Calpain-1 cleaves and activates caspase-7

Abstract

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V(max) when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca(2+) dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.

FIGURE 1.

Calpain-1 cleaves procaspases. In vitro-translated ³⁵S-labeled procaspases were treated with active calpain-1 to determine which caspases are calpain substrates. Each caspase, with and without calpain treatment, was run on a single gel and developed by autoradiography. All of the caspases were run on four separate gels.

FIGURE 2.

Calpain-1 pretreatment affects the activity of a small number of active caspases. Active caspases were incubated with calpain-1 and then caspase activity was measured using a fluorescently labeled substrate with the peptide sequence relevant to each caspase. Results are presented as the % activity of the calpain-1-pretreated sample relative to the activity of the untreated active caspase. Calpain pretreatment resulted in no change in activity for the majority of caspases (A; n = 3), a dramatic reduction in caspase-1 activity (B; n = 3, ***, p < 0.001) and a significant increase in caspase-7 and -10 activities (C; n = 3, ***, p < 0.001, **, p < 0.01).

FIGURE 3.

Calpain-1 pretreatment increases the activity of granzyme B-activated caspase-7. Caspase-7 was incubated with calpain-1 alone, granzyme B alone, or calpain-1 and granzyme B together, and activity was measured using a rhodamine-DEVD substrate. Granzyme B pretreatment increased the activity of procaspase-7 (A; n = 3, ###, p < 0.001) and truncated (ΔN) caspase-7 (B; n = 3, #, p < 0.05). In addition, calpain-1 pretreatment increased the activity of granzyme B-activated procaspase-7 (A; n = 3, ***, p < 0.001) and granzyme B-activated ΔN caspase-7 (B; n = 3, ***, p < 0.001). RFU is the relative fluorescent units.

FIGURE 4.

Calpain-1 cleavage of caspase-7 produces two novel cleavage products. A, purified procaspase-7 and truncated (ΔN) caspase-7 was treated with calpain-1 or caspase-3. Samples were run on a denaturing gel, and proteins were visualized using a Coomassie Blue stain. All samples were run on a single gel. B, activity of active caspase-7 ± calpain-1 treatment was analyzed by zymography, followed by protein quantitation using SYPRO Ruby Stain on the exact same gel. Top arrowhead indicates caspase-3-truncated caspase-7, and lower arrowhead indicates calpain-1-truncated caspase-7 (containing both large and small subunits). Upper arrow indicates caspase-3 derived caspase-7 large subunit and lower arrows indicate calpain-1-derived caspase-7 large subunits. C, calpain pretreatment of active caspase-7 increases the amount of caspase-7-mediated PARP cleavage. Arrow indicates caspase-7 cleavage product of PARP. Samples were run on two separate gels.

FIGURE 5.

Mass spectrometry predicts three calpain cleavage sites in caspase-7. A, active caspase-7 ± calpain-1 pretreatment was analyzed by MALDI-TOF mass spectrometry for protein fragmentation with the x axis representing protein size in Da. Insert above lower graph shows an expansion of the double peak observed at around 17,245 Da. The doubly charged molecular ions [MH²⁺] are indicated by asterisks, and a 1-point external calibration against cytochrome c was used. B, amino acid sequence of the caspase-7 large subunit with predicted calpain cleavage sites highlighted in blue, a putative nuclear localization signal highlighted in red and the active cysteine highlighted in green.

FIGURE 6.

Sequencing of the calpain-1 cleavage site in caspase-7. A, vMALDI-LTQ MS of ΔN caspase-7. B, vMALDI-LTQ MS of ΔN caspase-7 treated with calpain-1. A peptide resulting from calpain-1 cleavage appears at m/z 1450.83. C, vMALDI-LTQ MS/MS of the calpain-1 cleavage product, peptide AKPDRSSFVPSLF (m/z 1450.83). *, impurity in ΔN caspase-7 preparation. #, peak present in calpain-1 preparation.

FIGURE 7.

Calpain fragments of caspase-7 are observed in vivo in cortical neuronal cultures. Primary cortical cultures were treated with 0, 100, or 250 μm of NMDA for 3 h. Untreated lysates were exogenously treated with calpain-1 as a positive control. A caspase-7 antibody was used to detect full-length caspase-7, the caspase-3 fragment (arrowhead), and the calpain fragments (arrows). All samples were run on a single gel. Blots were reprobed with tubulin to demonstrate equal loading.

FIGURE 8.

Cleavage at the second calpain site prevents nuclear localization. Full-length caspase-7, the caspase-3 fragment (amino acids 24–303), the larger calpain-1 fragment (amino acids 37–303), and the smaller calpain-1 fragment (amino acids 48–303) were expressed in 293T cells. Cells were fractionated into cytoplasmic (C) and nuclear (N) components, and blots were probed with the caspase-7 antibody to visualize endogenous and expressed caspase-7 and a FLAG antibody to visualize expressed caspase-7 protein. Successful fractionation and equal loading were confirmed by reprobing the blots with tubulin, calreticulin, and PARP antibodies. Arrows indicate full-length caspase-7, and asterisks indicate the expressed C-terminal caspase-7 constructs. Control samples were run on a second gel.

FIGURE 9.

Diagram of the calpain cleavage sites of caspase-7. A, caspase propeptide is 34 kDa in length. Cleavage by caspase-3 after D23 and caspase-8/-10 or granzyme B after D198 releases the active large subunit (p20). Cleavage by calpains after Phe-36 produces an 18.5 kDa active large subunit. Further cleavage by calpains after Met-45 or Ser-47 produces a 17.4 or 17.2 kDa large subunit, respectively, which has increased activity and decreased nuclear localization. B, model of the activation process of caspase-7 by calpain and caspase-8/-10 or granzyme B.