Tat-calpastatin fusion proteins transduce primary rat cortical neurons but do not inhibit cellular calpain activity

Tomoko Sengoku, Vimala Bondada, Duane Hassane, Sam Dubal, James W. Geddes

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


Excessive activation of calpains (calcium-activated neutral proteases) is observed following spinal cord contusion injury, traumatic brain injury, stroke, and in neurodegenerative disorders including Alzheimer's disease. Calpain inhibition represents an attractive therapeutic target, but current calpain inhibitors possess relatively weak potency, poor specificity, and in many cases, limited cellular and blood-brain barrier permeability. We developed novel calpain inhibitors consisting of the endogenous inhibitor, calpastatin or its inhibitory domain I, fused to the protein transduction domain of the HIV trans-activator (Tat) protein (Tat47-57). The Tat-calpastatin fusion proteins were potent calpain inhibitors in a cell-free activity assay, but did not inhibit cellular calpain activity in primary rat cortical neurons when applied exogenously at concentrations up to 5 μM. The fusion proteins were able to transduce neurons, but were localized within endosome-like structures. A similar endosomal uptake was observed for Tat-GFP. Together, the results suggest that endosomal uptake of the Tat-calpastatin prevents its interaction with calpain in other cellular compartments. Endosomal uptake of proteins fused to the Tat protein transduction domain severely limits the applications of this methodology.

Original languageEnglish
Pages (from-to)161-170
Number of pages10
JournalExperimental Neurology
Issue number1
StatePublished - Jul 2004

Bibliographical note

Funding Information:
We thank Dr. Stephen Dowdy for the generous gift of the pTat and pTat-HA vectors and pTAT-βgal construct; Dr. Steve Estus for the pTAT-GFP construct; and Dr. Subarrao Bondada and Dr. Lakshman Chevalrin for assistance with the HPLC purification of Tat-GFP. This research was supported by NIH grant R01 NS45726-01A1 (JWG), an American Heart Association Fellowship (TS), and by grants from the Kentucky Spinal Cord and Head Injury Research Trust and the Kentucky Science and Engineering Foundation.


  • Cysteine proteases
  • Endosomes
  • Protein transduction
  • Spectrin

ASJC Scopus subject areas

  • Neurology
  • Developmental Neuroscience


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