Caspases,Paracaspases, and Metacaspases Methods and Protocols

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Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_4, © Springer Science+Business Media New York 2014

Chapter 4

Caspase-2 Protocols

Loretta Dorstyn and Sharad Kumar

Abstract

Caspase-2 has been shown to function in apoptosis and in some non-apoptotic pathways, including tumor
suppression and aging. Caspase-2 has some unique features and is the only caspase that constitutively local-
izes to the nucleus, although its nuclear function remains unknown. During apoptosis signaling, caspase-2
rapidly homodimerizes, which leads to its activation and proteolytic processing. The activation of caspase-2
can be measured by assessing its dimerization and/or cleavage of the caspase-2 zymogen and its substrates.
This chapter outlines commonly used methods to purify recombinant caspase-2 and assess its activity and
function in vitro and in cultured cells or tissue extracts.

Key words Apoptosis , Caspase-2 , Caspase activity , Peptide substrates , Recombinant protein ,

Immunoblotting

1 Introduction

Caspase-2, one of the most conserved caspases in metazoans, has

been shown to play redundant and non-redundant roles in apop-

totic and non-apoptotic processes [ 1 – 3 ]. Caspase-2 contains a cas-

pase activation and recruitment domain (CARD), which is a

protein–protein interaction domain required for the dimerization

of pro-caspase-2 molecules and for its activation [ 4 – 6 ]. The cas-

pase- 2 CARD also facilitates recruitment into large multiprotein

complexes [ 7 , 8 ]. For example, caspase-2 interacts with the CARD-

containing proteins RAIDD (RIP-associated Ich-1/CED homolo-

gous protein with a death domain) and PIDD (p53-induced

protein with a death domain) to form a protein complex called

“PIDDosome,” which is suggested to promote caspase-2 activa-

tion [ 8 ]. However, the role of the PIDDosome in cell death is

unclear, and knockout studies suggest that caspase-2 activation can

occur in the absence of PIDD [ 9 , 10 ].

Caspase-2 zymogen contains basal enzyme activity that is

enhanced by dimerization and further proteolytic processing into

subunits (p19 and p12) (Fig. 1 ) [ 4 , 7 , 11 – 13 ]. Structural studies