TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death.
Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Deltaloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Deltaloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Deltaloop occurs at 37 degrees C but not at 4 degrees C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Deltaloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.
Soane
Lucian
L
Department of Anesthesiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201, USA.
Fiskum
Gary
G
D000818:Animals; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004355:Drug Stability; D004705:Endocytosis / Q000502:physiology*; D015696:Gene Products, tat / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D019812:Heparan Sulfate Proteoglycans / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D021962:Membrane Microdomains / Q000502:physiology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D018696:Neuroprotective Agents / Q000378:metabolism* / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D014161:Transduction, Genetic
D015696:Gene Products, tat; D019812:Heparan Sulfate Proteoglycans; D018696:Neuroprotective Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins