101Peter 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_6, © Springer Science+Business Media New York 2014
Chapter 6
Caspase Protocols in Caenorhabditis elegans
Eui Seung Lee and Ding Xue
Abstract
Caenorhabditis elegans genome has four genes ( ced-3 , csp-1 , csp-2 , and csp-3 ) encoding caspase-like proteins.
Among these four proteins, CED-3 is the most well-known cell-killing caspase. Elucidation of the role of
CED-3 as a central component of the apoptotic pathway in C. elegans has contributed to the understand-
ing of the more complex apoptosis network in mammals and in other metazoa. In the highly conserved
pathway of programmed cell death in C. elegans , CED-3 functions at the terminal step of this cell-killing
pathway. Identifi cation of CED-3 caspase substrates is essential for bridging the gaps between CED-3
activation and various downstream cell death execution events. If a protein is cleaved by CED-3 in vitro,
this protein could be a potential CED-3 substrate in vivo. Here, we describe the method for purifi cation
of active CED-3 caspase. We will also describe in vitro assays for determining CED-3 proteolytic activity,
CED-3 substrates, and CED-3 cleavage sites in the substrates.
Key words CED-3 , CED-3 substrates , C. elegans apoptosis pathway , In vitro translation , In vitro
cleavage assay1 Introduction
Programmed cell death (PCD) or apoptosis was fi rst described in
C. elegans in the late 1970s [ 1 – 3 ], and many cell death genes iden-
tifi ed in C. elegans have been shown to be conserved throughout
metazoa [ 4 ]. Identifi cation and analyses of the C. elegans ced-3
gene and its homologues, caspases, in mammals and other organ-
isms demonstrate that caspases are responsible for most apoptotic
events [ 5 ]. Four genes encoding proteins with sequence similarity
to caspases have been found in the C. elegans genome: ced-3 , csp-1 ,
csp-2 , and csp-3 [ 6 , 7 ]. CED-3 is the key cell-killing caspase in
C. elegans [ 6 ], whereas CSP-2 and CSP-3 do not display any cas-
pase activity and, instead, function as caspase inhibitors [ 8 , 9 ]. A
recent study reports that csp-1 also promotes apoptosis in C. ele-
gans [ 10 ]. In this chapter, we will describe biochemical methods
important for the characterization of the C. elegans CED-3 caspase
and its substrates.