Expression and characterization of constitutively active human caspase-14

Area of Science:

• Biochemistry
• Molecular Biology
• Dermatology

Background:

• Caspase-14 is a cysteine endoproteinase primarily found in the epidermis.
• Its activation occurs during keratinocyte differentiation, but its specific function remains unclear.

Purpose of the Study:

• To produce an active form of human caspase-14 for functional analysis.
• To determine the substrate specificity and enzymatic activity of caspase-14.

Main Methods:

• Engineered and expressed a constitutively active form of human caspase-14 (Rev-hC14) in E. coli and mammalian cells.
• Assessed activity using the substrate WEHD and inhibition by zVAD-fmk.
• Determined substrate preference via positional scanning of synthetic tetrapeptide libraries.

Main Results:

• Rev-hC14 exhibited proteolytic activity without requiring zymogen processing.
• Caspase-14 showed strong activity against the WEHD substrate and was inhibited by zVAD-fmk.
• The substrate preference of human caspase-14 was identified as W(or Y)-X-X-D.

Conclusions:

• Caspase-14 possesses a substrate specificity akin to group I caspases.
• Active caspase-14 expression in mammalian cells did not disrupt normal cell adherence or morphology.
• Caspase-14 functions distinctly from executioner caspases in specific proteolytic events during keratinocyte differentiation.