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Title: On the sequential determinants of calpain cleavage
Authors: Tompa, P ×
Buzder-Lantos, Peter
Tantos, A
Farkas, A
Szilagyi, A
Banoczi, Z
Hudecz, F
Friedrich, P #
Issue Date: May-2004
Publisher: Amer soc biochemistry molecular biology inc
Series Title: Journal of Biological Chemistry vol:279 issue:20 pages:20775-20785
Abstract: The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action via site-directed mutagenesis. In general, our data underline the importance of the primary structure of the substrate around the scissile bond in the recognition process. Significant amino acid preferences were found to extend over 11 residues around the scissile bond, from P-4 to P-7'. In compliance with earlier data, preferred residues in the P-2 position are Leu, Thr, and Val, and in P-1 Lys, Tyr, and Arg. In position P-1', small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P-2-P-1' segment, i.e. positions P-3 and P-2'- P-4'; most notable is its occurrence 5.59 times above chance in P-3'. Intriguingly, the segment C-terminal to the cleavage site resembles the consensus inhibitory region of calpastatin, the specific inhibitor of the enzyme. Further, the position of the scissile bond correlates with certain sequential attributes, such as secondary structure and PEST score, which, along with the amino acid preferences, suggests that calpain cleaves within rather disordered segments of proteins. The amino acid preferences were confirmed by site-directed mutagenesis of the autolysis sites of Drosophila calpain B; when amino acids at key positions were changed to less preferred ones, autolytic cleavage shifted to other, adjacent sites. Based on these preferences, a new fluorogenic calpain substrate, DABCYL-TPLKSPPPSPR-EDANS, was designed and synthesized. In the case of mu- and m-calpain, this substrate is kinetically superior to commercially available ones, and it can be used for the in vivo assessment of the activity of these ubiquitous mammalian calpains.
URI: 
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Medicinal Chemistry (Rega Institute)
× corresponding author
# (joint) last author

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