Caspase-mediated degradation of human 5-lipoxygenase in B lymphocytic cells

Abstract

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 virustransformed 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.apoptosis ͉ leukotriene ͉ lymphocytes T he enzyme 5-lipoxygenase (5-LO) initiates the synthesis of the bioactive leukotrienes (LTs) from arachidonic acid (for review, see ref. 1). LTs cause constriction and mucus secretion in the lung, increase vascular permeability, and are potent vasoconstrictors of coronary arteries, but they also induce inflammatory reactions (2). In view of these properties, LTs are regarded as powerful mediators that may have crucial roles in asthma and inflammatory disorders (2), and in vascular diseases (3) and cancer (4). Expression and cellular catalysis of 5-LO is tightly regulated. Thus, 5-LO is present mainly in mature leukocytes or dentritic cells, and the capability of cells to express 5-LO is acquired during cell maturation (5). In cells that do not express 5-LO, transcription is blocked by DNA methylation (6).Compared with the intense efforts to elucidate 5-LO expression, investigation of the fate of cellular 5-LO protein has been addressed less. Pulse-chase studies in differentiated HL60 determined the t 1/2 of 5-LO to be 26 h (7). However, the components and mechanisms of 5-LO protein degradation are unknown. Among the degradative enzymes within the cell, caspases are highly regulated cysteine proteases that control critical biological processes by means of specific limited proteolysis (for review, see ref. 8). Caspases exist as latent zymogens, which, after activation, can cleave a huge number of proteins at specific consensus sites (after Asp residues). When activated, caspases transactivate other procaspases. For example, after autoactivation of procaspase-8, the downstream caspase-3 (casp-3) that activates casp-6 is recruited. Both active casp-3 and casp-6...

Two dimensional difference gel electrophoresis analysis of cerebrospinal fluid in tuberculous meningitis patients

“…This observation can be attributed to proteolytic cleavage of the protein. The caspase mediated degradation of ALOX-5 has been reported in B-lymphocyte cells [25]. ALOX-5 has been shown to have a direct role in TBM pathogenesis in mice experiments.…”

Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo

“…We used a refined version of our homology model of 5-LO as target [23], where several potential binding pockets were manually selected. GOLD parameter settings for the genetic algorithm were: number of generations = 100,000, population size = 100, selection pressure = 1.1, number of islands = 5, niche size = 2, migrate = 10, mutate = 95, crossover = 95.…”

“…Evaluation of binding of hyperforin to the active site region of 5-LO Automated molecular docking, using a refined version of a 5-LO homology model [23], was performed in order to probe hyperforin in comparison with BWA4C and ZM230487 in the active site of 5-LO. BWA4C is expected to bind to the active site of 5-LO [27] whereas ZM230487 might bind also to a distinct site [13].…”

Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI‐MS

“…The predicted kinases were first reduced to those with phosphorylation site(s) on the surface of 5-LO, as judged by the 5-LO homology model [25] (and later confirmed with the model of the human 5-LO crystal structure [26]), and only kinases corresponding to sites that were identified by at least three of the four prediction tools were considered further. Subsequently, the kinases were sorted according to their expression patterns reported in the literature.…”