The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.


Results
GRP75 interacted with BCL-RAMBO via its BHNo domain.To verify the interaction between BCL-RAMBO and GRP75, we overexpressed FLAG-BCL-RAMBO and VSV-GRP75 in 293T cells by transient transfection using pCR3 expression vectors, and performed co-immunoprecipitation assays using anti-FLAG antibody-conjugated agarose beads.VSV-GRP75 was co-immunoprecipitated with FLAG-BCL-RAMBO (Fig. 1a).
To confirm this, we also performed pull-down assays using the glutathione S-transferase (GST) protein and GST-BCL-RAMBO protein, both of which were produced in Escherichia coli, and cell lysates prepared from 293T cells transiently expressing FLAG-GRP75.The GST-BCL-RAMBO protein, but not the GST protein, pulled down FLAG-GRP75 (Fig. 1b).These results showed that GRP75 physically interacted with BCL-RAMBO.
Hsc70-5, a mammalian GRP75 ortholog, genetically interacted with BCL-RAMBO.We previously showed that the ectopic expression of BCL-RAMBO, but not BCL-RAMBO ΔTM, induced apoptosis and caused aberrant rough eye phenotypes in Drosophila 18 .To allow the ectopic expression of green fluorescence protein (GFP) and BCL-RAMBO in Drosophila eyes, UAS-GFP and UAS-BCL-RAMBO fly lines were crossed with glass multiple reporter (GMR)-GAL4 fly lines.The morphology of adult eyes was normal in fly lines expressing GFP (Fig. 2a-a″,f-f″).Consistent with our previous findings 18 , BCL-RAMBO caused a loss of pigmentation, a reduction in eye sizes, and marked morphological changes in ommatidia (Fig. 2c-c″,h-h″).Hsc70-5 is an ortholog of mammalian GRP75.The Hsc70-5 mutations, Hsc70-5 k06618 and Hsc70-5 k04907 , partly rescued the reduction in pigmentation in BCL-RAMBO fly lines (Fig. 2d-d″,i-i″), but did not affect the morphology of adult eyes in fly lines expressing GFP (Fig. 2b-b″,g-g″) and BCL-RAMBO ΔTM (Fig. 2e-e″,j-j″).These results showed that Hsc70-5 genetically interacted with BCL-RAMBO in Drosophila.
We further investigated the activation of procaspase-7 using Western blotting.FLAG-BCL-RAMBO and FLAG-GRP75 were detected to a similar degree when expressed in 293T cells for 16 h (Fig. 3c).Consistent with caspase-3/7 activity (Fig. 3b), BCL-RAMBO, but not GRP75, augmented the cleavage of procaspase-7 into the active p20 subunit (Fig. 3c,d).GRP75 promoted the augmentation of procaspase-7 cleavage induced by BCL-RAMBO (Fig. 3c,d).These results showed that GRP75 cooperated with BCL-RAMBO to promote the activation of executioner caspases.
We investigated whether the knockdown of GRP75 attenuated the BCL-RAMBO-induced apoptosis signaling pathway.Treatment of 293T cells with siGRP75 resulted in an approximately 70% reduction in the GRP75 protein compared to siControl (Fig. 3e,f).To induce sufficient activation of executioner caspases by BCL-RAMBO itself, we used a larger amount of DNA in the transient transfection experiments.Treatment with siGRP75 reduced the BCL-RAMBO-induced increase in caspase-3/7 activity (Fig. 3g).Caspase-7 cleavage was then assessed by Western blotting.The amount of cleaved caspase-7 was augmented by BCL-RAMBO in siControl-treated 293T  cells (Fig. 3h,j).The treatment of 293T cells with siGRP75 resulted in a decrease in the GRP75 protein from that with siControl by approximately 60% (Fig. 3h,i).Under these conditions, the augmentation of cleaved caspase-7 caused by BCL-RAMBO was attenuated by the siGRP75 treatment (Fig. 3h,j).These results indicated that GRP75 knockdown attenuated BCL-RAMBO-induced executioner caspase activation.
The knockdown of GRP75 attenuated the release of cytochrome c into the cytosol.In mitochondrial apoptosis, the release of cytochrome c into the cytosol is essential for the activation of caspases 11,12 .We previously showed that BCL-RAMBO induced the mitochondrial release of cytochrome c in the cytosol 15,18 .To evaluate the subcellular localization of cytochrome c, cell lysates were separated into cytosolic, nuclear, and organellar fractions by successive treatments with digitonin and Triton X-100, followed by centrifugation.The GRP75 protein was recovered in the organellar fraction, but not in the cytosolic fraction (Fig. 5a,b).The treatment of 293T cells with siGRP75 diminished the amount of the GRP75 protein from that with siControl by more than 80% (Fig. 5a,c).Cytochrome c levels in the organellar fraction were not markedly affected by BCL-RAMBO or siGRP75 (Fig. 5a,d).BCL-RAMBO augmented the amount of cytochrome c in the cytosolic fraction of siControl-treated cells (Fig. 5b,e).The BCL-RAMBO-induced augmentation of cytosolic cytochrome c was markedly diminished in siGRP75-treated cells (Fig. 5b,e).These results showed that the knockdown of GRP75 attenuated the BCL-RAMBO-induced release of cytochrome c into the cytosol.BCL-RAMBO formed a complex with GRP75 and VDAC1.We showed that GRP75 interacted with BCL-RAMBO via its BHNo domain (Fig. 1d).VDAC1 has been previously reported to interact with GRP75 30 .To test whether the complex formed with BCL-RAMBO and GRP75 contains additional factors such as VDAC1, we transfected 293T cells with expression vectors encoding FLAG-BCL-RAMBO, together with VSV-GRP75 and VSV-VDAC1, and cell lysates were used for immunoprecipitation assay using anti-FLAG antibody beads.As shown in Fig. 1a, VSV-GRP75 was immunoprecipitated with FLAG-BCL-RAMBO (Fig. 6).In contrast, VSV-VDAC1 was barely immunoprecipitated with FLAG-BCL-RAMBO (Fig. 6).However, VSV-VDAC1 was immunoprecipitated with FLAG-BCL-RAMBO when VSV-GRP75 was co-transfected (Fig. 6).These results suggest that GRP75 mediates the interaction between BCL-RAMBO and VDAC1.

Discussion
The overexpression of BCL-RAMBO has been shown to induce apoptosis in human embryonic kidney 293T cells, human breast cancer MCF-7 cells, and human prostate cancer PC3 cells 15,19,26 .A previous study demonstrated that BCL-RAMBO promoted taxol-induced cell death in 293A cells and etoposide-induced cell death in human cervical cancer HeLa cells 34 .In Drosophila S2 cells, the ectopic expression of BCL-RAMBO induced apoptosis 18 .Collectively, these findings showed that BCL-RAMBO is a pro-apoptotic protein capable of inducing apoptosis in multiple cell lines.In the present study, we identified GRP75 as an emerging partner capable of interacting with BCL-RAMBO.GRP75 promoted BCL-RAMBO-induced caspase activity and PARP-1 cleavage, while its knockdown attenuated BCL-RAMBO-induced caspase activity, PARP-1 cleavage, and the mitochondrial release of cytochrome c.The present results revealed that GRP75 functions as an important regulator of the BCL-RAMBO-dependent apoptosis signaling pathway.
The present study revealed that GRP75 interacted with BCL-RAMBO.In addition to the import of mitochondrial proteins, GRP75 exerts various functions by interacting with several binding partners [27][28][29] .Closely related to the present study, GRP75 was shown to become a bridge between the ER channel protein IP3R and the mitochondrial channel protein VDAC1 31 .The IP3R-GRP75-VDAC1 complex is present in MAMs and transfers Ca 2+ from the ER and mitochondria 37 .We previously showed that the GST-BCL-RAMBO protein interacted with FLAG-VDAC1 expressed in 293T cells 20 .In this study, the immunoprecipitation assay showed that BCL-RAMBO formed a complex with GRP75 and VDAC1.Taken together, the present results suggest that BCL-RAMBO associates with the IP3R-GRP75-VDAC1 complex and is an additional component that regulates its biological activity.
BCL-RAMBO has been shown to mediate pro-apoptotic activity via the BHNo domain anchored at the mitochondrial outer membrane, whereas the BH domain of BCL-RAMBO is not necessarily essential for proapoptotic activity 15 .To date, BCL-RAMBO has been shown to induce MOMP in a manner that is independent of its direct interactions with other BCL-2 family proteins.In the present study, we showed that GRP75 interacted with BCL-RAMBO via its BHNo domain.BCL-RAMBO and GRP75 cooperatively promoted caspase activation and PARP-1 cleavage, while the knockdown of GRP75 attenuated the BCL-RAMBO-induced mitochondrial release of cytochrome c, caspase activation, and PARP-1 cleavage.Therefore, GRP75 appears to interact with BCL-RAMBO, thereby promoting MOMP.However, future studies are needed to elucidate the mechanism by which BCL-RAMBO and GRP 75 induce cytochrome c release from mitochondria, including the multimerization of BAX and BAK.
GRP75 has been identified as an important regulator of apoptosis, which is mediated by the IP3R-GRP75-VDAC1 complex 38,39 .Adriamycin and angiotensin II were found to increase the expression of IP3R, GRP75, VDAC1, and mitochondrial calcium uniporter, and thereby induced apoptosis in mouse podocytes, while the knockdown of GRP75 inhibited adriamycin-and angiotensin II-induced apoptosis 38 .The knockdown of GRP75 also attenuated elevated mitochondrial Ca 2+ levels in retinal microvascular endothelial cells (RMECs) treated with high glucose or advanced glycosylation end products, which represent a cellular model for diabetic retinopathy 39 .BAPTA-AM, an intracellular calcium chelator, prevented apoptosis induced by tunicamycin, an ER stress inducer, in RMECs 39 .These findings indicate that GRP75 regulates mitochondrial Ca 2+ levels and thereby promotes Figure 3. BCL-RAMBO and GRP75 cooperatively promoted the activation of executioner caspases, while GRP75 knockdown suppressed the activation of executioner caspases induced by BCL-RAMBO.(a,b) 293T cells were transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO and/or FLAG-GRP75, together with an expression vector encoding the cytomegalovirus promoter-driven Renilla luciferase reporter, for 18 h.Cell lysates were assessed by Western blotting (a) and caspase-3/7 activity assay (b).Original blots are presented in Supplementary Fig. S4.Representative blots from three independent experiments are shown.Renilla luciferase activity was used to normalize caspase-3/7 activity.Caspase-3/7 activity (fold) is presented as the mean ± S.E. of three independent experiments.(c,d) 293T cells were transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO and/or FLAG-GRP75 for 16 h.Cell lysates were assessed by Western blotting (c).The asterisk indicated additional bands detected by the anti-caspase-7 antibody.Original blots are presented in Supplementary Fig. S5.Representative blots from three independent experiments are shown.The amount of β-actin was used to normalize cleaved caspase-7.Cleaved caspase-7 (fold) is presented as the mean ± S.E. of three independent experiments (d).(e-g) 293T cells were transfected without (−) or with (+) siControl or siGRP75 for 24 h, and then transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO together with an expression vector encoding the cytomegalovirus promoter-driven Renilla luciferase reporter for 16 h.Cell lysates were assessed by Western blotting (e) and caspase-3/7 activity assay (g).Original blots are presented in Supplementary Fig. S6.Representative blots from three independent experiments are shown.The amount of β-actin was used to normalize that of GRP75.GRP75 protein (%) is presented as the means ± S.E. of three independent experiments (f).Renilla luciferase activity was used to normalize caspase-3/7 activity.Caspase-3/7 activity (fold) is presented as the means ± S.E. of three independent experiments (g).(h-j) 293T cells were transfected without (−) or with (+) siControl or siGRP75 for 24 h, and then transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO for 16 h.Cell lysates were assessed by Western blotting (h).The asterisk indicates additional bands detected by the anti-caspase-7 antibody.Original blots are presented in Supplementary Fig. S7.Representative blots from three independent experiments are shown.The amount of β-actin was used to normalize those of GRP75 and cleaved caspase-7.GRP75 protein (%) (i) and cleaved caspase-7 (%) (j) are presented as the means ± S.E. of three independent experiments.GRP75 protein in siControl-transfected 293T cells is set to 100%.Cleaved caspase-7 in siControl-and BCL-RAMBO-transfected 293T cells is set to 100%.P values less than 0.05 are shown.Microscopic observations.GMR-GAL4 driver fly lines were crossed with the UAS fly lines.Fly lines were then crossed with Hsc70-5 mutant fly lines.The compound eyes of adult flies were observed using the stereomicroscope SZX10 (Olympus, Tokyo, Japan) and scanning electron microscope VE-7800 (Keyence, Osaka, Japan).

Figure 1 .
Figure 1.GRP75 interacted with BCL-RAMBO via its BHNo domain.(a) 293T cells were transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO and/or VSV-GRP75 for 18 h.Cell lysates were subjected to immunoprecipitation (IP) and assessed by Western blotting.The asterisk indicates nonspecific bands.The arrow corresponds to IgG heavy chains.β-actin (n = 2) or γ1-actin (n = 1) was used as the loading control.Representative blots, in which β-actin was used as the loading control, from three independent experiments are shown.Original blots are presented in Supplementary Fig. S1.(b) 293T cells were transfected with (+) expression vectors encoding FLAG-GRP75 for 18 h.Cell lysates were incubated without (−) or with (+) the GST protein or GST-BCL-RAMBO protein together with Glutathione Sepharose beads overnight.Pulldown fractions and cell lysates were assessed by Western blotting.Arrows indicate cleaved forms of the GST-BCL-RAMBO protein.Ponceau S staining was used to detect the GST protein and GST-BCL-RAMBO protein.Original blots and gels are presented in Supplementary Fig. S2.Representative results from three independent experiments are shown.(c) Structures of BCL-RAMBO and its deletion mutants.BH: BCL-2 homology motifs, BHNo: No BH motif domain, TM: transmembrane domain, LIR: MAP1LC3-interacting region.(d) 293T cells were transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO mutants and VSV-GRP75 for 18 h.Cell lysates were subjected to immunoprecipitation (IP) and assessed by Western blotting.The asterisk indicates non-specific bands.The arrow corresponds to IgG heavy chains.Original blots are presented in Supplementary Fig. S3.Representative blots from three independent experiments are shown.

Figure 4 .Figure 5 .
Figure 4. BCL-RAMBO and GRP75 cooperatively promoted PARP-1 cleavage, whereas GRP75 knockdown suppressed the BCL-RAMBO-induced PARP-1 cleavage.(a,b) 293T cells were transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO and/or FLAG-GRP75 for 24 h.Whole cell lysates were assessed by Western blotting (a).Original blots are presented in Supplementary Fig. S8.Representative blots from three independent experiments are shown.The amount of β-actin was used to normalize that of cleaved PARP-1.Cleaved PARP-1 (fold) is presented as the mean ± S.E. of three independent experiments (b).(c-e) 293T cells were transfected without (−) or with (+) siControl or siGRP75 for 24 h, and then transfected without (−) or with (+) expression vectors encoding FLAG-BCL-RAMBO for 24 h.Whole cell lysates were assessed by Western blotting (c).Original blots are presented in Supplementary Fig. S9.Representative blots from three independent experiments are shown.The amount of β-actin was used to normalize those of GRP75 (d) and cleaved PARP-1 (e).GRP75 protein (%) (d) and cleaved PARP-1 (%) (e) are presented as the means ± S.E. of three independent experiments.GRP75 protein in siControl-transfected 293T cells is set to 100%.Cleaved PARP-1 in siControl-and BCL-RAMBO-transfected 293T cells is set to 100%.P values less than 0.05 are shown.

Figure 6 .
Figure 6.BCL-RAMBO formed a complex with GRP75 and VDAC1.293T cells were transfected without (−) or with (+) expression vectors FLAG-BCL-RAMBO, VSV-GRP75, and/or VSV-VDAC1 for 18 h.Cell lysates were subjected to immunoprecipitation (IP) and assessed by Western blotting.The asterisk indicates nonspecific bands.The arrows correspond to IgG chains.Original blots are presented in Supplementary Fig. S12.Representative blots from three independent experiments are shown.