Expression and characterization of constitutively active human caspase-14.
Caspase-14 is a cysteine endoproteinase that is expressed in the epidermis and a limited number of other tissues. It is activated during keratinocyte differentiation by zymogen processing, but its precise function is unknown. To obtain caspase-14 for functional studies, we engineered and expressed a constitutively active form of human caspase-14 (Rev-hC14) in Escherichia coli and cultured mammalian cells. Rev-hC14 required no proteolytic processing for activity, showed strong activity against the caspase substrate WEHD, and was inhibited by the pan-caspase inhibitor zVAD-fmk. Mammalian cells that expressed active caspase-14 showed normal cell adherence and morphology. Using positional scanning of synthetic tetrapeptide libraries, we determined the substrate preference of human caspase-14 to be W (or Y)-X-X-D. These studies affirm that caspase-14 has a substrate specificity similar to the group I caspases, and demonstrate that it functions in a distinct manner from executioner caspases to carry out specific proteolytic events during keratinocyte differentiation.
Park
Kyewhan
K
Department of Oral Biology, University of Washington, Seattle, WA 98195, USA.
Kuechle
Melanie K
MK
Choe
Youngchool
Y
Craik
Charles S
CS
Lawrence
Owen T
OT
Presland
Richard B
RB
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D053456:Caspase 14; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis* / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D009842:Oligopeptides; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C513343:tryptophyl-glutamyl-histidyl-aspartic acid; C506446:CASP14 protein, human; D053456:Caspase 14; D020169:Caspases