Caspase-mediated degradation of human 5-lipoxygenase in B lymphocytic cells.
5-Lipoxygenase (5-LO) is a tightly regulated enzyme in the synthesis of bioactive lipids from arachidonic acid. Here, we demonstrate that 5-LO is regulated by caspases, which are signaling molecules that control critical biological processes by means of specific limited proteolysis. Cell splitting of the Epstein-Barr virus-transformed B lymphocytic cell line BL41-E95-A caused a pronounced, but transient, reduction of functional 5-LO protein, accompanied by the appearance of a 62-kDa 5-LO cleavage product. In parallel, splitting of BL41-E95-A cells induced activation of caspase-6 (casp-6) and casp-8. Caspase activation and 5-LO degradation were blocked by the protein-synthesis inhibitor cycloheximide, and cell-permeable peptide inhibitors of casp-6 and casp-8 prevented 5-LO cleavage. Activation of casp-6 and casp-8 was connected to subsequent enhancement of cell proliferation, whereas selective caspase inhibition blocked cell growth. Last, isolated human 5-LO was cleaved by recombinant casp-6 in vitro to a 58-kDa fragment. Based on site-directed mutagenesis studies, 5-LO is cleaved by casp-6 after Asp-170, which in a homology-based 3D model of 5-LO is located on the enzyme periphery. We suggest that splitting of BL41-E95-A cells induces de novo synthesis of a protein involved in the activation of casp-6, which cleaves 5-LO.
Werz
Oliver
O
Institutes of Pharmaceutical Chemistry and Organic Chemistry, University of Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany. o.werz@pharmchem.uni-frankfurt.edu
Tretiakova
Irina
I
Michel
Angela
A
Ulke-Lemee
Annegret
A
Hörnig
Michael
M
Franke
Lutz
L
Schneider
Gisbert
G
Samuelsson
Bengt
B
Rådmark
Olof
O
Steinhilber
Dieter
D
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D001665:Binding Sites; D053178:Caspase 6; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D014176:Protein Biosynthesis
D003513:Cycloheximide; D001094:Arachidonate 5-Lipoxygenase; C505514:CASP6 protein, human; C505530:CASP8 protein, human; D053178:Caspase 6; D053181:Caspase 8; D020169:Caspases