Caspases,Paracaspases, and Metacaspases Methods and Protocols

89

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_5, © Springer Science+Business Media New York 2014

Chapter 5

Caspase-14 Protocols

Mami Yamamoto-Tanaka and Toshihiko Hibino

Abstract

Unlike other caspase family members, caspase-14 shows restricted expression, being found mostly in
epidermis and its appendages. It has been suggested that caspase-14 is not involved in apoptosis or infl am-
mation, but participates in keratinocyte terminal differentiation. Its activation occurs at the corneocyte
formation. In previous work, we have purifi ed active caspase-14 from human corneocyte extracts. In
addition, we have clarifi ed activation mechanism of caspase-14, where kallikrein-related peptidase 7
(KLK7) generates an intermediate form from procaspase-14 and this form fi nally converts procaspase-14
to active, mature caspase-14. Here we describe techniques for measurement of caspase-14 activity using
synthetic substrate, purifi cation of caspase-14 from corneocyte extract, preparation of constitutively active
caspase- 14 and specifi c antibody, quantifi cation of total and active caspase-14 in corneocyte extracts using
ELISA, as well as methods for caspase-14 activation and its visualization by immunohistochemistry.

Key words Caspase-14 , Epidermis , Keratinocyte , Terminal differentiation , Corneocyte ,

Kallikrein- related peptidase

1 Introduction

Caspase-14 is a member of the caspase family, which is most

exclusively expressed in epidermis and its appendages [ 1 – 3 ].

Keratinocytes are the major components of epidermis and they

constitute 90 % of this protective tissue. Keratinocytes undergo

terminal differentiation that is precisely controlled, starting from

proliferating basal cells; progressing to the appearance of spinous,

granular cells; and ultimately leading to cornifi ed cells [ 4 ]. This

process results in formation of the permeability barrier that pro-

tects the body against hazardous environmental factors. Epidermal

barrier includes various components, such as natural moisturizing

factor (NMF), extracellular lipid membranes, cornifi ed envelope

(CE), and internal structure of macrofi brils to support and to

maintain fl exible yet sturdy architecture. Dramatic changes occur

at the transition phase from granular cells to cornifi ed cells. Cellular

organelles are digested and cytoplasm is occupied with keratin