Development of a Cell Permeable Competitive Antagonist of RhoA and CRMP4 binding, TAT-C4RIP, to Promote Neurite Outgrowth
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Neurons fail to re-extend their processes within the central nervous system environment in vivo, and this is partly because of inhibitory proteins expressed within myelin debris and reactive astrocytes that actively signal to the injured nerve cells to limit their growth. The ability of the trans-acting activator of transcription (TAT) protein transduction domain (PTD) to transport macromolecules across biological membranes raises the possibility of developing it as a therapeutic delivery tool for nerve regeneration. Most studies have produced TAT PTD fusion protein in bacteria, which can result in problems such as protein solubility, the formation of inclusion bodies and the lack of eukaryotic posttranslational modifications. While some groups have investigated the production of TAT PTD fusion protein in mammalian cells, these strategies are focused on generating TAT PTD fusions that are targeted to the secretory pathway, where furin protease as well as other proteases can cleave the TAT PTD. As an alternative to mutating the furin cleavage site in the TAT PTD, we describe a novel method to generate cytosolic TAT PTD fusion proteins and purify them from cell lysates. Here, we use this method to generate TAT-C4RIP, a cell permeable competitive antagonist of binding between the small GTPase RhoA and the cytosolic phosphoprotein Collapsin response mediator protein 4 (CRMP4). We demonstrate that TAT-C4RIP transduces cells in vitro and in vivo and retains its biological activity to attenuate myelin inhibition in an in vitro neurite outgrowth assay.
KeywordsTAT Protein purification Nerve regeneration CRMP4 C4RIP
The authors would like to thank Dr. Steven F Dowdy for providing reagents for TAT protein purification. This study was supported by a grant to AEF from the Canadian Institutes of Health Research (CIHR) and by a studentship to SOT from CIHR. AEF is a Tier 2 Canada Research Chair.
Localization of TAT mRFP in live HeLa cells. Time-lapse microscopy performed on HeLa cells incubated with 100 μg/mL TAT mRFP for 3 h, washed three times with media, and incubated in OptiMEM. TAT mRFP localizes to small vesicles, which are seen moving throughout the cells. A diffuse cytosolic distribution of TAT mRFP is observed throughout the cell (MOV 307 kb)
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