Quantitative evaluation of chaperone activity and neuroprotection by different preparations of a cell-penetrating Hsp70.
Cell-penetrating peptides (CPPs), such as the one derived from the human immunodeficiency virus Tat protein, facilitate the delivery of cargoes across cellular membranes. However, questions about the therapeutic potential of CPP-mediated delivery remain. For instance, the impact of the purification procedure on the functionality of Tat-fusion proteins has not been systematically examined. Here, we isolated fusion proteins of the chaperone heat shock protein 70 (Hsp70) and the Tat CPP under denaturing or native conditions. To investigate the therapeutic potential of different recombinant protein preparations, we examined Tat-Hsp70 transduction efficiency and quantified Tat-Hsp70-mediated folding of a chaperone-dependent yellow fluorescent protein in vitro. Transduction efficiency and chaperone activity of Tat-Hsp70-treated cells was significantly higher compared to cells treated with Hsp70. The application of native isolated Tat-Hsp70 had the strongest effect. This chaperone activity correlates with increased viability of cells treated with the recombinant protein after cell death induction with 6-hydroxydopamine. This suggests that the method of recombinant Tat-fusion protein purification influences its functionality. For Tat-Hsp70, the method of choice seems to be isolation under native conditions, for which we present a purification protocol. Our results may contribute to improve Tat-fusion protein application in basic research and may facilitate its use as therapeutic tool, for instance in Parkinson's disease.
Nagel
Florian
F
Department of Neurology, Waldweg 33, D-37073 Göttingen, Germany.
Dohm
Christoph P
CP
Bähr
Mathias
M
Wouters
Fred S
FS
Dietz
Gunnar P H
GP
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009447:Neuroblastoma; D016627:Oxidopamine / Q000633:toxicity; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013565:Sympatholytics / Q000633:toxicity; D014162:Transfection
D015696:Gene Products, tat; D018840:HSP70 Heat-Shock Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D013565:Sympatholytics; D016627:Oxidopamine