Ceramide phosphoethanolamine synthase SMSr is a target of caspase-6 during apoptotic cell death

Ceramides are essential precursors of sphingolipids with a dual role as mediators of apoptotic cell death. Previous work revealed that the ER-resident ceramide phosphoethanolamine (CPE) synthase SMSr/SAMD8 is a suppressor of ceramide-mediated apoptosis in cultured cells. Anti-apoptotic activity of SMSr requires a catalytically active enzyme but also relies on the enzyme’s N-terminal sterile α-motif or SAM domain. Here, we demonstrate that SMSr itself is a target of the apoptotic machinery. Treatment of cells with staurosporine or the death receptor ligand FasL triggers caspase-mediated cleavage of SMSr at a conserved aspartate located downstream of the enzyme’s SAM domain and upstream of its first membrane span. Taking advantage of reconstitution experiments with SMSr produced in a cell-free expression system, specific caspase-inhibitors and gene silencing approaches, we show that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Huntington’s and Alzheimer’s diseases. Our findings underscore a role of SMSr as negative regulator of ceramide-induced cell death and, in view of a prominent expression of the enzyme in brain, raise questions regarding its potential involvement in neurodegenerative disorders.


DNA constructs
For mammalian expression of C-terminal V5/His6-tagged human SMSr, the corresponding cDNA was PCR amplified and cloned into pcDNA3.1/V5-His TOPO (Invitrogen) according to the manufacturer's instructions. For cell-free expression, the ORF of SMSr was PCR-amplified in-frame with a C-terminal V5 epitope and cloned into the wheat germ pEU-Flexi expression vector (kind gift of Brian G. Fox and James D. Bangs, University of Wisconsin, Madison) [40]. For the establishment of HeLa cell lines stably transduced with SMSr expression constructs, the ORF of SMSr with a C-terminal V5 epitope was cloned into retroviral expression vector pLNCX2 (Clonetech). Single amino acid substitutions were introduced by site-directed mutagenesis using the megaprimer PCR method [41].

Generation of cell lines stably expressing SMSr-V5 and SMSr CR -V5
HeLa cells stably expressing V5-tagged SMSr and SMSr CR were created by retroviral transduction. To this end, low-passage human HEK293T cells (ATCC R CRL-3216 TM ) grown in DMEM supplemented with 10% serum were co-transfected with SMSr-V5/pLNCX2 or SMSr CR -V5/pLNCX2 and packaging vectors using Lipofectamine 2000 (Thermo Fisher). After 48 h, supernatants were harvested, passed through a 0.45 μm filter, and the virus-containing medium was used to transduce HeLa cells. The cells were cultured in DMEM supplemented with 10% serum and 0.8 mg/μl geneticin (G418). Expression of V5-tagged proteins was confirmed by indirect immunofluorescence and Western blotting using V5 antibody. For in vivo inhibition of CASP6-mediated SMSr cleavage, cells were treated with 1.5 μg/ml staurosporine for 6 h in the presence of 20 μM z-VAD-fmk, 4 μM z-VEID-fmk, or DMSO for 6 h. For the time course experiments of staurosporine-and FasL-induced apoptosis, cells were treated with 1μg/ml staurosporine or 50 ng/μl FasL and 18.75 μM cyclohexamide for indicated times. Floating and adherent cells were collected and lysed in lysis buffer. Nuclei were removed by centrifugation at 15000 g for 10 min at 4 • C. Post-nuclear supernatants were subjected to immunoblotting.

siRNA transfection and immunoblotting
RNA interference was performed on HeLa cells stably expressing V5-tagged SMSr using Oligofectamine (Invitrogen) in Opti-MEM I Glutamax medium (Invitrogen) as described previously [39]. Hs CASP6 8 (SI02662450) and Hs CASP3 7 (SI02654603) siRNAs were from QIAGEN. Nonsense (NS) siRNA (QIAGEN) target sequence was: 5 -AAUUCUCCGAACGUGUCACGU-3 . Seventy-two hours after the start of siRNA treatment, cells were treated with staurosporine (1.5 μg/ml) for an additional 6 h. Next, cells were lysed in lysis buffer and nuclei were separated by centrifugation at 15000 g for 10 min at 4 • C. Post-nuclear supernatant samples were mixed with 2× SDS sample buffer (125mM Tris (pH 6.8), 4% SDS, 20% glycerol, 0.01% Bromophenol Blue) and incubated at 95 • C for 5 min. Samples were subjected to SDS/PAGE and proteins were transferred on PVDF membranes. Membranes were incubated with blocking, primary antibody and HRP-conjugated secondary antibody solutions respectively. Images were acquired with BIO-RAD ChemiDoc XRS+ system and quantified using Image Lab 5.0 (BIO-RAD) software. Statistical analysis was performed using MATLAB (MathWorks) software.

Cell-free production and caspase-mediated cleavage of SMSr
Cell-free production of SMSr-V5 in proteoliposomes was described previously [40]. Briefly, wheat germ expression vector SMSr-V5/pEU-Flexi was treated with Proteinase K to remove trace amounts of RNAse, purified by phenol/chloroform extraction, and dissolved at 1 μg/μl in water. To confirm the activity of recombinant human caspases, HeLa cells were lysed in 0.5% CHAPS, 42 mM KCl, 5 mM MgCl 2 , 50 mM HEPES (pH 7.4), 50 mM NaCl, and 10 mM EDTA. Nuclei were removed by centrifugation at 15000 g for 10 min at 4 • C and DTT was added to a final concentration of 10 mM. Twenty microliters of post-nuclear supernatant (corresponding to ∼600,000 cells) was incubated with one unit active recombinant human caspase for 2 h at 37 • C. Reactions were subjected to immunoblot analysis using anti-PARP1 and Lamin A/C antibodies.

Immunofluorescence microscopy
HeLa cells stably transduced with V5-tagged SMSr expression constructs were seeded on glass coverslips. Cells were fixed using 4% (w/v) paraformaldehyde in PBS at RT and quenched using 25 mM NH 4 Cl in PBS. Cells were permeabilized using PBS containing 0.1% (w/v) saponin and 0.2% (w/v) BSA. Coverslips were immunostained using mouse anti-V5, sheep anti-TGN46, rabbit anti-calnexin primary antibodies followed by donkey anti-rabbit Cy5, donkey anti-mouse Cy3, and donkey anti-sheep/goat FITC antibodies. Coverslips were mounted with Prolong Gold Antifade Reagent (Thermo Fisher Scientific). Images were captured using a confocal microscope (Olympus LSM FV1000) equipped with two spectral and a single standard detector, an UPLSAPO 60x/NA 1.35 oil immersion objective (Olympus) and an Olympus laser box with AOTF laser combiner. Fluorophores were excited using 488, 559, and 635 nm lasers. Excitation light was reflected by a 405/488/559/635nm dichroic mirror. Emitted light was collected using secondary dichroic mirrors SDM-560 and SDM-640 and a barrier filter BA 655-755 for FITC, Cy3, and Cy5 respectively. To avoid cross-talk between image channels, images for different fluorophores were collected sequentially in a descending order of wavelength, i.e. longer to shorter wavelengths. Image analysis was performed using ImageJ (NIH) software.

SMSr undergoes caspase-mediated cleavage at Asp120 in staurosporine-treated HeLa cells
To determine whether SMSr is a target of the apoptotic machinery, HeLa cells expressing human SMSr with a C-terminal polyHis and V5 epitope were treated with staurosporine, lysed and then subjected to immunoblot analysis using anti-V5 antibody. Stauroporine-induced proteolytic cleavage of SMSr, yielding a V5-tagged fragment of ∼33 kDa ( Figure 1A and B). Cleavage of SMSr was blocked by incubating cells with the pan-caspase inhibitor z-VAD-fmk, indicating that this process is dependent on caspases. Immunoblot analysis of staurosporine-treated cells expressing SMSr with an N-terminal V5 tag suggested that cleavage occurred at a single site downstream of the N-terminal SAM domain, but upstream of the first predicted membrane span (data not shown). This region contains two predicted    Figure 1E). To test which of these sites is cleaved in staurosporine-treated cells, each of the corresponding aspartates was substituted for an alanine. While substitution of Asp118 had no obvious impact, substitution of Asp120 virtually abolished staurosporine-induced cleavage of SMSr ( Figure 1D). We noticed that substitution of Asp118 affected the apparent gel mobility of the V5-tagged cleavage fragment, which is remarkable as cleavage at Asp120 should give rise to the same C-terminal fragment in both SMSr-V5 and SMSr D118A -V5. Thus, it appears that substitution of Asp118 causes a shift in the capsase cleavage site. Moreover, we observed some residual cleavage of SMSr D120A -V5 in staurosporine-treated cells, which was eliminated when both Asp118 and Asp120 were substituted. The caspase-resistant form of SMSr, SMSr D118A/D120A -V5, is henceforth termed SMSr CR . From these data, we conclude that SMSr is a substrate of caspases that undergoes caspase-mediated cleavage primarily at Asp120 during staurosporine-induced apoptosis.

Caspase-6 cleaves SMSr in vitro
Having established that SMSr is a caspase substrate, we next set out to determine which caspase(s) is responsible for SMSr cleavage. As caspases are part of proteolytic cascades characterized by countless cleavage events, identification of specific pairs of caspases and substrates is a challenging task. To circumvent this complexity, we used a previously established wheat germ-based system for the cell-free production of V5-tagged human SMSr [40]. In this system, mRNA encoding SMSr-V5 is translated in wheat germ extract in the presence of unilamellar liposomes (Figure 2A). Next, SMSr-containing proteoliposomes are incubated with active recombinant human caspases and then subjected to immunoblotting using anti-V5 antibody. Importantly, this approach takes advantage of the fact that plants are devoid of any structural and functional homologs of animal caspases [43]. Figure 2(B), incubation of SMSr-V5 proteoliposomes with recombinant caspase-6 yielded a V5-tagged cleavage product of ∼33 kDa. No cleavage product was observed when SMSr-V5 proteoliposomes were incubated with recombinant caspase-2, -3, -7, -8, -9, or -10, or when incubations were performed with proteoliposomes produced in the absence of SMSr-V5 mRNA. Proteolytic activity of recombinant caspase-3, -6, -7, and -8 was verified by testing their ability to induce cleavage of the caspase substrate PARP1 in HeLa cell lysates. Moreover, immunoblot analysis of caspase-treated HeLa cell lysates indicated that Lamin A/C is a specific substrate of caspase-6 ( Figure 2C), in line with previous reports [44][45][46]. To confirm that SMSr is a substrate of caspase-6, SMSr-V5 proteoliposomes were treated with caspase-6 in the presence or absence of various caspase inhibitors. Pan-caspase inhibitor zVAD-fmk and the caspase-6-specific inhibitor z-VEID-fmk in each case completely blocked caspase-6-mediated cleavage of SMSr ( Figure 2D). Together, these results indicate that SMSr is a specific substrate of caspase-6 in vitro.

SMSr is a substrate of caspase-6 in cellulo
We next addressed whether SMSr also represents a physiological target of caspase-6 during apoptotic cell death. To this end, we created HeLa cell lines stably expressing V5-tagged versions of SMSr or the caspase-resistant mutant, SMSr CR . Expression of SMSr-V5 and SMSr CR -V5 was confirmed by immunoblot analysis ( Figure 3A). Immunofluorescence microscopy revealed that both enzymes predominantly localized to the ER ( Figure 3B), indicating that the mutations that render SMSr caspase-resistant do not interfere with its subcellular distribution. Next, we analyzed the fate of SMSr-V5 and SMSr CR -V5 during staurosporine-induced apoptosis. Treatment of cells with staurosporine resulted in proteolytic cleavage of SMSr-V5 but not of its caspase-resistant counterpart, SMSr CR -V5 ( Figure 3C), hence confirming that Asp118 and Asp120 are the principle caspase cleavage sites in SMSr.
We then asked whether staurosporine-induced proteolytic cleavage of SMSr in cells is mediated by caspase-6. Addition of pan-caspase inhibitor zVAD-fmk completely abolished cleavage of both SMSr and the caspase-6 substrate Lamin A/C in staurosporine-treated cells, while reducing cleavage of PARP1 ( Figure 3D). Addition of caspase-6-specific inhibitor z-VEID-fmk, on the other hand, significantly reduced cleavage of SMSr and Lamin A/C without affecting cleavage of PARP1. Importantly, siRNA-mediated knockdown of caspase-6 also reduced cleavage of SMSr in staurosporine-treated cells ( Figure 4A and B). Knockdown of caspase-3 diminished cleavage of SMSr as well; however, this effect is likely indirect given that caspase-3 mediates proteolytic activation of caspase-6 [47][48][49] and does not recognize SMSr as substrate in vitro ( Figure 2B). Together, these results indicate that caspase-6 mediates cleavage of SMSr during staurosporine-induced apoptosis.

Caspase-mediated cleavage of SMSr does not sensitize cells toward apoptotic stimuli
Previous works revealed that cellular ceramide levels rise concomitantly with apoptosis induction in response to staurosporine and various other apoptotic stimuli through activation of sphingomyelinases, stimulation of de novo ceramide synthesis, or both [12,20,50]. Interventions that suppress ceramide accumulation render cells resistant to these apoptotic stimuli, suggesting that ceramides play a central role in sensitizing cells to stress-induced apoptosis. Our present data indicate that SMSr loses its N-terminal SAM domain due to cleavage by caspases in staurosporine-treated cells. As SMSr requires its SAM domain to suppress ceramide-induced cell death [39], we wondered whether caspase-mediated cleavage of the enzyme serves to sensitize HeLa cells to staurosporine and other apoptotic stimuli. However, stable expression of the caspase-resistant form of SMSr had no obvious impact on the time-resolved progression of caspase-9 and PARP1 cleavage in staurosporine-treated HeLa cells, indicating that the rate of apoptosis is unaffected by blocking caspase-mediated SMSr cleavage ( Figure 5A).
Since SMS1 undergoes caspase-mediated cleavage in leukemia cells treated with the death receptor ligand FasL and because this process has been reported to contribute to FasL-induced apoptosis [35], we next addressed whether SMSr is also cleaved in FasL-treated HeLa cells. As shown in Figure 5(B), treatment with FasL resulted in proteolytic cleavage of SMSr-V5, yielding a V5-tagged fragment of ∼33 kDa. Moreover, cleavage of SMSr was blocked by treating cells with z-VAD-fmk, indicating that this process is mediated by caspases. However, progression of FasL-induced apoptosis was not affected by stable expression of the caspase-resistant form of SMSr ( Figure 5C). Collectively, these data suggest that caspase-mediated cleavage of SMSr does not significantly contribute to either staurosporine-or FasL-induced apoptosis in HeLa cells.

Discussion
CPE synthase SMSr/SAMD8 is an ER-resident suppressor of ceramide-mediated mitochondrial apoptosis that requires its N-terminal SAM domain to exert its anti-apoptotic activity. As several negative regulators of apoptosis are cleaved by caspases during apoptosis induction, we addressed whether SMSr is a target of the apoptotic machinery.
In here, we demonstrate that SMSr is a novel and specific substrate of the executioner caspase-6. We provide evidence that in HeLa cells undergoing staurosporine or FasL-induced apoptosis, SMSr is cleaved by caspase-6 at a conserved aspartate located downstream of the SAM domain and upstream of the enzyme's first membrane span. While this finding is in line with a role of SMSr as negative regulator of ceramide-induced cell death, addressing the physiological relevance of caspase-6-mediated cleavage of SMSr will require further investigations.
Both staurosporine and FasL trigger a caspase-mediated release of the N-terminal SAM domain of human SMSr in HeLa cells. Site-directed mutagenesis revealed that cleavage primarily occurs at Asp120, a residue conserved in SMSr homologs from human to zebrafish. In human SMSr, some residual cleavage also occurs at Asp118. However, this residue is not conserved in the mouse homolog. Taking advantage of human SMSr produced in a wheat germ-based cell-free translation system that lacks endogenous caspase-like activities [43], we were able to reconstitute proteolytic release of the enzyme's N-terminal SAM domain upon external addition of recombinant caspase-6, but not when adding recombinant caspase-2, -3, -7, -8, -9, or -10. Furthermore, addition of caspase-6-targeting siRNAs or the caspase-6 inhibitor z-VEID-fmk to staurosporine-treated HeLa cells specifically reduced cleavage of SMSr and the caspase-6 substrate Lamin A/C [44]. Together, these findings establish SMSr as a novel target of caspase-6. Whether caspase-6 is the sole caspase responsible for cleaving SMSr during apoptotic cell death remains to be established.
SMSr belongs to the SM synthase family, which also includes the Golgi-resident enzyme SMS1 and the plasma membrane-resident enzyme SMS2 [29,31]. SMS1 has previously been recognized as a target of caspases during FasL-induced apoptosis in Jurkat cells. Contrary to SMSr, SMS1 in FasL-treated cells is cleaved at multiple sites, abolishing the enzyme's catalytic activity, disrupting de novo SM synthesis, and resulting in an accumulation of ceramides [35]. Whereas SMS1 knockdown sensitized cells to FasL-induced apoptosis, SMS1 overexpression had the opposite effect, suggesting that caspase-mediated inhibition of SM production and associated ceramide accumulation are involved in the regulation of FasL-triggered cell death [35]. As SMSr relies on its N-terminal SAM domain to suppress ceramide-induced cell death [39], we considered whether caspase-6-mediated release of this domain may accelerate apoptosis in FasL-or staurosporine-treated cells. However, we did not find any evidence that heterologous expression of a caspase-resistant SMSr mutant influenced progression of apoptotic cell death triggered by these stimuli. While these data suggest that caspase-mediated inactivation of SMSr is functionally unrelated to the regulation of apoptotic cell death, we cannot rule out that overexpression of the enzyme or the coexistence of caspase-resistant and caspase-sensitive SMSr pools in apoptotic cells mask the effect. Therefore, our ongoing efforts are aimed at generating cell-lines in which the proteolytic release of SMSr-SAM can be specifically induced at the level of the endogenous enzyme. Caspase-6, along with caspase-3 and caspase-7, is classified as an executioner caspase. However, its substrate specificity and activation mechanism are unique among executioner caspases. While caspase-3 and -7 use similar recognition sites to cleave their targets, the recognition sites used by caspase-6 share a high degree of similarity with those of the initiator caspases caspase-8 and -9 [51]. Unlike caspase-3 and -7, caspase-6 can undergo self-activation [ 52,53]. During apoptosis, caspase-6 is translocated to and activated in the nucleus where it cleaves various transcription factors as well as nuclear structural proteins such as lamins, resulting in shrinkage and fragmentation of the nucleus [44,54]. This implies that of all SMSr molecules that populate the ER, only those that reside in the nuclear envelope and expose their SAM-domain containing N-terminal tails in the nuclear matrix would initially be subjected to caspase-6-mediated proteolysis. This may explain our finding that only a relative minor portion of SMSr molecules is cleaved in staurosporine-or FasL-treated cells.
Unlike caspase-3 and -7, caspase-6 activity does not always contribute to apoptotic cell death. For instance, numerous studies point at a critical role of caspase-6 in the development of neurodegenerative diseases [55]. Caspase-6-mediated cleavage of mutant huntingtin protein and amyloid precursor protein (APP) is critical for the onset of Huntington's disease [56] and Alzheimer's disease [57] respectively. Moreover, the brains of Alzheimer's patients have been reported to contain 2-3-fold elevated levels of active caspase-6 [57]. As SMSr constitutes the principle CPE synthase in brain [38], it would be of interest to explore whether its proteolytic cleavage by caspase-6 has any relevance in the pathogenesis of neurodegenerative diseases. In this respect, it is of interest to note our recent observation that the SAM domain of SMSr drives self-assembly of the enzyme into ER-resident trimers and hexamers [58,58]. Moreover, when expressed on its own, SMSr-SAM readily self-associates into stable polymers [59,60]. Whether such polymers also form in the brain of patients with elevated levels of active caspase-6 merits further investigation.