Caspases,Paracaspases, and Metacaspases Methods and Protocols

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Caspases, Paracaspases, and Metacaspases: Methods and ProtocolsPeter V. Bozhkov and Guy Salvesen (eds.), ,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_1, © Springer Science+Business Media New York 2014

Chapter 1

General In Vitro Caspase Assay Procedures

Dave Boucher, Catherine Duclos, and Jean-Bernard Denault

Abstract

One of the most valuable tools that have been developed for the study of apoptosis is the availability of

recombinant active caspases. The determination of caspase substrate preference, the design of sensitive

substrates and potent inhibitors, the resolution of caspase structures, the elucidation of their activation

mechanisms, and the identification of their substrates were made possible by the availability of sufficient

amounts of enzymatically pure caspases. The current chapter describes at length the expression, purifica-

tion, and basic enzymatic characterization of apoptotic caspases.

Key words Caspase, Purification, Active-site titration, Enzymatic assays

1 Introduction

Since the identification of the first caspase in humans more than

20 years ago [ 1 , 2 ], we have seen the unraveling of a new field of

research that year after year still unveils fascinating new discoveries.

By the same token, we have gained new understanding of many

physiological and pathological processes, the most prominent

being apoptotic cell death. This success is in part due to the avail-

ability of enzymatically pure recombinant caspase preparations.

Moreover, the ever-growing recognition of the involvement of cas-

pases in cellular processes [ 3 ] will require the use of recombinant

caspases for years to come to understand the subtleties implied by

this involvement. Earlier works using purified enzymes involved

the characterization of the substrate preference of caspases [ 4 , 5 ],

which allowed the development of reliable peptidic substrates and

potent inhibitors [ 6 , 7 ], and the determination of many caspase

structures in various molecular forms and complexes [ 8 ]. Through

this work, important insight was also gained into the intricacies of

caspase activation mechanisms.

In the past decade, the availability of recombinant caspases

permitted the development of several proteomic approaches involv-

ing peptidases (degradomics) [ 9 , 10 ], and along with several