Role of proteolysis in caspase-8 activation and stabilization.
Caspase-8 is an apoptotic protease that is activated at the cytosolic face of the cell membrane. Activation relies on adaptor-induced dimerization of monomeric caspase-8 and is followed by specific limited autoproteolysis of the linker which separates the two subunits of the catalytic domain. However, the role of this autoproteolysis, which directly activates executioner caspases-3 and -7, is unknown for the apical caspase-8. We have generated linker mutants of caspase-8 that can be proteolyzed in a controlled manner by thrombin or tobacco etch mosaic virus protease, and we use these to define the role of proteolysis in the activation and stability of the enzyme. We show that proteolysis is insufficient for generating enzymatic activity in recombinant caspase-8. Kinetic activation studies using Hoffmeister salts demonstrate that activation is the result of caspase dimerization. However, linker proteolysis significantly enhances the equilibrium for caspase-8 dimerization, thereby increasing the stability of the dimer. Kinetic and fluorescence measurements demonstrate that caspase-8 activation by Hoffmeister salts is at least a two-step event, with the required step being dimerization, followed by an intramolecular event that further stabilizes the catalytic conformation. Autoproteolysis of caspase-8 may be a mechanism for increasing the lifetime of the dimeric enzyme following dissociation from its activating complex at the cell membrane.
Pop
Cristina
C
Program in Apoptosis and Cell Death Research, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037, USA.
Fitzgerald
Patrick
P
Green
Douglas R
DR
Salvesen
Guy S
GS
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002021:Buffers; D053181:Caspase 8 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002850:Chromatography, Gel; D002951:Citrates / Q000494:pharmacology; D019281:Dimerization; D010450:Endopeptidases / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000077559:Sodium Citrate; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology
D002021:Buffers; D002951:Citrates; D011994:Recombinant Proteins; D000077559:Sodium Citrate; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease; D013917:Thrombin; D053181:Caspase 8; D000409:Alanine