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Journal of Molecular Neuroscience

, Volume 55, Issue 2, pp 406–415 | Cite as

Development of a Cell Permeable Competitive Antagonist of RhoA and CRMP4 binding, TAT-C4RIP, to Promote Neurite Outgrowth

  • Mohammad R. Khazaei
  • Samuel Montcalm
  • Adriana Di Polo
  • Alyson E. Fournier
  • Yves DurocherEmail author
  • Stephan Ong ToneEmail author
Article
  • 431 Downloads

Abstract

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.

Keywords

TAT Protein purification Nerve regeneration CRMP4 C4RIP 

Notes

Acknowledgments

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.

Supplementary material

12031_2014_350_MOESM1_ESM.tif (2.2 mb)
Supplemental Fig. 1 Proteolytic cleavage of TAT mRFP targeted to the secretory pathway in CHO cells. A FC-TAT mRFP plasmid containing an Igκ signal sequence and a TEV cleavage site was transfected into CHO cells, and the conditioned media was collected and concentrated. The condition media was incubated with GST-TEV (1:50 ratio) overnight at room temperature. GST-TEV was removed from the conditioned media with a GSTrap column and the N-terminal FC fragment was removed with mab beads. Collected fractions were run on a SDS-polyacrylamide gel and Coomassie stained or immunoblotted with an anti-histidine antibody. Both Coomassie staining and immunoblotting revealed two TAT mRFP products: a TEV-cleavage product and a furin-cleavage product (TIFF 2224 kb)
12031_2014_350_Fig6_ESM.gif (2.2 mb)

High resolution image (GIF 135 kb)

Supplemental Video 1

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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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mohammad R. Khazaei
    • 1
  • Samuel Montcalm
    • 2
  • Adriana Di Polo
    • 3
  • Alyson E. Fournier
    • 1
  • Yves Durocher
    • 2
    Email author
  • Stephan Ong Tone
    • 1
    Email author
  1. 1.Department of Neurology and NeurosurgeryMontreal Neurological InstituteMontrealCanada
  2. 2.Human Health Therapeutics PortfolioNational Research Council CanadaMontréalCanada
  3. 3.Department of Neuroscience and Groupe de Recherche sur le Système Nerveux CentralUniversité de MontréalMontréalCanada

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