Synthesis of Cell-Penetrating Peptides and Their Application in Neurobiology

Dietz, Gunnar P.H.; Bähr, Mathias

doi:10.1007/978-1-59745-504-6_13

Cited by 32 publications

(21 citation statements)

References 55 publications

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“…novagen.com) and proteins were isolated in 10 mM Tris, pH 10, 20% glycerol, 274 mM NaCl, 0.1% Pluronic, and 0.02% Tween 80. Bacterial debris was removed by centrifugation and the cell extracts were purified by affinity chromatography using Ni-triscarboxymethyl-ethylene-diamine [37]. Protein was eluted by stepwise addition of binding buffer containing increasing concentrations of imidazole.…”

Section: Preparation Of Recombinant Proteinsmentioning

confidence: 99%

Transduction of Neural Precursor Cells with TAT-Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

et al. 2012

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Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection.

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Section: Preparation Of Recombinant Proteinsmentioning

confidence: 99%

Transduction of Neural Precursor Cells with TAT-Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

et al. 2012

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“…In brief, Tat-Hsp70, Tat-HA and Hsp70 were expressed in E. coli strain BL21 (DE3) pLysS (Novagen, Madison, WI) and were isolated in their native conformation (in 10 mM Tris pH 10, 20% glycerol, 274 mM NaCl, 0.1% Pluronic, 0.02% Tween-80 buffer) (Dietz and Bähr, 2007) or under denaturing conditions in binding buffer (8 M urea, 274 mM NaCl, 20 mM Hepes, pH 8.0, 5 mM imidazole) as reported previously (Becker-Hapak et al, 2001;Dietz and Bähr, 2007;Vocero-Akbani et al, 2000. Cell debris was removed by centrifugation and the cell extracts were purified by metal-affinity chromatography using either Ni-tris-carboxymethyl-ethylene-diamine (TED) for purification under native conditions or a Ni-nitrilotriacetic-acid (NTA) for denaturing protocols (Fig.…”

Section: Synthesis and Purification Of Tat-fusion Proteinsmentioning

confidence: 99%

Quantitative evaluation of chaperone activity and neuroprotection by different preparations of a cell-penetrating Hsp70

Nagel

Dohm

Bähr

et al. 2008

Journal of Neuroscience Methods

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“…The discovery that the transactivator of transcription (TAT) 2 protein of human immunodeficiency virus type 1 was able to traverse cellular membranes and subsequently affected gene transcription (1,2) led to the emergence of a new research field on cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs) or membrane transduction peptides (3). CPPs opened up the possibility to effectively deliver cell-impermeable hydrophilic compounds into living cells.…”

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confidence: 99%

Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis

Ter-Avetisyan

Tünnemann

Nowak

et al. 2009

Journal of Biological Chemistry

203

174

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Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4°C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.

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Synthesis of Cell-Penetrating Peptides and Their Application in Neurobiology

Cited by 32 publications

References 55 publications

Transduction of Neural Precursor Cells with TAT-Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

Transduction of Neural Precursor Cells with TAT-Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

Quantitative evaluation of chaperone activity and neuroprotection by different preparations of a cell-penetrating Hsp70

Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis

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