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

Nagel, Florian; Dohm, Christoph P.; Bähr, Mathias; Wouters, Fred S.; Dietz, Gunnar P.H.

doi:10.1016/j.jneumeth.2008.03.008

Cited by 15 publications

(10 citation statements)

References 38 publications

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“…It had been reported that TAT-Hsp70 was able to be delivered into cells and mice efficiently [46], [47]. We examined the effect of TAT-Hsp70 on viral infection in BALB/c mice.…”

Section: Resultsmentioning

confidence: 99%

Heat Shock Protein 70 Inhibits the Activity of Influenza A Virus Ribonucleoprotein and Blocks the Replication of Virus In Vitro and In Vivo

Zhang

Tong

et al. 2011

PLoS ONE

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BackgroundHeat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated.Principal FindingsHere we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice.SignificanceOur study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.

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“…It had been reported that TAT-Hsp70 was able to be delivered into cells and mice efficiently [46], [47]. We examined the effect of TAT-Hsp70 on viral infection in BALB/c mice.…”

Section: Resultsmentioning

confidence: 99%

Heat Shock Protein 70 Inhibits the Activity of Influenza A Virus Ribonucleoprotein and Blocks the Replication of Virus In Vitro and In Vivo

Zhang

Tong

et al. 2011

PLoS ONE

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“…The column eluate was purified from imidazole by gel filtration (Sephadex G-25 M, GE Healthcare BioSciences AB, Munich, Germany, www.gehealthcare.com). According to this procedure, TAT-Hsp70 is highly stable after cell transduction as no protein degradation could be detected 24 hours after protein application, suggesting that the intracellular half-life time is at least a few days [36]. For transplantation of NPCs, in vitro incubation period with either TAT-Hsp70 (250 nM) or TAT-HA (250 nM) was 4 hours.…”

Section: Preparation Of Recombinant Proteinsmentioning

confidence: 99%

“…TAT-Hsp70 and TAT-hemagglutinin (TAT-HA), which is also included in the TAT-Hsp70 construct serving as a negative control, were prepared under native conditions as described [36]. Briefly, the recombinant genes were expressed in Escherichia coli strain BL21 (DE3) pLysS (Novagen, Madison, WI, www.…”

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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“…Values measured for CDK5 band intensities were normalized by dividing them by the corresponding intensities of the same blot probed with an anti-actin antibody. To induce apoptotic cell death, cells were treated with 400 nM staurosporine for 24 h. 6-OHDA toxicity assays were performed as described [17], with 80 µM 6-OHDA used here. Statistics were performed using the two-tailed student’s t-test.…”

Section: Methodsmentioning

confidence: 99%

Cell-Penetrating Fragments of the Cdk5 Regulatory Subunit Are Protective in Models of Neurodegeneration

et al. 2010

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Cdk5 is essential for neuronal differentiation processes in the brain. Activation of Cdk5 requires the association with the mostly neuron-specific p35 or p39. Overactivation of CDK5 by cleavage of p35 into p25 is thought to be involved in neurodegenerative processes. Here, we have tested an approach to inhibit pathological Cdk5 activation with a Tat-linked dominant-negative fragment of p25. It reduced cell death induced by staurosporine and showed a tendency to alleviate manganese-induced cell death, while it did not protect against 6-OHDA toxicity. Our results suggest that the Tat technique is a suitable tool to inhibit dysregulated CDK5.

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

Cited by 15 publications

References 38 publications

Heat Shock Protein 70 Inhibits the Activity of Influenza A Virus Ribonucleoprotein and Blocks the Replication of Virus In Vitro and In Vivo

Heat Shock Protein 70 Inhibits the Activity of Influenza A Virus Ribonucleoprotein and Blocks the Replication of Virus In Vitro and In Vivo

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

Cell-Penetrating Fragments of the Cdk5 Regulatory Subunit Are Protective in Models of Neurodegeneration

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