Novel cell-penetrating calpain substrate

Z. Bánóczi, Anita Alexa, Attila Farkas, P. Friedrich, F. Hudecz

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The calpain enzymes play important roles in numerous processes in the cell. In vivo analysis of calpain activity might be useful for clarification of their role in different diseases. Our early results suggested that a peptide substrate, Dabcyl-TPLKSPPPSPR-EDANS, based on the calpain cleavage sequences is suitable for developing a new cell-penetrating calpain substrate. This conjugate with the Dabcyl and EDANS fluorophores as a FRET pair is specific for calpain even in cell lysate, but unfortunately has poor cell uptake. Therefore, we have modified this sequence by C-terminal elongation with heptaarginine unit possessing cell-penetrating activity. In order to preserve the necessary distance between the two FRET partners, we inserted a Glu residue between the substrate and heptaarginine parts of the peptide. Thus, the cell-penetrating substrate Dabcyl-TPLKSPPPSPRE(EDANS)R7 was synthesized. This peptide not only retained the substrate property, but was a better substrate of Calpain B enzyme. The cell uptake of the substrate conjugate was studied by fluorescence microscopy and flow cytometry. The results showed that the conjugate enters COS-7 cells more efficiently than the peptide substrate without heptaarginine. The uptake occurs already at low concentration and the compound is distributed homogeneously inside cells. These observations might indicate that this new cell-penetrating substrate could be useful for determining calpain activity in cell lysate or in intact cells of various origins.

Original languageEnglish
Pages (from-to)1375-1381
Number of pages7
JournalBioconjugate Chemistry
Volume19
Issue number7
DOIs
Publication statusPublished - Jul 2008

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Calpain
Substrates
Peptides
Enzymes
Flow cytometry
Fluorophores
Fluorescence microscopy
COS Cells
Elongation
Fluorescence Microscopy
Flow Cytometry

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Novel cell-penetrating calpain substrate. / Bánóczi, Z.; Alexa, Anita; Farkas, Attila; Friedrich, P.; Hudecz, F.

In: Bioconjugate Chemistry, Vol. 19, No. 7, 07.2008, p. 1375-1381.

Research output: Contribution to journalArticle

Bánóczi, Z. ; Alexa, Anita ; Farkas, Attila ; Friedrich, P. ; Hudecz, F. / Novel cell-penetrating calpain substrate. In: Bioconjugate Chemistry. 2008 ; Vol. 19, No. 7. pp. 1375-1381.
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