E3 ligase adaptor FBXO7 contributes to ubiquitination and proteasomal degradation of SIRT7 and promotes cell death in response to hydrogen peroxide
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.102909
ISSN1083-351X
AutoresSu Hyoun Lee, Yun Ju Lee, Sungyeon Jung, Kwang Chul Chung,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoParkinson's disease (PD) is a degenerative disorder of the central nervous system that affects 1% of the population over the age of 60. Although aging is one of the main risk factors for PD, the pathogenic mechanism of this disease remains unclear. Mutations in the F-box-only protein 7 (FBXO7) gene have been previously found to cause early onset autosomal recessive familial PD. FBXO7 is an adaptor protein in the SKP1–Cullin–1–F-box (SCF) E3 ligase complex that facilitates the ubiquitination of substrates. Sirtuin 7 (SIRT7) is an NAD+-dependent histone deacetylase that regulates aging and stress responses. In this study, we identified FBXO7 as a novel E3 ligase for SIRT7 that negatively regulates intracellular SIRT7 levels through SCF-dependent Lys-48-linked polyubiquitination and proteasomal degradation. Consequently, we show that FBXO7 promoted the blockade of SIRT7 deacetylase activity, causing an increase in acetylated histone 3 levels at the Lys-18 and Lys-36 residues and the repression of downstream RPS20 gene transcription. Moreover, we demonstrate that treatment with hydrogen peroxide triggered the FBXO7-mediated degradation of SIRT7, leading to mammalian cell death. In particular, the PD-linked FBXO7-R498X mutant, which reduced SCF-dependent E3 ligase activity, did not affect the stability of SIRT7. Collectively, these findings suggest that FBXO7 negatively regulates SIRT7 stability and may suppress the cytoprotective effects of SIRT7 during hydrogen peroxide–induced mammalian cell death. Parkinson's disease (PD) is a degenerative disorder of the central nervous system that affects 1% of the population over the age of 60. Although aging is one of the main risk factors for PD, the pathogenic mechanism of this disease remains unclear. Mutations in the F-box-only protein 7 (FBXO7) gene have been previously found to cause early onset autosomal recessive familial PD. FBXO7 is an adaptor protein in the SKP1–Cullin–1–F-box (SCF) E3 ligase complex that facilitates the ubiquitination of substrates. Sirtuin 7 (SIRT7) is an NAD+-dependent histone deacetylase that regulates aging and stress responses. In this study, we identified FBXO7 as a novel E3 ligase for SIRT7 that negatively regulates intracellular SIRT7 levels through SCF-dependent Lys-48-linked polyubiquitination and proteasomal degradation. Consequently, we show that FBXO7 promoted the blockade of SIRT7 deacetylase activity, causing an increase in acetylated histone 3 levels at the Lys-18 and Lys-36 residues and the repression of downstream RPS20 gene transcription. Moreover, we demonstrate that treatment with hydrogen peroxide triggered the FBXO7-mediated degradation of SIRT7, leading to mammalian cell death. In particular, the PD-linked FBXO7-R498X mutant, which reduced SCF-dependent E3 ligase activity, did not affect the stability of SIRT7. Collectively, these findings suggest that FBXO7 negatively regulates SIRT7 stability and may suppress the cytoprotective effects of SIRT7 during hydrogen peroxide–induced mammalian cell death. Parkinson's disease (PD) is a common neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra of the midbrain (1Samii A. Nutt J.G. Ransom B.R. Parkinson's disease.Lancet. 2004; 363: 1783-1793Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). Multiple factors, including abnormal protein aggregation, oxidative damage, brain inflammation, and mitochondrial dysfunction, have been suggested to play critical roles in PD pathogenesis (2Antony P.M. Diederich N.J. Kruger R. Balling R. The hallmarks of Parkinson's disease.FEBS J. 2013; 280: 5981-5993Crossref PubMed Scopus (193) Google Scholar, 3Hindle J.V. 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Although most cases of PD are sporadic, 5 to 10% of cases develop at a much younger age, which are caused by mutations in one of several familial genes, including α-synuclein, PTEN-induced kinase 1 (PINK1), parkin, LRRK2, DJ-1, ATP13A2, and FBXO7 (6Houlden H. Singleton A.B. The genetics and neuropathology of Parkinson's disease.Acta Neuropathol. 2012; 124: 325-338Crossref PubMed Scopus (245) Google Scholar, 7Li W. Fu Y. Halliday G.M. Sue C.M. PARK genes link mitochondrial dysfunction and alpha-synuclein pathology in sporadic Parkinson's disease.Front. Cell Dev. Biol. 2021; 9: 612476Crossref PubMed Scopus (33) Google Scholar). FBXO7 is an F-box protein (FBP) containing an F-box domain and acts as a substrate recognition adaptor protein of Skp1–Cullin-1 (CUL1)–F-Box (SCF) E3 ligases complexes (8Nelson D. Randle S.J. Laman H. Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins.Open Biol. 2013; 3: 130131Crossref PubMed Scopus (66) Google Scholar). 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Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region.J. Biol. Chem. 2004; 279: 32592-32602Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Kuiken H.J. Egan D.A. Laman H. Bernards R. Beijersbergen R.L. Dirac A.M. Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.J. Cell. Mol. Med. 2012; 16: 2140-2149Crossref PubMed Scopus (32) Google Scholar, 18Kang J. Chung K.C. The F-box protein FBXO7 positively regulates bone morphogenetic protein-mediated signaling through Lys-63-specific ubiquitination of neurotrophin receptor-interacting MAGE (NRAGE).Cell. Mol. Life Sci. 2015; 72: 181-195Crossref PubMed Scopus (25) Google Scholar, 19Vingill S. Brockelt D. Lancelin C. Tatenhorst L. Dontcheva G. 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Small-molecule inhibitors of FBXO7 that interfere with the FBXO7–PINK1 interaction reduce mitochondrial injury and inflammation, leading to neuroprotection in primary cortical neurons, human neuroblastoma cells, and PD patient–derived cells (21Liu Y. Lear T.B. Verma M. Wang K.Z. Otero P.A. McKelvey A.C. et al.Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.JCI Insight. 2020; 5e131834Crossref Scopus (35) Google Scholar). Silent information regulator 2 is an NAD+-dependent protein deacetylase and extends the life span of yeast (22Blander G. Guarente L. The Sir2 family of protein deacetylases.Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1335) Google Scholar). Sirtuins are mammalian homologs of silent information regulator 2, which share a conserved NAD+-dependent catalytic domain, localize to different subcellular compartments, and regulate various physiological processes, including cell cycle progression, proliferation, apoptosis, metabolism, genomic stability, and aging (23Haigis M.C. Sinclair D.A. Mammalian sirtuins: biological insights and disease relevance.Annu. Rev. Pathol. 2010; 4: 253-295Crossref Scopus (1677) Google Scholar). Therefore, dysregulation of proper sirtuin functions is implicated in the pathogenesis of many human diseases, such as cancer, type II diabetes, and neurodegenerative diseases. Among the seven mammalian sirtuins, SIRT7 predominantly localizes to the nucleus and is involved in various cellular processes, including metabolism, ribosome biogenesis, and stress resistance (24Blank M.F. Grummt I. The seven faces of SIRT7.Transcription. 2017; 8: 67-74Crossref PubMed Scopus (84) Google Scholar, 25Wu Y. Li Y. Zhu K.S. Wang H. Zhu W.G. Advances in cellular characterization of the sirtuin isoform, SIRT7.Front. Endocrinol. (Lausanne). 2018; 9: 652Crossref PubMed Scopus (64) Google Scholar). Many studies have revealed that sirtuins affect several life extension pathways as well as brain function, which could offer new targets in the treatment of neurodegenerative diseases, including Alzheimer's disease and PD (26Carafa V. Rotili D. Forgione M. Cuomo F. Serretiello E. Hailu G.S. et al.Sirtuin functions and modulation: from chemistry to the clinic.Clin. Epigenet. 2016; 8: 61Crossref PubMed Scopus (241) Google Scholar, 27Tang B.L. Sirtuins as modifiers of Parkinson's disease pathology.J. Neurosci. Res. 2017; 95: 930-942Crossref PubMed Scopus (33) Google Scholar). For example, SIRT1 was found to protect human neuroblastoma SH-SY5Y cells by downregulating the expression of NF-κB and cleaved poly(ADP-ribose) polymerase 1 and by reducing the formation of phospho-α-Syn aggregates, whereas SIRT2 promoted toxic protein aggregation by deacetylating α-Syn and α-tubulin, thereby exacerbating the pathogenesis of PD (28Manjula R. Anuja K. Alcain F.J. SIRT1 and SIRT2 activity control in neurodegenerative disease.Front. Pharmacol. 2021; 11: 585821Crossref PubMed Scopus (73) Google Scholar). Furthermore, SIRT3 was reported to protect cells against 1-methyl-4-phenylpyridinium+ (MPP+)-induced neuronal injury (29Cui X.X. Li X. Dong S.Y. Guo Y.J. Liu T. Wu Y.C. SIRT3 deacetylated and increased citrate synthase activity in PD model.Biochem. Biophys. Res. Commun. 2017; 484: 767-773Crossref PubMed Scopus (51) Google Scholar). However, little is known about the functionality of SIRT7 with respect to cell viability and its implication in the progression of PD. Based on accumulating evidence that several FBPs and sirtuins are functionally linked, in this study, we further examined whether and how mammalian sirtuins and FBXO7 are closely associated. We here show that FBXO7 specifically binds to SIRT7. In addition, we found that FBXO7 acts as a novel ubiquitin E3 ligase that targets SIRT7, promoting its ubiquitination and subsequent SCF-dependent proteasomal degradation. Furthermore, FBXO7-mediated degradation of SIRT7 was observed during hydrogen peroxide (H2O2)–induced cell death in SH-SY5Y cells, which considerably promoted the cytotoxicity. Taken together, our study implies that the functional relationship between FBXO7 and cytoprotective SIRT7 in a reverse manner may play a role in H2O2-induced cell death and could contribute to the pathogenesis of PD. Based on our previous finding that FBXO7 exacerbates 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by interacting with aging-related FOXO4 and promoting its degradation (15Lee S.H. Jung S. Lee Y.J. Hyun M. Chung K.C. FBXO7 triggers caspase 8-mediated proteolysis of the transcription factor FOXO4 and exacerbates neuronal cytotoxicity.J. Biol. Chem. 2021; 297: 101426Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), we speculated that FBXO7 may also be physically and/or functionally related to sirtuins, another family of crucial aging-related genes. To investigate whether this hypothesis is valid, we first checked whether FBXO7 affects the levels of the seven sirtuins when they were coexpressed. When human embryonic kidney 293 (HEK293) cells were transiently transfected with a plasmid encoding one of the seven sirtuins with a FLAG tag at the C terminus alone or together with HA-FBXO7, ectopically expressed FBXO7 effectively reduced the level of SIRT7, whereas the other six sirtuin levels were unaffected (Fig. 1A). Unlike other sirtuins, the putative role of SIRT7 in cell viability and its functional regulation have not been elucidated; therefore, we further assessed the link between FBXO7 and SIRT7 and particularly focused on whether SIRT7 may act as a new target of FBXO7. We confirmed that ectopically expressed SIRT7 interacted with FBXO7 in HEK293 cells (Fig. 1B). The biochemical interaction between endogenous FBXO7 and SIRT7 was also confirmed in HEK293 cells (Fig. 1C). In addition, the interaction between endogenous FBXO7 and SIRT7 was verified in mouse whole-brain lysates (Fig. 1D). These results suggested that the observed FBXO7–SIRT7 interaction was not an artifact of DNA transfection in immortalized cancer cells but rather represents a specific interaction in mammalian cells. Subsequently, we examined whether the two proteins colocalized in human neuroblastoma SH-SY5Y cells. Since endogenous SIRT7 is quickly degraded, the cells were treated with MG132, and immunohistochemical analysis of SH-SY5Y cells revealed that endogenous FBXO7 and SIRT7 colocalized mainly in the nucleus (Fig. 1E). To clarify the FBXO7 domain(s) responsible for the SIRT7 interaction, several FLAG-tagged FBXO7-deletion mutants were generated (Fig. S1A). Each of these mutants lacked one of the following wildtype protein domains: the Ubl domain (▵U), F-box domain (▵F), C-terminal region of amino acids 376 to 522 (▵C), or N-terminal region of amino acids 1 to 333 (▵N). HEK293 cells were then transfected with the plasmid encoding FLAG-tagged wildtype FBXO7 alone, together with wildtype SIRT7-V5/His or one of the deletion mutants. After coimmunoprecipitation analysis of cell lysates with an anti-V5 antibody, immunoblotting analysis revealed that SIRT7 bound to FBXO7-▵U, FBXO7-▵F, and FBXO7-▵C, as well as its binding to full-length FBXO7. However, this interaction was not observed for FBXO7-▵N (Fig. S1B). Taken together, these results suggested that FBXO7 binds to SIRT7 and affects its expression level in mammalian cells and that the region spanning amino acid residues 92 to 333 of FBXO7, including the FP domain, is important for SIRT7 binding. To gain further insight into the link between FBXO7 and SIRT7, we examined whether FBXO7, as a component of SCF E3 ligase, negatively regulates the level of SIRT7. Similar to the pattern shown in Figure 1, A and B, exogenous and endogenous SIRT7 levels were significantly reduced in the presence of FBXO7 in a dose-dependent manner (Fig. 2, A and B). In contrast, siRNA-mediated FBXO7 knockdown in HEK293 cells significantly increased the endogenous SIRT7 levels (Fig. 2C). In addition, endogenous SIRT7 levels were significantly increased in FBXO7-knockout HAP1 cells (Fig. 2D). Moreover, reintroduction of wildtype FBXO7 into FBXO7-knockout cells reduced endogenous SIRT7 levels (Fig. 2D). To determine whether the catalytic activity of FBXO7 is required for SIRT7 downregulation, HEK293 cells were transfected for 24 h with plasmids encoding SIRT7-V5/His alone or together with FLAG-FBXO7-WT or FLAG-FBXO7-▵U, which lacks the Ubl domain and thus acts as a catalytically inactive mutant in SCF. The FBXO7-▵U mutant had no effect on SIRT7 levels, in contrast to the effect of FBXO7-WT (Fig. 2E). These results suggested that the catalytic activity of FBXO7 is critical for SIRT7 reduction. Next, we assessed whether FBXO7 regulates the stability of SIRT7 by measuring the SIRT7 half-life. HEK293 cells were transfected with plasmids encoding V5/His-tagged SIRT7, either alone or in combination with FLAG-FBXO7, and treated with 25 μM cycloheximide for various times. Immunoblot analysis revealed that the half-life of SIRT7 was significantly decreased by FBXO7 (Fig. 2F). In contrast, the half-life of endogenous SIRT7 was stabilized by siRNA-mediated FBXO7 knockdown (Fig. 2G). Taken together, these results suggested that FBXO7 promoted the degradation of SIRT7. Next, we determined whether FBXO7-mediated SIRT7 degradation occurs through one of two intracellular proteolysis systems: the ubiquitin proteasome system (UPS) or the lysosome-involved autophagy pathway. HEK293 cells were transfected for 24 h with plasmid encoding SIRT7-V5/His or FLAG-FBXO7 alone or in combination and then further treated with either a proteasome inhibitor (MG132 or epoxomicin) or a lysosomal autophagy inhibitor (NH4Cl). As shown in Figure 3A, cells treated with the proteasome inhibitor exhibited restored SIRT7 levels. However, treatment with NH4Cl had no effect on the FBXO7-mediated reduction of SIRT7 levels (Fig. 3B), suggesting that the degradation of SIRT7 occurs through the UPS. We then examined whether the proteolysis of SIRT7 by FBXO7 occurred through the SCF complex–dependent pathway using MLN4924, a commonly employed chemical inhibitor of SCF. Specifically, MLN4924 acts as an inhibitor of the NEDD8-activating enzyme, preventing the NEDDylation of cullin and thereby inactivating cullin-RING E3 ligase activity (30Lan H. Tang Z. Jin H. Sun Y. Neddylation inhibitor MLN4924 suppresses growth and migration of human gastric cancer cells.Sci. Rep. 2016; 6: 24218Crossref PubMed Scopus (74) Google Scholar). When HEK293 cells were pretreated with MLN4924, endogenous SIRT7 accumulated in the presence of FBXO7 in a dose-dependent manner (Fig. 3C). To verify the involvement of the SCF complex in FBXO7-mediated SIRT7 degradation, HEK293 cells were transfected with either control siRNA or siRNA-FBXO7 for 48 h, followed by treatment with vehicle or MLN4924. Consistent with the results shown in Figure 3C, MLN4924 treatment increased SIRT7 levels compared with those of cells transfected with control siRNA (Fig. 3D). However, this effect was not observed in cells transfected with FBXO7-siRNA (Fig. 3D). Likewise, the accumulation of SIRT7 was not seen in FBXO7-knockout cells (Fig. 3E). These results revealed that SIRT7 expression was reduced through SCFFBXO7-dependent proteasomal degradation. As SIRT7 levels were significantly reduced through SCFFBXO7-dependent proteasome degradation (Fig. 3), we determined that it is highly probable that SIRT7 could be polyubiquitinated by FBXO7. To explore this possibility, HEK293 cells were transfected for 24 h with plasmids encoding SIRT7-V5/His alone or together with FLAG-FBXO7-WT or FLAG-FBXO7-▵U, followed by immunoprecipitation of the cell lysates with anti-V5 antibody. Immunoblotting of the samples with anti-FLAG antiserum revealed that wildtype FBXO7, but not FBXO7-▵U mutant, promotes the polyubiquitination of SIRT7 (Fig. 4A). We then examined whether the FBXO7-mediated polyubiquitination of SIRT7 specifically targets either K48- or K63-linked chains. As expected, FBXO7-mediated SIRT7 polyubiquitination was significantly increased by proteasome-linked anti-K48-linked-ubiquitin antibody but not by anti-K63-linked-ubiquitin antibody (Fig. 4, B and C). In addition, SIRT7 polyubiquitination was reduced in FBXO7-knockout cells (Fig. 4D). These results demonstrated that FBXO7 directly mediates the polyubiquitination of SIRT7 by targeting K48-linked polyubiquitin chains. We further examined whether the FBXO7-mediated polyubiquitination of SIRT7 was dependent on the SCF complex. When HEK293 cells were transfected with FLAG-FBXO7, the polyubiquitination of SIRT7 was markedly increased in cells transfected with control siRNA, whereas this increase was not observed in cells transfected with CUL1-siRNA, targeting a component of the SCF complex (Fig. S2). These results further suggested that FBXO7 promotes polyubiquitination of SIRT7 in an SCF complex–dependent manner. Next, we attempted to identify the site(s) of SIRT7 that undergo FBXO7-meditated ubiquitination. A total of 21 lysine sites within SIRT7 were individually replaced with arginine one or two sites at a time, and we examined whether FBXO7-mediated SIRT7 ubiquitination could be still observed using those SIRT7 mutants. Among the 19 mutants, we found that SIRT7 mutants with substitutions at K292R, K395R, and K397/K398R displayed significantly reduced FBXO7-mediated ubiquitination (Fig. S3, A and B) compared with that of wildtype SIRT7. These four residues were then mutated to arginine (SIRT7-4KR), and the same experiments were performed (Fig. 4E). Compared with that of SIRT7-WT, the SIRT7-4KR mutant level was not considerably reduced by FBXO7 (Fig. 4F). In addition, FBXO7-mediated ubiquitination of the SIRT7-4KR mutant was markedly reduced compared with that of SIRT7-WT (Fig. 4G). These results suggested that FBXO7 mediates the polyubiquitination of SIRT7 by targeting at least four lysine sites of SIRT7: K292, K395, K397, and K398. In addition to multiple histones, the deacetylase SIRT7 binds to DNA polymerase I, II, III, and several transcription factors, facilitating DNA transcription (31Barber M. Michishita-kioi E. Xi Y. Tasselli L. Kioi M. Moqtaderi Z. et al.SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.Nature. 2012; 487: 114-118Crossref PubMed Scopus (463) Google Scholar). In addition, SIRT7 has a specific activity to catalyze deacetylation of H3K18 and H3K36 (31Barber M. Michishita-kioi E. Xi Y. Tasselli L. Kioi M. Moqtaderi Z. et al.SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.Nature. 2012; 487: 114-118Crossref PubMed Scopus (463) Google Scholar, 32Wang W.W. Angulo-Ibanez M. Lyu J. Kurra Y. Tong Z. Wu B. et al.A click chemistry approach reveals the chromatin-dependent histone H3K36 deacelyase nature of SIRT7.J. Am. Chem. Soc. 2019; 141: 2362-2473Google Scholar). Based on this report, we investigated whether FBXO7 controls the acetylation of H3K18 via SIRT7 degradation. To examine whether the FBXO7-mediated degradation of SIRT7 regulates the endogenous acetylated H3K18 level, HEK293 cells were transfected with plasmids encoding SIRT7-V5/His alone or in combination with FLAG-FBXO7-WT or FLAG-FBXO7-▵U. Immunoblot analysis of cell lysates using an anti-H3K18ac antibody revealed that acetylated H3K18 levels were decreased by SIRT7 alone (Fig. 5A). In contrast, FBXO7-WT, but not FBXO7-▵U, increased acetylated H3K18 levels via the reduction of SIRT7 (Fig. 5A). Moreover, among FBXO7-specific siRNA #1, #2, and #3, downregulation of acetylated H3K18 and upregulation of SIRT7 were observed most strongly in cells transfected with FBXO7-siRNA #2 (Fig. 5B). Likewise, acetylated H3K36 levels were also decreased by siRNA-mediated FBXO7 knockdown (Fig. 5C). Based on the report that SIRT7 induces the deacetylation of H3K18 at the promoter region of RPS20, resulting in the repression of its transcription (31Barber M. Michishita-kioi E. Xi Y. Tasselli L. Kioi M. Moqtaderi Z. et al.SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.Nature. 2012; 487: 114-118Crossref PubMed Scopus (463) Google Scholar), we further assessed and compared the changes in RPS20 mRNA expression in the absence and presence of FBXO7. Real-time PCR analysis revealed that cells overexpressing exogenous SIRT7, which was used as a positive control, had approximately 40% lower RPS20 mRNA expression levels than those of controls (Fig. 5D). By contrast, cells overexpressing exogenous FBXO7 showed no change in RPS20 mRNA levels compared with those of mock-transfected cells (Fig. 5D). Furthermore, the cells coexpressing exogenous SIRT7 and FBXO7 showed significantly increased RPS20 mRNA levels when compared with those of cells overexpressing SIRT7 alone (Fig. 5D). To verify the effect of FBXO7-mediated polyubiquitination and the proteolysis of SIRT7 on RPS20 transcription, we further examined the influence of SIRT7-4KR on RPS20 mRNA expression. After endogenous SIRT7 expression was depleted using SIRT7-specific siRNA, either SIRT7-WT or SIRT7-4KR was reintroduced into SIRT7-depleted cell. Similar to the patterns shown in Figure 5D, real-time PCR analyses demonstrated that RPS20 mRNA expression levels were significantly increased by siRNA-mediated SIRT7 knockdown. In addition, SIRT7-knockdown cells reintroduced with SIRT7-4KR mutant displayed more strongly decreased RPS20 mRNA expression compared with that of SIRT7-knockdown cells reintroduced with SIRT7-WT (Fig. 5E). These data indicated that endogenous FBXO7 might not affect the function of the SIRT7-4KR mutant. Collectively, these results suggested that FBXO7 negatively regulates the deacetylase activity of SIRT7 via ubiquitination and degradation. Since several studies have reported that both FBXO7 and SIRT7 act as regulators of the stress response (15Lee S.H. Jung S. Lee Y.J. Hyun M. Chung K.C. FBXO7 triggers caspase 8-mediated proteolysis of the transcription factor FOXO4 and exacerbates neuronal cytotoxicity.J. Biol. 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Deng J. et al.H2O2 down-regulates SIRT7's protective role of endothelial premature dysfunction via microRNA-335-5p.Biosci. Rep. 2022; 42 (BSR20211775)Crossref Scopus (8) Google Scholar), we investigated whether FBXO7-induced SIRT7 degradation affects cell viability in response to various stress inducers. First, SH-SY5Y cells were treated with various neurotoxic stimuli, including MPP+, 6-OHDA, rotenone, staurosporine, and H2O2, which cause neuronal cell death. Treatment with H2O2 caused a strong reduction in endogenous SIRT7 levels (Fig. 6A). H2O2 is commonly used to cause cellular oxidative stress and damage to cells, which also contributes to the progression of PD. The rate of reduction in endogenous SIRT7 rapidly decreased, which was completed by 4 h after H2O2 treatment (Fig. 6B). In addition, we found that endogenous SIRT7 expression was decreased in a dose-dependent manner by H2O2 in HEK293 cells (Fig. S4A). Next, we examined whether
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