Human Scythe contains a functional nuclear localization sequence and remains in the nucleus during staurosporine-induced apoptosis

Human Scythe (also known as BAT3) has been implicated in the control of apoptosis and regulating heat shock protein (HSP) 70 activity. We have attempted to further characterize the role of human Scythe in HeLa cells by studying the cellular localization and functional domains of a hemagglutinin (HA) epitope-tagged Scythe protein. Several HA-Scythe deletion mutant proteins were expressed in HeLa cells and their localization was detected using indirect immunofluorescence. Our data demonstrate that full-length human Scythe is a nuclear protein that contains an active C-terminal nuclear localization sequence (NLS). Site-directed mutagenesis of the NLS leads to complete nuclear exclusion of full-length Scythe. Furthermore, induction of apoptosis by staurosporine does not cause redistribution or cleavage of Scythe, suggesting that Scythe remains localized in the nucleus during apoptosis. These results provide evidence that Scythe is a nuclear protein that probably does not interact with elements of the apoptotic machinery in the cytosol.     

Inhibition of translation and induction of apoptosis by Bunyaviral nonstructural proteins bearing sequence similarity to reaper

Members of the California serogroup of bunyaviruses (family Bunyaviridae) are the leading cause of pediatric viral encephalitis in North America. Significant cell death is observed as part of the infection pathology. We now report that a Bunyaviral nonstructural protein termed NSs shows sequence similarity to Reaper, a proapoptotic protein from Drosophila. Although NSs proteins lack the Reaper N-terminal motif critical for IAP inhibition, they do retain other functions of Reaper that map to conserved C-terminal regions. Like Reaper, NSs proteins induce mitochondrial cytochrome c release and caspase activation in cell-free extracts and promote neuronal apoptosis and mortality in a mouse model. Independent of caspase activation, Bunyavirus NSs proteins also share with Reaper the ability to directly inhibit cellular protein translation. We have found that the shared capacity to inhibit translation and induce apoptosis resides in common sequence motifs present in both Reaper and NSs proteins. Data presented here suggest that NSs induce apoptosis through a mechanism similar to that used by Reaper, as both proteins bind to an apoptotic regulator called Scythe and can relieve Scythe inhibition of Hsp70. Thus, bunyavirus NSs proteins have multiple Reaper-like functions that likely contribute to viral pathogenesis by promoting cell death and/or inhibiting cellular translation.     

Scythe cleavage during Fas (APO-1)-and staurosporine-mediated apoptosis

Scythe is a nuclear protein that has been implicated in the apoptotic process in Drosophila melanogaster; however, its role in apoptosis of mammalian cells is not fully elucidated. Here we show that cleavage of Scythe by caspase-3 occurs after activation of both the extrinsic (i.e. Fas/APO-1-mediated) and the intrinsic (i.e. staurosporine-induced) apoptosis pathway. Moreover, this caspase-dependent cleavage correlates with Scythe translocation from the nucleus to the cytosol. We also show that cytosolic re-localization of Scythe is required for Fas/APO-1-triggered phosphatidylserine (PS) exposure, a signal for macrophage clearance of apoptotic cells. Our data suggest that Scythe cleavage may represent a marker for caspase-3 activation and implicate cytosolic re-localization of Scythe in the pathway of PS exposure.     

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.     

Human SGT interacts with Bag-6/Bat-3/Scythe and cells with reduced levels of either protein display persistence of few misaligned chromosomes and mitotic arrest

The human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division since RNA-interference-mediated strong reduction of hSGT protein levels causes mitotic arrest (M. Winnefeld, J. Rommelaere, and C. Cziepluch, The human small glutamine-rich TPR-containing protein is required for progress through cell division, Exp. Cell Res. 293 (2004), 43-57). Analysis of HeLa cells expressing a histone 2A-YFP fusion protein revealed the continuous presence of few mislocalized chromosomes close to the spindle poles as possible cause for hSGT depletion-dependent prometaphase arrest. Cells unable to rescue these mislocalized chromosomes into the metaphase plate died at this stage through apoptosis. In order to address hSGT function at the molecular level, mass spectrometry analysis of proteins which co-immunoprecipitated with Flag-tagged hSGT was performed. Thereby, Hsp70 and Bag-6/Bat-3/Scythe were identified as novel hSGT interaction partners while interaction with Hsc70 was confirmed. Results obtained with truncated versions of the hSGT protein revealed that Bag-6/Bat-3/Scythe and Hsp70 or Hsc70 were independently able to form complexes with hSGT. Interaction of hSGT with Hsc70, Hsp70 or Bag-6/Bat-3/Scythe was demonstrated in prometaphase, thereby suggesting a possible role for complexes containing hSGT and distinct (co)-chaperones during mitosis. Finally, cells from populations with reduced levels of Bag-6/Bat-3/Scythe also displayed persistence of mislocalized chromosomes and mitotic arrest, which strongly indicated that hSGT-Bag-6/Bat-3/Scythe complexes could be directly or indirectly required for complete chromosome congression.     

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development.     

Scythe: a novel reaper-binding apoptotic regulator.

Reaper is a central regulator of apoptosis in Drosophila melanogaster. With no obvious catalytic activity or homology to other known apoptotic regulators, reaper's mechanism of action has been obscure. We recently reported that recombinant Drosophila reaper protein induced rapid mitochondrial cytochrome c release, caspase activation and apoptotic nuclear fragmentation in extracts of Xenopus eggs. We now report the purification of a 150 kDa reaper-interacting protein from Xenopus egg extracts, which we have named Scythe. Scythe is highly conserved among vertebrates and contains a ubiquitin-like domain near its N-terminus. Immunodepletion of Scythe from extracts completely prevented reaper-induced apoptosis without affecting apoptosis triggered by activated caspases. Moreover, a truncated variant of Scythe lacking the N-terminal domain induced apoptosis even in the absence of reaper. These data suggest that Scythe is a novel apoptotic regulator that is an essential component in the pathway of reaper-induced apoptosis.     

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action.

Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.     

Low Levels of GSTA1 Expression Are Required for Caco-2 Cell Proliferation

The colonic epithelium continuously regenerates with transitions through various cellular phases including proliferation, differentiation and cell death via apoptosis. Human colonic adenocarcinoma (Caco-2) cells in culture undergo spontaneous differentiation into mature enterocytes in association with progressive increases in expression of glutathione S-transferase alpha-1 (GSTA1). We hypothesize that GSTA1 plays a functional role in controlling proliferation, differentiation and apoptosis in Caco-2 cells. We demonstrate increased GSTA1 levels associated with decreased proliferation and increased expression of differentiation markers alkaline phosphatase, villin, dipeptidyl peptidase-4 and E-cadherin in postconfluent Caco-2 cells. Results of MTS assays, BrdU incorporation and flow cytometry indicate that forced expression of GSTA1 significantly reduces cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increases cells in S-phase and associated cell proliferation. Sodium butyrate (NaB) at a concentration of 1 mM reduces Caco-2 cell proliferation, increases differentiation and increases GSTA1 activity 4-fold by 72 hours. In contrast, 10 mM NaB causes significant toxicity in preconfluent cells via apoptosis through caspase-3 activation with reduced GSTA1 activity. However, GSTA1 down-regulation by siRNA does not alter NaB-induced differentiation or apoptosis in Caco-2 cells. While 10 mM NaB causes GSTA1-JNK complex dissociation, phosphorylation of JNK is not altered. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.     

WRN-targeted therapy using inhibitors NSC 19630 and NSC 617145 induce apoptosis in HTLV-1-transformed adult T-cell leukemia cells

Background

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells.

Methods

Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential.

Results

Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145.

Conclusions

WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.

     

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.     

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells

Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15(INK4b)), and Gas3/PMP22. The expression of p21 and p15(INK4b) contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death.     

Unique proteasome subunit Xrpn10c is a specific receptor for the antiapoptotic ubiquitin-like protein Scythe

The Rpn10 subunit of the 26S proteasome can bind to polyubiquitinoylated and/or ubiquitin-like proteins via ubiquitin-interacting motifs (UIMs). Vertebrate Rpn10 consists of five distinct spliced isoforms, but the specific functions of these variants remain largely unknown. We report here that one of the alternative products of Xenopus Rpn10, named Xrpn10c, functions as a specific receptor for Scythe/BAG-6, which has been reported to regulate Reaper-induced apoptosis. Deletional analyses revealed that Scythe has at least two distinct domains responsible for its binding to Xrpn10c. Conversely, an Xrpn10c has a UIM-independent Scythe-binding site. The forced expression of a Scythe mutant protein lacking Xrpn10c-binding domains in Xenopus embryos induces inappropriate embryonic death, whereas the wild-type Scythe did not show any abnormality. The results indicate that Xrpn10c-binding sites of Scythe act as an essential segment linking the ubiquitin/proteasome machinery to the control of proper embryonic development.     

Bag6/Bat3/Scythe: A novel chaperone activity with diverse regulatory functions in protein biogenesis and degradation

Upon emerging from the ribosome exiting tunnel, polypeptide folding occurs immediately with the assistance of both ribosome‐associated and free chaperones. While many chaperones known to date are dedicated folding catalysts, recent studies have revealed a novel chaperoning system that functions at the interface of protein biogenesis and quality control by using a special “holdase” activity in order to sort and channel client proteins to distinct destinations. The key component, Bag6/Bat3/Scythe, can effectively shield long hydrophobic segments exposed on the surface of a polypeptide, preventing aggregation or inappropriate interactions before a triaging decision is made. The biological consequences of Bag6‐mediated chaperoning are divergent for different substrates, ranging from membrane integration to proteasome targeting and destruction. Accordingly, Bag6 can act in various cellular contexts in order to execute many essential cellular functions, while dysfunctions in the Bag6 system can cause severe cellular abnormalities that may be associated with some pathological conditions.     

Down regulation of the proliferation and apoptotic pathways in the embryonic brain of diabetic rats

Compelling evidence shows that the offspring subjected to uncontrolled hyperlycemia during gestation display behavioral, neurochemical, and cellular abnormalities during adulthood. However, the molecular mechanisms underlying these defects remain elusive. Previous studies have shown an increased rate of apoptosis and a decreased index of neuronal proliferation associated with diabetic embryopathy. The aim of the present study was to determine whether impairments in apoptotic related proteins also occur in the developing central nervous system from non-malformed embryos exposed to uncontrolled gestational hyperglycemia. Pregnant rats injected with either streptozotocin or vehicle were killed on gestational day 19. Offspring brains were quickly removed to evaluate protein expression by Western blotting. Embryonic brains from diabetic rats exhibited a decrease in the cell survival p-Akt expression (52.83 ± 24.35%) and in the pro-apoptotic protein Bax (56.16 ± 6.47%). Moreover, the anti-apoptotic protein Bcl-2 showed a non-significant increase while there were no changes in Procaspase 3 or cleaved Caspase 3 proteins. The cytoskeleton proteins NF-200 and GFAP were also examined. Neither NF-200 nor GFAP showed differences in embryonic brains from diabetic rats compared to controls. Altogether, these results indicate that both proliferation and apoptotic pathways are decreased in the brain from the developing offspring of diabetic rats. Since selective neuronal apoptosis, as well as selective cell proliferation, are specifically involved in brain organogenesis, it is possible that simultaneous impairments during the perinatal period contribute to the long lasting alterations observed in the adult brain.     

A novel anti-proliferative property of clusterin in prostate cancer cells

Clusterin is a ubiquitous secretory glycoprotein that is known to suppress certain forms of apoptosis. Since apoptosis and proliferation are two opposing cellular events, it remains unclear if clusterin has any effect on cellular proliferation. The objective of the present study was to examine the effects of clusterin on proliferation in a prostate cancer cell line, LNCaP. We found that clusterin inhibited EGF-mediated proliferation in these cells, as measured by (3)H-thymidine incorporation and by cell counting. Clusterin did not bind with EGF nor did it block phosphorylation of the EGF receptor. Treatment of LNCaP cells with EGF resulted in a transient increase in the expression of both c-Fos and c-Jun. Addition of clusterin to these cultures significantly down-regulated the protein level of c-Fos, but not c-Jun. These results demonstrated a novel biological role for clusterin. Clusterin is not only anti-apoptotic but also anti-proliferative. The anti-proliferative event maybe associated with a down-regulation of c-Fos.     

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.