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Proc Natl Acad Sci U S A. Sep 13, 2005; 102(37): 13164–13169.
Published online Aug 31, 2005. doi:  10.1073/pnas.0505991102
PMCID: PMC1201604
Cell Biology

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 virus-transformed B lymphocytic cell line BL41-E95-A caused a pronounced, but transient, reduction of functional 5-LO protein, ac-companied 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.

Keywords: apoptosis, leukotriene, lymphocytes

The 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 t1/2 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 then cleave diverse proteins categorized into functional groups, including proteins involved in scaffolding of the cytosplasm and nucleus, signal transduction and transcription regulatory proteins, and cell-cycle and DNA repair-related proteins (9). Activation of caspases occurs mainly in response to apoptotic stimuli, and apoptotic cell death is executed by caspase-mediated protein cleavage (10). Caspases have also been linked to nonapoptotic biological responses, such as inflammation by cytokine (IL-1β) maturation, proliferation and cellular differentiation, spreading, and receptor internalization (1113).

BL41-E95-A cells, which were obtained by infection of the Burkitt's lymphoma line BL41 with the B95-8 Epstein–Barr virus strain (14), constitutively express 5-LO and, in comparison with various B cell lines, exhibit high 5-LO protein levels (15). We (16) have observed that 5-LO activity is transiently decreased in BL41-E95-A cells after splitting. In this study, we sought to investigate the mechanisms reducing 5-LO in BL41-E95-A cells. Our data provide evidence that functional 5-LO is cleaved by casp-6.

Materials and Methods

Cells and Cell Culture. BL41-E95-A cells were seeded in RPMI medium 1640 with glutamine (GIBCO) [supplemented with 10% FCS (Roche Molecular Biochemicals), 100 μg/ml streptomycin, and 100 units/ml penicillin (culture medium)] at a density of 0.2 × 106 cells per ml and grown at 37°C in a 5% CO2/95% air atmosphere. For cell splitting, cell suspensions were diluted into fresh culture medium to 0.2 × 106 cells per ml. For harvesting or transfer into fresh medium, cells were centrifuged (200 × g for 10 min at room temperature) and then resuspended as indicated.

RNA Extraction and RT-PCR Analysis. Total RNA was isolated from BL41-E95-A cells by the guanidinium thiocyanate method (17), and RT-PCR analysis was performed as described (18). The following PCR primers were used at a concentration of 5 ng/μl: β-actin (24 cycles), 5′-GAGGAGCACCCCGTGCTGCTGA-3′ and 5′-CTAGAAGCATTTGCTGTGGACGATGGAGGGGCC-3′; 5-LO (30 cycles), 5′-ACCATTGAGCAGATCGTGGACACGC-3′ and 5′-GCAGTCCTGCTCTGTGTAGAATGGG-3′. Signal intensities of ethidium bromide-stained DNA bands were quantitated by densitometry (Gel Doc 1000 system, Bio-Rad) and analyzed with the program molecular analyst (Bio-Rad). Results are expressed as relative changes in RNA amounts normalized to β-actin as an internal standard.

Determination of 5-LO Activity in Crude Homogenates. BL41-E95-A cells (1 × 107) were resuspended in 1 ml of PBS (pH 7.4) containing 1 mM EDTA, kept on ice for 5 min, and sonicated (3 × 10 s), and 1 mM ATP was then added. The samples were preincubated for 30 s at 37°C, and 2 mM CaCl2 and 40 μM arachidonic acid were added. After 10 min at 37°C, the incubation was stopped, and the formed 5-LO products were extracted and analyzed by HPLC as described (19). 5-LO product formation is expressed as nanograms of 5-LO products per 106 cells, which includes LTB4 and its all-trans isomers and 5(S)-hydro(peroxy)-6-trans-8,11,14-cis-eicosatetraenoic acid. Cysteinyl LTs (LTC4, D4, and E4) were not detected, and oxidation products of LTB4 were not determined.

SDS/PAGE and Western Blot Analysis (WB). BL41-E95-A cells (4 × 106) were lysed in 50 μl of lysis buffer (10 mM Tris·HCl, pH 7.5/50 mM NaCl/30 mM sodium pyrophosphate/10 mM 4-nitrophenyl phosphate/50 mM NaF/20 mM β-glycerophosphate/2 mM Na3VO4/0.5 mM PMSF/10 μg/ml leupeptin/60 μg/ml soybean trypsin inhibitor) at 4°C. Supernatants (10,000 × g for 10 min at 4°C) of the lysates were mixed with one volume of 2 × SDS/PAGE sample loading buffer [SDS-b; 20 mM Tris·HCl, pH 8/2 mM EDTA/5% (mass/vol) SDS/10% β-mercaptoethanol] and boiled for 5 min at 95°C. Aliquots (20 μl) corresponding to equivalents of exactly 0.8 × 106 cells (≈15–20 μg per well) were mixed with 4 μl of glycerol/0.1% bromphenol blue (1:1, vol/vol) and analyzed by SDS/PAGE. After electroblotting to nitrocellulose membrane (Amersham Biosciences) and blocking with 5% nonfat dry milk for 1 h at room temperature, membranes were washed and incubated with primary Abs, overnight at 4°C. The polyclonal anti-5-LO antiserum (AK7, 1551) was affinity-purified on a 5-LO column and used at 1:10 dilution. Abs against the active form of casp-6 and casp-8 (New England Biolabs) were used at a 1:2,000 dilution. The membranes were washed and incubated with a 1:1,000 dilution of alkaline phosphatase-conjugated IgGs (Sigma) for 2 h at room temperature. After washing, proteins were visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma).

Determination of Caspase Activity. BL41-E95-A cells (5 × 106) were resuspended in 100 μl of ice-cold lysis buffer {10 mM Hepes, pH 7.4/0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)/0.1% Triton X-100/0.1 mM EDTA/1 mM DTT/1 mM PMSF/1 μg/ml leupeptin}. After 30 min on ice, the lysates were centrifuged (10,000 × g, 10 min) and 30 μl of the supernatants was added to 195 μl of assay buffer (50 mM Hepes, pH 7.4/100 mM NaCl/0.1% CHAPS/1 mM EDTA/10 mM DTT/10% glycerol). The reaction was started at room temperature by addition of 200 nM (final concentration) of the colorimetric substrate peptides Ac-VEID-p-nitroaniline (pNA) (Ac-Val-Glu-Ile-Asp-pNA; for casp-6) and Ac-IETD-pNA (Ac-Ile-Glu-Thr-Asp-pNA; for casp-8) (Calbiochem). The absorption at 405 nm was measured every 2 min within a total period of 2 h.

In Vitro Cleavage of 5-LO by Caspases. Purified recombinant 5-LO (1 μg) was incubated with the indicated amounts of purified recombinant casp-6 (Sigma) or casp-8 (Upstate Biotechnology, Lake Placid, NY) in assay buffer {50 mM Hepes, pH 7.4/100 mM NaCl/0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)/1 mM EDTA/10 mM DTT/10% glycerol} in a final volume of 100 μl. The samples were incubated at 37°C, and aliquots (10 μl) were assayed for 5-LO protein after 3 and 16 h by WB.

Site-Directed Mutagenesis, Expression, and Purification of 5-LO Proteins. The codons for selected Asp residues in the plasmid pT3–5LO were mutated by using the QuikChange kit from Stratagene as described (20). The mutated DNA was confirmed by using the ABI PRISM dye terminator cycle-sequencing ready-reaction kit (PerkinElmer), followed by analysis on a PRISM 377 sequencer (Applied Biosystems). Escherichia coli MV1190 was transformed with mutated and WT DNA, and recombinant 5-LO proteins were expressed at 27°C and purified as described (20).

Construction of a 5-LO Homology Model. A homology model of human 5-LO was constructed from the x-ray structure of 15-LO (PDB ID code 1lox). According to the work of Hemak et al. (21), the amino acid sequence of human 5-LO (SwissProt accession no. P09917) was aligned to rabbit 15-LO, human 15-LO, mouse 5-LO, rat 5-LO, and hamster 5-LO (SwissProt accession nos. P12530, P16050, P48999, P12527, and P51399, respectively) by using the multiple alignment tool of moe (version 2004.03; Chemical Computing Group, Montreal) (gap-open penalty, 12; gap-extension penalty: 2; blosum62; otherwise, default options). The alignment flanking the casp-6 recognition site was adjusted manually so that Lys-158 of human 5-LO and Lys-157 of rabbit 15-LO matched (the original alignment showed a shift of the 5-LO sequence by one residue). Then, side chains were energy-minimized by using the amber94 force field. The resulting 5-LO model was analyzed by what if (June 2004) indicating no clashes. Final relaxation of residues 155–195 was performed (amber94, moe default options).

Statistics. The program prism (version 3.0; GraphPad, San Diego) was used for statistical comparisons. Statistical evaluations of the data were performed by using Student's t test for unpaired observations. P < 0.05 was considered significant.

Results

5-LO in BL41-E-95-A Cells Is Down-Regulated by Cell Splitting. BL41-E95-A cells, grown to 1.5–2 × 106 cells per ml within 4 days, were diluted into fresh medium to 0.2 × 106 cells per ml. Aliquots of the cell suspension were assayed for 5-LO activity in cell homogenates after various time periods. 5-LO activity was already impaired significantly at 12 h after cell splitting, with minimal levels after 24–48 h, but it then started to recover and was restored at days 4–5 (Fig. 1A). Subsequently, 5-LO activity further increased up to day 7 but again declined until day 10.

Fig. 1.
5-LO in BL41-E95-A cells is down-regulated by splitting. BL41-E95-A cells, grown to 1.5–2 × 106 cells per ml within 4 days, were diluted into fresh medium to 0.2 × 106 cells per ml and harvested after the indicated times. The data ...

Next, 5-LO protein and mRNA levels were assessed. In all WB, total cell lysates corresponding to the same cell number (0.8 × 106) were applied. Cell size did not vary appreciably during different culture conditions; the samples contained 15–20 μg of protein. Splitting of BL41-E95-A cells to 0.2 × 106 cells per ml into fresh medium caused a reduction of 5-LO protein (Fig. 1B). Interestingly, low 5-LO protein levels were accompanied by the appearance of a 62-kDa immunoreactive 5-LO fragment. Another immunoreactive band (≈45 kDa) was consistently detected in samples from various cells and cell types and did not correlate to 5-LO expression. In parallel, within 14 h, 5-LO mRNA decreased to ≈30% vs. parental cells (grown for 4 days) and remained low (40%) in cells grown for up to 48 h (Fig. 1C).

Splitting of cells into fresh medium is accompanied by two major changes in the culture conditions: reduction of the cell density and alterations of the composition of the culture medium. Cells grown to 1.5–2 × 106 cells per ml within 4 days were left untreated (Fig. 2, I), diluted into the same medium (Fig. 2, II), transferred into fresh medium without dilution (Fig. 2, III), or diluted into fresh medium (Fig. 2, IV). After 36 h, 5-LO protein and activity in cell homogenates were assessed. In contrast to cells diluted into fresh medium (Fig. 2, IV), no significant alterations in 5-LO activity and protein levels were determined for cells that were diluted in the same medium (Fig. 2, II) or transferred into fresh medium without dilution (Fig. 2, III). Thus, reduction of the cell density and alterations of the composition of the growth medium are required for down-regulation of 5-LO. These changes in the growth conditions may be regarded as cellular stress, which requires the induction of adaptive mechanisms of the cell. Interestingly, down-regulation of 5-LO was connected to cell proliferation, whereas cell numbers remained the same under the conditions shown in Fig. 2, I–III, in which 5-LO protein levels were unchanged (data not shown).

Fig. 2.
Transfer to fresh medium and dilution of the cell density is required for down-regulation of 5-LO. BL41-E95-A cells, grown to 1.5–2 × 106 cells per ml within 4 days in regular growth medium, were left untreated (I), diluted into the same ...

De Novo Protein Synthesis Is Required for Degradation of 5-LO by Cell Splitting. To test whether cell-splitting-induced suppression of 5-LO requires de novo protein synthesis, BL41-E95-A cells, grown for 4 days, were split into fresh medium or left untreated and cultured in the presence or absence of the protein-synthesis inhibitor cycloheximide (CHX; 50 μM). After 24 h, 5-LO activity and 5-LO protein levels were determined. Whereas CHX had no significant effects on 5-LO in cells cultured in the same medium for 5 days, it almost completely prevented the suppression of 5-LO activity or protein levels in split cells (Fig. 3). Note that CHX treatment also abolished the appearance of the 62-kDa 5-LO fragment. In contrast, CHX did not restore the impaired levels of 5-LO mRNA of split cells (data not shown), implying that suppressed mRNA levels are not primarily responsible for cell-splitting-induced loss of 5-LO protein. Thus, the consistent 5-LO protein level caused by CHX is not related to increased de novo 5-LO expression. In contrast, CHX apparently prevents the synthesis of a protein that is involved in the degradation of 5-LO.

Fig. 3.
Effects of CHX on 5-LO activity and protein levels. BL41-E95-A cells, which were grown for 4 days to 1.5–2 × 106 cells per ml, were split into fresh medium to 0.2 × 106 cells per ml or left untreated. CHX (50 μM) was added ...

Casp-6 and Casp-8 Are Activated in BL41-E95-A Cells by Cell Splitting. It seemed to be reasonable that 5-LO protein could be degraded by caspases. In BL41-E95-A cells that were split into fresh medium and grown for 24 h, enzymatic activity of casp-8, as well as processing to its active fragment, were considerably increased as compared with cells that were grown continuously in the same medium for 5 days (Fig. 4A). Also, the activity of the downstream casp-6 was enhanced, and CHX prevented activation of both caspases. Activation of casp-6 and casp-8 correlated with the down-regulation of 5-LO and the appearance of the 62-kDa 5-LO fragment. As observed for 5-LO degradation, splitting-induced caspase activation required both reduction of the cell density and fresh growth medium (data not shown). Analysis of the time course revealed a significant correlation between the activation of casp-6 and casp-8 and 5-LO degradation (Fig. 4B). In contrast, activation of casp-3 and casp-9 was not detectable (data not shown). BL41-E95-A cells started to proliferate 24–36 h after splitting, thus after caspase activation and 5-LO degradation (Fig. 4C). Note that treatment of BL41-E95-A cells with typical proapoptotic stimuli [30 μM cis-platinum(II)-diamine dichloride, 5 or 50 ng/ml TNF-α, 2 ng/ml TGF-β, 3 μM daunomycine, or 1 μM staurosporine], which has been reported to activate casp-8 in other cell types, caused neither casp-8 activation nor 5-LO degradation; also, in agreement with other studies (22), it failed to induce apoptosis of BL41-E95-A cells (data not shown). Also, splitting of other cell lines (MM6 and RBL-1) and cultivation of primary PMNL from human peripheral blood for 36 h caused no activation of casp-6 or casp-8, and 5-LO cleavage was not detected (data not shown).

Fig. 4.
Cell splitting induces activation of casp-6 and casp-8. Effects of CHX are shown. (A) BL41-E95-A cells, grown to 1.5–2 × 106 cells per ml within 4 days, were split into fresh medium in the presence or absence of 50 μM CHX, or left ...

Inhibition of Casp-6 or Casp-8 Prevents 5-LO Cleavage and Reduced Cell Growth. Next, studies were conducted by using VEID-CHO and IETD-CHO (Calbiochem), which are cell-permeable inhibitors of casp-6 and casp-8, respectively. Both inhibitors, at reasonable concentrations (10 and 6 μM, respectively), prevented the loss of 5-LO and the appearance of the 62-kDa 5-LO fragment in BL41-E95-A cells that had been split and grown for 48 h (Fig. 5A). In accordance with ref. 13, these caspase inhibitors also strongly reduced cell proliferation in a concentration-dependent manner (Fig. 5B).

Fig. 5.
Effects of caspase inhibitors on 5-LO cleavage and proliferation of BL41-E95-A cells. (A) BL41-E95-A cells, grown to 1.5–2 × 106 cells per ml within 4 days, were split into fresh medium and grown for 48 h in the presence or absence of ...

5-LO Is Cleaved by Casp-6 at Asp-170 in Vitro. Isolated human recombinant 5-LO was incubated with casp-6 or casp-8. 5-LO protein integrity was assessed after 3 and 16 h by WB. No cleavage of 5-LO by casp-8 was detectable (Fig. 6A), although a synthetic peptide containing the casp-8 cleavage site IETD was rapidly converted under the same assay conditions (data not shown). However, incubation of 5-LO with casp-6 led to a 5-LO fragment with an apparent molecular mass of ≈58 kDa (Fig. 6A). These concentration-dependent effects of casp-6 (Fig. 6B) were prevented by the casp-6 inhibitor VEID-CHO (1 μM; data not shown).

Fig. 6.
5-LO is cleaved by casp-6 in vitro. (A) Purified 5-LO protein (1 μg) was incubated with 0.3 units of either isolated recombinant casp-6 or casp-8 as described. After 16 h, aliquots were analyzed for full-length (78 kDa) and cleaved (58 kDa) 5-LO ...

The preferred motif within substrates for casp-6 is the tetrapeptide VEID (23, 24), where the Asp is absolutely essential for substrate recognition (8). Thus, mutation of Asp to Ala prevents cleavage. Putative consensus tetrapeptides within 5-LO were selected based on (i) the presence of an Asp within the primary sequence, (ii) the calculated mass of the resulting cleavage fragment, and (iii) the surface exposition of the Asp and the accessibility for the caspase (depicted from a computational model). Accordingly, the Asp residues within the following tetrapeptides were mutated to Ala: LARD121 (63,855 Da), ARDD122 (63,739 Da), LSID156 (59,805 Da), LPRD166 (58,649 Da), IQFD170 (58,187 Da), KGVD176 (57,493 Da), DFAD206 (54,022 Da), EVVD490 (56,682 Da), YEGD496 (57,376 Da), and VEED502 (58,071 Da) (Fig. 6C). Among these mutant 5-LO proteins, D170A-5-LO was resistant to cleavage by casp-6, whereas all other 5-LO proteins were cleaved (Fig. 6C). Thus, casp-6 seemingly recognizes the consensus tetrapeptide IQFD170 in 5-LO as a cleavage site. In control experiments, the synthetic peptide Ac-IQFD-pNA was processed by casp-6, whereas Ac-IQFA-pNA was not a substrate (data not shown).

Last, a homology-based model of the 3D structure of 5-LO was calculated based on the x-ray structure template of rabbit 15-LO (Fig. 7). In this model, the Asp-170 of the putative cleavage site is located on the surface of the protein in the direct neighborhood of the C2-like domain. It seems to be freely accessible to proteolytic attack by casp-6. Interestingly, cleavage of the putative fragment leads to separation of the N-terminal C2-like domain from the 5-LO catalytic domain. A similar 5-LO model was published recently (25, 26), substantiating these findings.

Fig. 7.
Homology-based model of the 3D structure of 5-LO. (A) Model of 5-LO constructed from an x-ray structure of 15-LO (Protein Data Bank ID code 1lox). (B) Close-up model of the putative casp-6 cleavage site region in 5-LO, viewed along the direction of the ...

Discussion

BL41-E95-A cells constitutively express 5-LO and, in comparison with other B cell lines, exhibit high 5-LO protein levels (15). We show that cell splitting strongly reduces the capacity of BL41-E95-A cell homogenates to produce 5-LO products, which could be clearly connected to the loss of functional 5-LO protein. Interestingly, the loss of full-length 5-LO protein was accompanied by the appearance of a 62-kDa 5-LO fragment, which, strongly correlated to the activation of casp-6 and casp-8. Previously, pulse–chase studies determined the t1/2 of 5-LO protein in differentiated HL60 cells at 26 h (7). In BL41-E95-A cells grown for 4 days, inhibition of de novo protein biosynthesis by CHX prevented reduction of functional 5-LO protein levels within 24 h, indicating that in BL41-E95-A cells, 5-LO protein stability per se is much higher (t1/2 > 26 h) as compared with HL60 cells. Because splitting of BL41-E95-A cells led to a rapid loss of functional 5-LO protein (80% within 24 h), we conclude that cell splitting activates an efficient 5-LO degradation machinery. Accordingly, increased degradation of the 5-LO protein pool, and not reduced biosynthesis, appears to be the major reason for the reduction of 5-LO protein, although cell split also resulted in reduction of 5-LO mRNA. If reduced biosynthesis would be the major reason, CHX would be expected to reduce 5-LO protein content to a similar extent as cell split, which was not the case. Instead, both the loss of 5-LO protein and the appearance of the 62-kDa 5-LO fragment was prevented by CHX, indicating that de novo protein biosynthesis is required for 5-LO degradation. CHX also prevented processing and activation of casp-6 and casp-8, implying that the de novo protein initiates caspase activation, which then cleaves 5-LO. The nature of this protein(s) and the detailed mechanisms are not known.

Note that splitting of other 5-LO-positive cell types (e.g., human PMNL, MM6, or RBL-1 cells) caused neither degradation of 5-LO nor activation of casp-6 or casp-8. It is known that certain protein cleavages by caspases can be cell-type specific, probably because of variations in the occurrence of individual caspases (9). For example, β-actin is cleaved by caspases in PC12 and ovarian carcinoma cells but not in other cell types (27). Accordingly, 5-LO protein metabolism by caspases may be unique for BL41-E95-A cells or B lymphocytes.

Although recruitment of caspases is generally considered as an initiation step in apoptotic cell death (8), activation of casp-6 and casp-8 in BL41-E95-A cells was not associated with apoptosis but, instead, was connected to subsequent cell proliferation. BL41-E95-A cells were originally obtained by in vitro infection of the Burkitt's lymphoma line BL41 with the B95-8 Epstein–Barr virus (EBV) strain (14), which confers Burkitt's lymphoma cells a malignant phenotype and resistance to apoptosis, because of EBV-encoded small RNAs, an inactive casp-3 pathway, and elevated bcl-2 expression (22, 28, 29). In view of the lack of casp-3 activity, it is unclear how casp-6 becomes activated in EBV-transformed Blymphocytes (13). Of interest, typical proapoptotic agents (e.g., staurosporine, daunomycine, cis-platin, or TNF-α) failed to reduce proliferation of BL41-E95-A cells and did not activate casp-6 or casp-8. However, inhibitors of casp-6 and casp-8 strongly reduced cell-splitting-induced proliferation of BL41-E95-A cells. This result concurs with the finding that in dense human tonsillar B cells, casp-6 and casp-8, but not casp-3, are activated by proliferative stimuli, which is required for entry into the cell cycle (13). Also, deletion of casp-6 in a chicken cell line reveals the importance of casp-6 for B cell proliferation and/or cell survival (30), and for normal human T lymphocytes, a positive role for caspases during proliferation could be demonstrated (31). Thus, besides executing initial steps in apoptosis in most cell types, caspases may have a unique role in lymphocyte activation and proliferation.

There are >280 caspase targets that have been identified, most of which are cleaved by casp-3, and there are strong differences in the efficacy of substrate cleavage (9). Caspases hydrolyze peptide bonds on the carboxyl side of an Asp, accomplished by recognizing a consensus tetrapeptide (P4 to P1, where P1 is the Asp) of the substrate. The requirement for Asp is absolute for hydrolysis by all caspases, whereas the P2 and P3 residues have a rather limited effect. The P4 residue appears to account for the substrate specificity. The optimal peptide sequence for casp-6 is VEID, which is present for example in lamin A (23), but also other motifs are recognized, such as VEVD in lamin B1 (32) and VEMD in SATB1 (33). In our study, site-directed mutagenesis revealed IQFD170 as a recognition motif in 5-LO for casp-6, at least in vitro. Importantly, in the predicted secondary structure of 5-LO, IQFD is on the periphery of the protein that may allow free access for casp-6. Intriguingly, a discrepancy in the molecular mass of the 5-LO fragment found in BL41-E95-A cells (≈62 kDa) and the fragment obtained after processing by casp-6 in vitro (≈58 kDa) was apparent. In intact cells, the 5-LO cleavage product is possibly further modified, for example, by acylation, ubiquitination, glycosylation, phosphorylation (34, 35), etc., which could alter its weight and/or its electrophoretic mobility. Studies are needed to elucidate possible posttranslational modifications.

Caspases may have an important role in the metabolism of 5-LO protein in B lymphocytic cells. The recent finding that 5-LO antagonized genotoxic stress-induced apoptosis (36) is of interest. 5-LO products have many functions, such as control of cell survival and proliferation, host defense, immune reactions, onset and propagation of inflammation, and functions in cardiovascular pathophysiology. Whether regulation of 5-LO by caspases has a discrete function related to these (patho)physiological events in B lymphocytes, and possibly also in other cell types, remains to be elucidated.

Acknowledgments

This study was supported by European Union Grant QLRT-2000-01521 (to LEUCHRON), European Union Grant LSHM-CT-2004-0050333 (to EICOSANOX), and the Beilstein-Institut zur Förderung der Chemischen Wissenschaften.

Notes

Author contributions: O.W., I.T., B.S., and D.S. designed research; O.W., I.T., A.M., A.U.-L., M.H., L.F., and G.S. performed research; O.R. contributed new reagents/analytic tools; O.W. and I.T. analyzed data; O.W., O.R., and D.S. wrote the paper; and I.T. performed most of the experiments.

Abbreviations: 5-LO, 5-lipoxygenase; casp-n, caspase-n; CHX, cycloheximide; LT, leukotriene; WB, Western blot analysis; pNA, p-nitroaniline; BL, Burkitt's lymphoma.

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