Cytosolic pro-apoptotic SPIKE induces mitochondrial apoptosis in cancer

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Biochemical and Biophysical Research Communications 395 (2010) 225–231

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Cytosolic pro-apoptotic SPIKE induces mitochondrial apoptosis in cancer Ivana Nikolic a,*, Tatjana Kastratovic b, Ivanka Zelen a, Aleksandar Zivanovic b, Slobodan Arsenijevic b, Marina Mitrovic a a b

Department of Biochemistry, Medical Faculty, University of Kragujevac, Kragujevac, Serbia Department of Gynecology and Obstetrics, Medical Faculty, University of Kragujevac, Kragujevac, Serbia

a r t i c l e

i n f o

Article history: Received 24 March 2010 Available online 31 March 2010 Keywords: Apoptosis SPIKE Cytochrome c Caspase 3 BCL-2 family

a b s t r a c t Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic ‘‘BH3-only” BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare proapoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase’s downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction For any healthy organism, an apoptotic cell death is required to maintain tissue homeostasis. In pathogenesis, apoptosis contributes to diseases including cancer, neurodegenerative disorders, autoimmune diseases, and viral infection [7]. Proteins of the BCL2 family are central regulators of apoptosis. Members of BCL-2 family possess either anti-apoptotic or pro-apoptotic function. They are characterized by the presence of conserved motifs, known as BCL-2 homology [BH] domains, and according to these domains, they are divided into three subgroups [1]. The BCL-2 family of proteins consists anti-apoptotic molecules that share all four BH domains, called BH1–4, and include BCL-2, BCL-XL, MCL-1, BCL-w, BFL-1 in mammals, and Ced-9 in Caenorhabditis elegans. The proapoptotic molecules include BAX, BAK, and BOK in mammals that contain BH1–3 domains and compete with each other by dimerization. Of note, interestingly, there is no homologue of multi-domain pro-apoptotic BCL-2 member yet identified in C. elegans. BH3-only pro-apoptotic BCL-2 proteins, following the activation of apoptosis form heterodimers with both anti-apoptotic and multi-domain pro-apoptotic members [15]. The BH3 domains of pro-apoptotic proteins are critical for their killing and heterodimerization func-

* Corresponding author. Address: Department of Biochemistry, Medical Faculty of Kragujevac, Svetozara Markovica 69, 34 000 Kragujevac, Serbia. Fax: +381 34306800x112. E-mail address: [email protected] (I. Nikolic). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.168

tion [13]. In fact, BH3 domain of pro-apoptotic members is sufficient for mediating killing, which is supported by identification of BH3 only BCL-2 family members, including BID, BIK, BAD, BIM, HRK, BMF, PUMA, NOXA, BNIP3 and SPIKE in mammals, and Egl1 in C. elegans [13,25,26,36]. Anti-apoptotic BCL-2 members interact with exposed BH3 domain of pro-apoptotic members resulting in inhibition of their pro-apoptotic activity. Besides to the wellknown three groups of the Bcl-2 subfamilies, some Bcl-2 members demonstrate the uncommon characteristics to both the pro-apoptotic and the anti-apoptotic Bcl-2 molecules. Bcl-GL is classified as weak pro-apoptotic Bcl-2 member that is distinguished from the other pro-apoptotic molecules because it contains both BH2 and BH3 domains, but it lacks BH1 domain [12]. On the contrary, Bclrambo clearly demonstrates all pro-apoptotic features even though it contains all four BH domains that are characteristic for the antiapoptotic Bcl-2 family members [14]. Furthermore, Bfk is weakly pro-apoptotic protein containing only BH3 and BH2 domains similarly to Bcl-GL [6]. Moreover, both Bfk and Bcl-GL weakly induce apoptosis localized mainly in the cytosol. Many different methods have been used to identify the new members of BCL-2 family. We utilized the method called Position Weight Matrix analysis [35] to identify the new BCL-2 family members by bioinformatics (Fig. 1). By using consensus sequences of the known BCL-2 members, we searched for the new BCL-2 proteins, including the earlier identified BH3-only protein SPIKE. In this search, SPIKE scored high for the BH3-only BCL-2 family member, compared to the known BH3-only proteins including BAD, HRK, PUMA, BIM, BIK, BMF,


I. Nikolic et al. / Biochemical and Biophysical Research Communications 395 (2010) 225–231

Fig. 1. Position Weight Matrix search results. In the search and identification of new BCL-2 proteins, SPIKE (highlighted in the red box) scored high for a new predicted BH3only protein compared to other known BH3-only proteins. The Name column represents the abbreviations of the names found at the NCBI protein database. The columns labeled gen, only, anti, and multi represent the scores obtained by construction of the matrix using all known Bcl2-related proteins, BH3-only proteins, anti-apoptotic proteins and multi pro-apoptotic proteins, respectively. The column Cons shows the scores calculated using alignment positions 1, 5, 8, 9, 10, 11, 12, and 15.

BID, and the multi-domain pro-apoptotic protein BAX. Previously, SPIKE was identified as a new BH3-only BCL-2 family member proposed to be recruited at the ER [26]. Its primary sequence is conserved from C. elegans to human and its expression is downregulated in certain tumors, such as kidney, breast and lung. Mund et al. demonstrated that overexpressed SPIKE interacted with ectopically expressed BAP31 and prevented the association between BAP31 and its interacting partner BCL-XL. The endoplasmic reticulum (ER), in addition to mitochondrial apoptotic pathway, has also been implicated as an organelle that regulates apoptosis [23,24]. BAP31 forms apoptosome-like complex at the ER that also includes pro-caspase-8L and BCL-XL [3,28]. Following induction of apoptosis by the FAS ligand, BAP31 is cleaved by caspase-8 resulting in the membrane p20 cleaved product that further contributes to the execution of apoptosis. In view of fact that SPIKE was predicted as the new BH3-only protein and the potential interacting partner of BAP31, we further characterized SPIKE and its relation to BAP31 and its potential involvement in the regulation of apoptosis. Here, we applied the Position Weight Matrix method to identified SPIKE to be a BH3only pro-apoptotic protein mainly localized in the cytosol, contrary to the previous report [26]. Enforced expression of SPIKE weakly induced apoptosis by promoting the mitochondrial cytochrome c release, the activation of caspase 3 and the caspase cleavage of both BAP31 and p130CAS. 2. Materials and methods

TOM20 (sc-11415; Santa Cruz Biotechnology); rabbit anti-calnexin (gift from Dr. Bergeron); rabbit or chicken anti-human BAP31 [31] anti-HA monoclonal antibody 12CA5 (Babco, Richmond, Calif.); anti-Flag M2 monoclonal antibody (Sigma); mouse anti-p130CAS (BD Transduction Laboratories); mouse anti-cytochrome c (Pharmingen); rabbit immunoglobulin G-horseradish peroxidases conjugate (Cappel, ICN Pharmaceuticals); zVAD-fmk (Enzyme System Products). 2.2. DNA construct and transfection cDNA encoding human SPIKE, containing the FLAG peptide epitope, was incorporated into pcDNA3.1 expression vector by PCR. The authenticity of SPIKE-Flag expression vector was confirmed by DNA sequencing. All transfections were performed using LipofectAMINETM Plus (Invitrogen). 2.3. Fluorescence microscopy Cells were seeded at 60% confluency on glass coverslips and 24 h later washed in PBS and fixed in 4% paraformaldehyde, 23 mM NaH2PO4, and 77 mM Na2HPO4, pH 7.3. Cells were permeabilized in PBS/0.2% Triton X-100, and incubated in blocking buffer (10% FCS, 0.1% Triton X-100 in PBS). Primary and secondary antibodies-anti-mouse IgG coupled to either red or green Alexa’s (Molecular Probes, Inc.) were incubated for 1 h at RT. Cells were visualized by fluorescence microscopy using an inverted microscope (TE-FM Epi-fl; Nikon) [31].

2.1. Cell cultures, reagents, and antibodies Human 293T embryonic kidney, H1299 lung carcinoma, HeLa cervical carcinoma, and KB oral epithelial carcinoma either stable expressing or not ectopic HA-BCL-2 (B23) cells were cultured in alpha-MEM medium supplement with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in humidified 5% CO2. The used antibodies: rabbit anti-SPIKE was generated in our laboratory against a recombinant GST-SPIKE fusion protein and absorbed on a GST column; mouse anti-actin (ICN Biomedical); rabbit anti-

2.4. Subcellular fractionation, cytochrome c release, and apoptotic assay For both subcellular fractionation and cytochrome c release, cells (4  106) were washed in PBS and suspended in 0.1 ml of 200 mN mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM HEPES, pH 7.5. After one cycle of freeze and thaw, the cells were homogenized at 2000 rpm, and centrifuged at 1000g for 10 min to remove nuclei and cell debris. The supernatant was centrifuged at 10,000g

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to get pellet (HM) containing mainly mitochondria and supernatant was centrifuged at 100,000g for 10 min to get pellet (LM) containing mainly ER and supernatant (S100) is a mainly cytosol. All fractions were analysed by immunoblotting. DEVD-amc caspase activity (Upstate Biothechnology, Lake Placid, NY) was conducted according to the manufacturer’s protocols.

of the caspase cleavage of BAP31 compared to BIK transfected cells. Therefore, SPIKE seemed to be a weaker pro-apoptotic protein than the known BH3-only pro-apoptotic protein, BIK.

2.5. Immunoblotting

Cytochrome c release from the mitochondria is a decisive step in apoptosis induction which leads to the assembly of apoptosome, a pro-apoptotic complex of Apaf-1, pro-caspase-9, and cytochrome c, and further execution of death [29]. Next, we investigated the ability of exogenous SPIKE to induce cytochrome c release by performing both the subcellular fractionation and the immunofluorescence microscopy. HeLa cells overexpressing SPIKE, but not control vector, showed diffuse cytosolic staining of cytochrome c indicative of its release from mitochondria to the cytosol. Consistent with immunofluorescence results, cytochrome c is recovered in the post-membrane fraction only when cells overexpressed SPIKE (Fig. 3B and C). Thus, SPIKE is pro-apoptotic protein which acts upstream of mitochondria to induce the egress of cytochrome c from this organelle. BAP31 has been implicated to play a key role in regulating apoptosis as downstream substrate of caspase 8 in FAS mediated apoptosis, whereas p130CAS demonstrated an important role in regulating the etoposide induced death as a direct substrate of caspase 3 [33]. Earlier, SPIKE was identified as a possible interacting partner of BAP31 and a regulator of FAS mediated death [26]. Therefore, we further investigated the possible effect of the overexpressed SPIKE in cancer cells to induce the caspase cleavage of both BAP31 and p130CAS. HeLa cells were transfected either with control or SPIKE-flag expression vectors and 24 h later the expression patterns of BAP31 and p130CAS was analyzed by immunoblotting (Fig. 3D). Our results showed that enforced expression of SPIKE in HeLa cancer cell induced the significant proteolytic cleavage of both p130CAS and BAP31 compared to the vector transfected cells. Following induction of apoptosis by the SPIKE-flag stimulation, BAP31 is proteolytically cleaved resulting in the membrane p20 cleaved product that is known to further contribute to the execution of apoptosis. Thus, the possible role, if any, of the pro-apoptotic SPIKE in the apoptotic pathways regulated by the downstream substrates of caspases, p130CAS and BAP31 is yet to be elucidated.

All cells (4  106) were washed in PBS and suspended in the lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Hepes, 10% glycerol, and 1 mM EDTA) for 1 h at 4 °C and eluted in SDS sample buffer. All samples were analyzed by SDS–polyacrylamide gel electrophoresis, probed with appropriate primary and secondary antibodies and visualized using ECL reagent (Amersham Biosciences). 3. Results 3.1. SPIKE is widely expressed protein with cytosolic localization To asses the expression of the endogenous SPIKE in various cancer cell lines, including 293T, Hela, H1299, KB, and B23, we generated an antibody against SPIKE. Rabbits were immunized with recombinant GST-SPIKE fusion protein and SPIKE specific antibody was purified from their serum by affinity chromatography on a GST-Sepharose column. The specificity of the SPIKE antibody was confirmed by Western blot analysis of lysates from cancer cells against endogenous SPIKE (Fig. 2A). SPIKE was widely expressed protein in all cancer cell lines tested with its predicted molecular size of 31 kDa. Next, we analyzed the subcellular localization of the endogenous SPIKE in cancer cells that expressed relatively abundant amount of endogenous SPIKE, such as 293T, Hela, and H1299 (Fig. 2B). Using the subcellular fractionation, we determined that the endogenous SPIKE was mainly localized in the soluble fraction (S100) containing cytosol with the insignificant amount of SPIKE presented in the light membrane fraction (LM) consisting of ER. This is in contrast to the earlier results that claimed that SPIKE was localized mainly in the ER in MCF-7 cells [26]. The efficacy of fractionation was confirmed by immunoblots with antibodies to mTOM20, which appeared only in the pellet (p) fraction (containing mitochondria), and to calnexin, which was largely recovered in the ER fraction. To confirm our findings, we performed the immunofluorescence microscopy to analyze the localization of both exogenous and endogenous SPIKE. Here, we demonstrated that HeLa cells transfected with either vector or SPIKE-flag, showed the exogenous SPIKE to be distributed throughout the cells and co-localized with cytosolic localization of GFP (Fig. 2C), clearly distinct from the punctuate mitochondrial localization of cytochrome c (Fig. 2C). Moreover, we also confirmed the cytosolic subcellular localization of the endogenous SPIKE in HeLa cells by the immunofluorescence microscopy. Consistent with fractionation results, SPIKE was mainly cytosolic, but not an ER protein as described previously (Fig. 2D).

3.2. SPIKE weakly induces apoptosis and caspase 3 activation in comparison to BH3-only protein BIK Earlier, SPIKE was proposed to be a pro-apoptotic protein [26]. We further investigated SPIKE’s killing ability in our system and compared it to the pro-apoptotic ability of known BH3-only BCL2 member, BIK. Thus, HeLa cells were transfected with either vector, SPIKE-FLAG or BIK-HA, and both caspase-3 activation and the caspase cleavage of BAP31 were assessed as markers of cell death (Fig. 3A). SPIKE’s transfected cells showed reduced but significant level of the caspase 3 activity that corresponded to the lower level

3.3. SPIKE induces cytochrome c release, and caspase cleavage of BAP31 and p130CAS

4. Discussion Until now, it has been well characterized that BH3-only proteins connect proximal death signals to the core mitochondrial pathway [13]. Importantly, the death activities of BH3-only proteins are regulated by both distinct apoptotic signals and mechanisms. Healthy cells apply different transcriptional and posttranslational mechanisms to regulate pro-apoptotic activity of BH3-only proteins to prevent inappropriate cell death. Although, there is now a strong support that BH3-only proteins, such as BIK, can stimulate apoptosis from the ER [23,24] SPIKE, as majority of BH3-only proteins, is located in the cytosol prior to apoptosis. In response to specific apoptotic signals, still unidentified for SPIKE, BH3-only proteins are regulated by a diverse array of mechanisms and targeted to specific organelle, such as ER or mitochondria, for apoptotic execution. For example, the genes of BIK, PUMA, NOXA, and HRK/DP5, BH3-only proteins, are transcriptional regulated by p53 during E1A oncogenic, and DNA damage-induced apoptosis, serum and nerve growth factor withdrawal, as well as treatments with tunicamycin, and thapsigargin [21,23,24,27]. Furthermore, in healthy cells, other BH3-only proteins, including BAD, BID, BIM-EL, BIM-L, and BMF are kept inactive in the cytosol and they are post-translational modified in response to their specific


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Fig. 2. Cellular expression and cytosolic distribution of SPIKE. (A) Equivalent samples of cell lysates from 293T, H1299, HeLa, KB, and B23 were analyzed by immunoblotting with rabbit anti-human SPIKE antibody and equal loading confirmed by anti-b-actin. (B) Cellular localization of endogenous SPIKE detected by subcellular fractionation of intact 293T, H1299, and HeLa cells using rabbit anti-human SPIKE, and proper fractionation confirmed by rabbit anti-TOM20, and rabbit anti-calnexin antibodies. (C) Cellular localization of transfected SPIKE-flag analyzed by fluorescence microscopy of HeLa cells and stained with anti-Flag M2 monoclonal antibody. Cytosolic localization of SPIKE was compared to cytosolic distribution of GFP and mitochondrial distribution of cytochrome c. (D) Cellular distribution of endogenous SPIKE detected by immunofluorescence of intact HeLa cells using rabbit anti-human SPIKE and confirmed by GFP and anti-cytochrome c.

apoptotic stimuli to target mitochondria to induce apoptosis. For example, inactive and cytosolic BAD is dephosphorylated in response to growth factor removal and translocated to mitochondria to interact with anti-apoptotic BCL-XL [34]. In healthy cells, BIMEL, BIM-L, and BMF, are maintained in inactive conformation by cytoskeletal proteins and upon serum removal or loss in cell attachment to extracellular matrix, they are unleashed to target mitochondria and bind pro-survival BCL-2 proteins [35]. BID is kept inactive in the cytosol by intramolecular interaction between its C-terminus and the N-terminal-located BH3 domain [30] and FAS – death receptor stimulation induce the activation of cas-

pase-8 that cleaves and converts inactive BID into active t-BID [19]. Although we have not yet tested the possibility of sequestration of SPIKE by cytoskeletal proteins, overexpression of SPIKE in our system induced significant caspase cleavage of p130CAS, a well-known focal adhesion molecule involved in etoposide, and nocodazole induced cell suicides [16,31]. Therefore, it is yet to be determined if SPIKE would play a role in etoposide, and nocodazole stimulated death pathways. However, we still can not exclude other proteases, calpain, or c-Jun N-terminal kinases that have been reported to cleave and activate the pro-apoptotic proteins upon specific apoptotic stimulus [8,10]. Unlike BIK, BIM, BMF,

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Fig. 3. SPIKE is a ‘‘weak” pro-apoptotic protein that induces cytochrome c. (A) Killing ability of transfected SPIKE-flag were compared to those of transfected BIK-HA in equal amount of cell extracts from HeLa cells by immunoblots stained with anti-BAP31 and anti-p130CAS antibodies. Transfection of proteins and equal loading were confirmed by anti-Flag M2, anti-HA 12CA5, and anti-b-actin antibodies, respectively. Caspase 3 activity was determined by incubating cell lysates with caspase 3 substrate, DEVDaminomethylcoumarin. (B,C) Release of cytochrome c by exogenous SPIKE was detected in Hela cells transfected either by vector alone or SPIKE-flag, both by subcellular fractionation and immunofluorescence using anti-Flag M2 and anti-cytochrome c antibodies. Vector alone or SPIKE-flag overexpressed cells that showed diffuse cytochrome c staining were scored and expressed as % of total cells number. (D) The effect of transfected SPIKE-flag into HeLa cells on the induction of the caspase cleavage of the downstream substrates of caspases, BAP31, and p130CAS, detected by immunoblotting of total cell lysates using the antibody against BAP31 and p130CAS. Transfection and equal loading were confirmed by anti-Flag M2, anti-SPIKE, and anti-b actin antibodies, respectively.

and HRK, SPIKE does not contain C-terminal transmembrane domain to facilitate interaction with intracellular membranes, simi-

larly to BID, BAD, NOXA, and PUMA that required some form of cellular modification to target specific organelle. BAX is also pre-


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dominantly cytosolic in healthy cells and translocates to mitochondria after death signal stimulation [37]. On the other hand, the proapoptotic Bcl-GL and Bfk, similarly as SPIKE, induce apoptosis without prior delocalization from the cytosol to the specific organelle [12]. Therefore, there are other pro-apoptotic proteins, including SPIKE, for which is not yet known how its apoptotic activities are regulated and which apoptotic signals it senses. SPIKE might have very distinct modes of apoptotic regulation than other BH3-only proteins in response to specific death stimulus. It is very important for the given cell to have a very distinct regulation of different BH3-only proteins in response to different conditions of apoptotic stress. Also, different cell types may require different BH3-only proteins to activate apoptosis. What are the apoptotic stresses that regulate BH3-only protein SPIKE to influence specific apoptotic pathway is left to be elucidated. Different BCL-2 members might play tissue-specific roles in the regulation of apoptosis. Although SPIKE is widely expressed in many tissues, it is highly downregulated in kidney and lung tumors [26]. However, in this report we mainly focused on the analysis of SPIKE apoptotic activity in HeLa cells generated from cervical cancer tissue. Until now, many BCL-2 proteins were identified in the lymphoid system, in which no detectible expression of SPIKE was observed [26]. Therefore, using different identification methods, such as yeast two-hybrid, novel BCL-2 homologues could be isolated that might be essential for apoptosis regulation in different tissue, including kidney and lung. Importantly, SPIKE is highly conserved through evolution. Besides Egl-1 [5] and ceBNip3 [25], SPIKE is so far only third known to be described BH3-only protein with a homologue in C. elegans. Thus, further studies in C. elegans would help us understand to role of SPIKE in apoptosis. Moreover, it is important to note that BH3-only proteins do not only function in apoptosis regulation, but they also play a significant role in the regulation of another critical pathway involved in cellular and tissue homeostasis called autophagy, a catabolic process characterized by the degradation of intracellular components via the lysosome [17]. Recently, a novel BH3-only protein Beclin-1 was identified to interact efficiently with the anti-apoptotic proteins including Bcl-2 and Bcl-XL at the ER, that failed to induce apoptosis, but rather this interaction is essential for Bcl-2 mediated inhibition of starvation-induced autophagy [2,4,9,22,32]. In addition, the other BH3-only proteins known to play a key role in apoptosis, such as BIM, BNIP3, PUMA, NOXA, BIK, BAD, also function through their BH3-only domains as important regulators of autophagy [11,18,20,38]. Therefore, it would be interesting to investigate if BH3-only SPIKE would play a possible role, if any, in regulating the molecular crosstalk between the autophagy and apoptosis. Therefore, SPIKE was identified by the Position Weight Matrix method to be a novel pro-apoptotic BCL-2 member localized in the cytosol and capable of inducing weakly apoptosis by promoting the release of cytochrome c from mitochondria, activating the caspase 3 and subsequently inducing the caspase cleavage of BAP31 and p130CAS. This cytosolic BH3-only protein, whose expression is conserved from C. elegans to mammals and downregulated in kidney, breast and lung tumors, provides a unique model for further studies of apoptotic mechanisms regulated by BCL-2 family proteins. References [1] J.M. Adams, S. Cory, Life-or-death decisions by the Bcl-2 protein family, Trends Biochem. Sci. 26 (2001) 61–66. [2] P. Boya, G. Kroemer, Beclin 1: a BH3-only protein that fails to induce apoptosis, Oncogene 28 (21) (2009) 2125–2127. [3] D.G. Breckenridge, M. Nguyen, S. Kuppig, M. Reth, G.C. Shore, The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum, Proc. Natl. Acad. Sci. USA 99 (2002) 4331–4336.

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