원문정보
초록
영어
Caspase-12, mainly detected in endoplasmic reticulum (ER), has been suggested to play a role in ER- mediated apoptosis and inflammatory caspase activation pathway. Removal of the prodomain by caspase-3/-7 at Asp94 or m -calpain at Lys158 was reported to be a part of caspase-12-involved (in) apoptosis. To provide biochemical background for the processes, we purified and characterized three forms of caspase-12; Δpro1(G95-D419), rev-Δpro1[(T319-N419)- (G95-D318), a reverse form of Δpro1] and rev-Δpro2[(T319-N419)-(T159-D318)]. Activity of Δpro1, comparable (in compare) to those of the other two, decreased significantly under the physiological salt concentration and pH or by mutations at both Asp318 and Asp320. These indicated (indicate) that activation of caspase-12 may need pH drop and ion efflux observed during apoptotic process, and auto-proteolytic cleavage at the sites. Furthermore, constitutively active forms of caspase-12 could induce cell death, but no cleavage of caspase-3, DFF45 and Bid was observed, implying involvement of caspase-12 in a distinct way rather than in the well-known pathway.
목차
1. Introduction
2. Experimental Section
2.1. Materials
2.2. Construction of Recombinant Caspase-12
2.3. Purification of Recombinant Caspase-12 and Caspase-7
2.4. Assay of Caspase Activity
2.5. Cell Culture, Transfection, and Cell Viability Assay
2.6 Immunoblot Analysis
2.7. DNA Fragmentation Assay
2.8. Caspase-12 Assay with Cell Extract
3. Results and Discussion
3.1. Construction and Purification of Caspase-12 Variants
3.2. Effects of pH and Ionic Strength on Caspase-12 Activity
3.3. Procaspase-12 Cleavage by Auto-proteolysis and Caspase-7, and Mapping the Cleavage Sites
3.4. Comparison of Activity of Caspase-12 Variants
3.5. Death of Cells Transfected with Variable Caspase-12 Constructs
Conclusion
References