On the Sequential Determinants of Calpain Cleavage

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, DABCYLTPLKSPPPSPR-EDANS, was designed and synthesized. In the case of micro- 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.

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

“…Second, calpain cleaves activated Bak at R42/H43 toward the end of a1 (ref. 12), and calpain rarely cleaves in helical regions 33 . Third, if a1 did remain helical, the hydrophobic surface (for example, the BH4 domain) that normally faces the hydrophobic core of Bak might be expected to make further protein-protein or protein-lipid interactions.…”

“…1a). For three of these antibodies , the immunogen contained a stretch of residues (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) conserved in mouse and human Bak (Table 1). Another two antibodies (4B5 and G23) had previously been mapped to a conserved region 9 .…”

BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation

“…The opposite result (calpain ncBID is not processed into tBID after MNNG treatment and is unable to restore PCD) would indicate that BID is directly cleaved into tBID by calpains to promote BAX activation. To design the calpain ncBID vectors, we took into consideration theoretical sequential/structural determinants of calpainmediated cleavage 33 and previous work suggesting that calpains processed BID into tBID at position Gly70. 34 Thus, we generated two constructs: BID-G70A, which contains the Gly70 mutated to Ala, and BID-D68-71, in which the entire 'calpain-cleavable region of BID' has been deleted.…”

Regulating cell migration: calpains make the cut

“…However, no single consensus sequence has been found to have significant value for predicting whether a protein can be proteolyzed by calpains or even where calpains cleave a known substrate. Instead, recognition and proteolysis seem to be controlled by multiple determinants, including but not limited to secondary structure and PEST score † (Tompa et al, 2004). This suggests that calpains cleave their substrates in disordered regions between structured domains.…”

“…This suggests that calpains cleave their substrates in disordered regions between structured domains. Nevertheless, despite this complex set of determinants, there is a significant preference for particular sequences immediately surrounding the site of proteolysis, and studies that have elucidated these preferences have provided valuable tools with which the calpain systems may be specifically and efficiently manipulated (Tompa et al, 2004).…”