Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis

Abstract

During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K 38 KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.caspase substrate | molecular recognition | enzymology A poptosis employs a family of cysteinyl peptidases, the caspases, to integrate and propagate various signals to cause cell demise. The latter step is governed by a subgroup of caspases named executioners that are responsible for the cleavage of a plethora of cellular proteins. The cleavage of some of these proteins provokes the associated hallmarks of apoptosis (1, 2). A survey of the published literature suggests that, among executioners, caspase-3 performs the bulk of the cleavage events. These data are in agreement with biochemical studies suggesting that this caspase is highly active and is present at the highest concentration among all caspases in most cells (3)(4)(5). For these reasons, caspase-3 supersedes other caspases in most biochemical readouts (4).Caspase-3 and -7 share 57% sequence identity throughout their catalytic domains. Additionally, they have the same substrate preference based on studies using peptide substrate libraries (6, 7). Despite this apparent redundancy, these two capases have an overlapping but nonidentical substrate repertoire. For example, Rho kinase ROCK1, α-fodrin, and Rho-GDI are cleaved efficiently by both caspases (8, 9). However, inhibitor of caspase-activated Dnase (ICAD) (9), the X-linked inhibitor of apoptosis protein (XIAP) (10), and initiator caspase-9 (11) are preferred by caspase-3, whereas Nogo-B (12), ataxin-7 (13), and the p23 cochaperone (9) are cleaved more efficiently by caspase-7. Poly(ADP ribose) polymerase 1 (PARP), the first caspase substrate identified (14), is a particularly interesting death substrate, and its cleavage is now recognized as a hallmark of apoptosis. PARP proteolysis is essential for an adequate energetic balance during apoptosis and protects against necrosis (15, 16). Previous work has suggested that caspase-7 is responsible for PARP inactivation during apoptosis (17), but no mechanism for such selectivity has been propos...

Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates

“…6,19 Caspase 3 and 7 are very closely related enzymes sharing 57% sequence identity and very similar substrate specificity. 20 At the P4 position, caspase 3 favored Asp B10-fold over other amino acids but caspase 7 was more catholic, preferring Asp but also tolerating aspartic acid methyl, cyclohexyl and benzyl esters, and also histdine benzyl ester, thioproline. The broader tolerance of caspase 7, also seen at the P3 position (see below), is the key distinguishing feature between the closely related paralogs caspases 3 and 7.…”

Zinc-mediated Allosteric Inhibition of Caspase-6

“…exosite for substrate binding has been identified in any caspase. In caspase-7 a tetra-lysine sequence at residue 38 ( 38 KKKK) is critical for caspase-7 to bind and distinguish protein substrates (54). Lys-36 is near the location of the caspase-7 exosite, is close to the N terminus of caspase-6, and is one of the first residues to be crystallographically ordered following the disordered prodomain, leading us to ponder whether this zinc-binding site may be involved in regulating substrate recognition.…”

Caspases rule the intracellular trafficking cartel

“…Indeed, the engineering of the 'best' motif in a structurally well-exposed loop in a model protein resulted in a cleavage rate that still falls short of some known endogenous substrates, which has led to the proposal that other determinants, such as exosites, allow further improvement of catalysis [19]. In fact, usage of exosites by caspases has been demonstrated for cleavage of PARP-1 by caspase-7 [20], and there is evidence that cleavage of lamin A/C by caspase-6 also employs an exosite [21]. Taken together, features including adequate primary and secondary structures and the potential use of exosites allow tailoring (speed, completeness, timing) of substrate cleavage efficacy.…”