Purification of catalytically active caspase-12 and its biochemical characterization.
Caspase-12, mainly detected in endoplasmic reticulum (ER), has been suggested to play a role in ER-mediated apoptosis and inflammatory caspase activation pathway. Cleavage of the prodomain by caspase-3/-7 at the carboxyl terminus of Asp94 or m-calpain at the carboxyl terminus of Lys158 was reported to be a part of caspase-12-involved apoptosis. We biochemically characterized the prodomain-free forms of caspase-12 and the equivalent enzymes; Deltapro1(G95-D419), rev-Deltapro1[(T319-N419)-(G95-D318), a reverse form of Deltapro1] and rev-Deltapro2[(T319-N419)-(T159-D318)]. The three variants showed comparable activities which were dependent on salt concentration and pH. Auto-proteolytic cleavage was observed at two sites (carboxyl termini of Asp318 and Asp320) in Deltapro1. Constitutively active forms of caspase-12 (rev-Deltapro1 and rev-Deltapro2) could induce cell death in cells transfected with the corresponding expression vectors, but no cleavage of caspase-3, DFF45 or Bid was observed, indicating caspase-12 may mediate a distinct apoptotic pathway rather than caspase-8 or -9-mediated cell death.
Lee
Hyun-Jung
HJ
Department of Biology, Ewha Womans University, Seoul, Republic of Korea.
Lee
Sung Haeng
SH
Park
Sung-Hee
SH
Sharoar
M Golam
MG
Shin
Song Yub
SY
Lee
Jung Sup
JS
Cho
Byungyun
B
Park
Il-Seon
IS
D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D053454:Caspase 12 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; C506444:CASP12 protein, human; C505520:CASP7 protein, human; D053454:Caspase 12; D053179:Caspase 7