 1998;He et al, 2002). Bacterial culture (500 ml) was homogenized in 20 ml of lysis buffer (8 M urea, 200 mM NaCl, and 20 mM HEPES pH 8.0) followed by sonication for 2 min.…”

Section: Construction Of Tat-fusion Proteinmentioning

confidence: 99%

Homer Isoforms Differentially Regulate Cocaine-Induced Neuroplasticity

Szumlinski

Abernathy

Oleson

et al. 2005

Neuropsychopharmacol

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Homer proteins modulate neuroplasticity in excitatory synapses and are dynamically regulated by cocaine. Whereas acute cocaine elevates immediate-early gene (short) isoforms of Homer1 in the nucleus accumbens, withdrawal from repeated cocaine administration downregulates the expression of constitutive Homer1 isoforms. The present study determined whether or not this downregulation in constitutive Homer expression in the accumbens is necessary for enduring alterations in cocaine-induced changes in the brain and behavior. The long vs short Homer isoforms were overexpressed in the rat nucleus accumbens during drug abstinence, and the adaptations elicited by repeated cocaine on glutamate transmission and motor behavior were measured. It was found that both chronic and acute overexpression of constitutive, but not short, Homer isoforms abolished cocaine-induced sensitization of locomotor hyperactivity and prevented the development of glutamate abnormalities in the accumbens, including the reduction in basal extracellular glutamate content and the sensitized glutamate response to a subsequent cocaine challenge injection. Together, these data indicate that the enduring reduction of long Homer isoforms in the nucleus accumbens of cocaine-withdrawn rats is necessary for the expression of cocaine-induced neuroplasticity.

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Section: Construction Of Tat-fusion Proteinmentioning

confidence: 99%

Homer Isoforms Differentially Regulate Cocaine-Induced Neuroplasticity

Szumlinski

Abernathy

Oleson

et al. 2005

Neuropsychopharmacol

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“…14 It has been shown that proteins are directly and readily introduced into cells regardless of their molecular size when fused with the PTD (protein transduction domain) of HIV/Tat protein. 15 PTD-fused proteins can be delivered to several tissues, including the brain, when injected into mice systemically. 16 In addition, PTD-FNK, a fusion protein of the PTD and FNK, penetrates the dense matrix of cartilage to reach chondrocytes.…”

Section: Introductionmentioning

confidence: 99%

Zonal necrosis prevented by transduction of the artificial anti-death FNK protein

et al. 2005

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Protection of cells from necrosis would be important for many medical applications. Here, we show protein transduction domain (PTD)-FNK therapeutics based on protein transduction to prevent necrosis and acute hepatic injury with zonal death induced by carbon tetrachloride (CCl 4 ). PTD-FNK is a fusion protein comprising the HIV/Tat PTD and FNK, a gain-offunction mutant of anti-apoptotic Bcl-x L . PTD-FNK protected hepatoma HepG2 from necrotic death induced by CCl 4 , and additionally, increased the apoptotic population among cells treated with CCl 4 . A concomitant treatment with a pancaspase inhibitor Z-VAD-FMK (N-benzyloxycarbonyl-Val-AlaAsp-fluoromethylketone), which alone could not prevent the necrosis, protected these cells from the apoptosis. When preinjected intraperitoneally, PTD-FNK markedly reduced zonal liver necrosis caused by CCl 4 . Moreover, injection of PTD-FNK accompanied by Z-VAD-FMK suppressed necrotic injury even after CCl 4 administration. These results suggest that PTD-FNK has great potential for clinical applications to prevent cell death, whether from apoptosis or necrosis, and organ failure.

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“…From this point of view, recombinant viral vectors have a great advantage by depending on their intrinsic machinery for infection. Basic peptides derived from human immunodeficiency virus type 1 (HIV-1) Tat protein and Drosophila Antennapedia protein have been reported to translocate through the cell membrane and to carry exogenous molecules into the cytoplasm and nucleus (Derossi et al, 1994(Derossi et al, , 1996Vivès et al, 1997;Nagahara et al, 1998;Schwartz et al, 1999;Dostman et al, 2000). HIV-1 Tat is an 86 amino-acid protein, and aa 47 -57 of Tat (YGRKKRRQRRR) possess a high net positive charge at physiological pH, with nine of its 11 amino acids being either arginine or lysine.…”

mentioning

confidence: 99%

“…Thus, this 11 aa region is considered a protein transduction domain (PTD). A 119-kDa protein, b-galactosidase, genetically fused to HIV-1 Tat PTD, was successfully carried into various mouse tissues, including the brain, following intraperitoneal injection (Nagahara et al, 1998).…”

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confidence: 99%

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

et al. 2004

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Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.

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Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration

Cited by 893 publications

References 19 publications

Homer Isoforms Differentially Regulate Cocaine-Induced Neuroplasticity

Homer Isoforms Differentially Regulate Cocaine-Induced Neuroplasticity

Zonal necrosis prevented by transduction of the artificial anti-death FNK protein

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

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