Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

Magdalena Wejda,Francis Impens,Nozomi Takahashi,Petra Van Damme,Kris Gevaert,Peter Vandenabeele

doi:10.1074/jbc.m112.384552

Magdalena Wejda, Francis Impens + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m112.384552

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Abstract

Caspase-2 is considered an initiator caspase because its long prodomain contains a CARD domain that allows its recruitment and activation in several complexes by homotypic death domain-fold interactions. Because little is known about the function and specificity of caspase-2 and its physiological substrates, we compared the cleavage specificity profile of recombinant human caspase-2 with those of caspase-3 and -7 by analyzing cell lysates using N-terminal COmbined FRActional DIagonal Chromatography (COFRADIC). Substrate analysis of the 68 cleavage sites identified in 61 proteins revealed that the protease specificities of human caspases-2, -3, and -7 largely overlap, revealing the DEVD↓G consensus cleavage sequence. We confirmed that Asp(563) in eukaryotic translation initiation factor 4B (eIF4B) is a cleavage site preferred by caspase-2 not only in COFRADIC setup but also upon co-expression in HEK 293T cells. These results demonstrate that activated human caspase-2 shares remarkably overlapping protease specificity with the prototype apoptotic executioner caspases-3 and -7, suggesting that caspase-2 could function as a proapoptotic caspase once released from the activating complex.

Highlights

To study the potential role of caspase-2, we determined its cleavage site specificity and compared it with that of caspase-3 and -7
We confirmed that Asp563 in eukaryotic translation initiation factor 4B is a cleavage site preferred by caspase-2 in COFRADIC setup and upon co-expression in HEK 293T cells
These results demonstrate that activated human caspase-2 shares remarkably overlapping protease specificity with the prototype apoptotic executioner caspases-3 and -7, suggesting that caspase-2 could function as a proapoptotic caspase once released from the activating complex

Summary

Background

To study the potential role of caspase-2, we determined its cleavage site specificity and compared it with that of caspase-3 and -7. We confirmed that Asp563 in eukaryotic translation initiation factor 4B (eIF4B) is a cleavage site preferred by caspase-2 in COFRADIC setup and upon co-expression in HEK 293T cells These results demonstrate that activated human caspase-2 shares remarkably overlapping protease specificity with the prototype apoptotic executioner caspases-3 and -7, suggesting that caspase-2 could function as a proapoptotic caspase once released from the activating complex. The prodomain of caspase-2 contains a CARD domain that allows formation of a protein complex of high molecular mass (Ͼ670 kDa), called the PIDDosome, involved in proximity-induced activation of procaspase-2 [9]. Upon overexpression in HEK 293T cells, we confirmed that one of two cleavage sites identified in eukaryotic translation initiation factor 4B (eIF4B) is preferred by caspase-2, as observed in our COFRADIC results. To the best of our knowledge, this is the first study reporting the use of a comprehensive proteomic approach to profile caspase-2 cleavage specificity

EXPERIMENTAL PROCEDURES

RESULTS

98 ASSAVKVKR

14 GIVPDIAVGTKR

DISCUSSION