RESUMEN
Mutations in the DDHD2 (DDHD domain containing 2) gene cause autosomal recessive spastic paraplegia type 54 (SPG54), a rare neurodegenerative disorder characterized by the early childhood onset of progressive spastic paraplegia. DDHD2 is reported as the principal brain triacylglycerol (TAG) lipase whose dysfunction causes massive lipid droplet (LD) accumulation in the brains of SPG54 patients. However, the precise functions of DDHD2 in regulating LD catabolism are not yet fully understood. In a recent study, we demonstrate that DDHD2 interacts with multiple members of the Atg8-family proteins (MAP1LC3/LC3s, GABARAPs), which play crucial roles in lipophagy. DDHD2 possesses two LC3-interacting region (LIR) motifs that contribute to its LD-eliminating activity. Moreover, DDHD2 enhances the colocalization between LC3B and LDs to promote lipophagy. LD·ATTEC, a compound that tethers LC3 to LDs to enhance their macroautophagic/autophagic clearance, effectively counteracts DDHD2 deficiency-induced LD accumulation. These findings provide insights into the dual functions of DDHD2 as a TAG lipase and cargo receptor for lipophagy in neuronal LD catabolism, and also suggest a potential therapeutic approach for treating SPG54 patients.
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Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.
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Factor 4A Eucariótico de Iniciación , Diana Mecanicista del Complejo 1 de la Rapamicina , Biosíntesis de Proteínas , Proteínas Quinasas S6 Ribosómicas 70-kDa , Transducción de Señal , Ubiquitinación , Animales , Humanos , Ratones , Línea Celular Tumoral , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/genéticaRESUMEN
Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative disorders characterized by progressive lower limb spasticity and weakness. One subtype of HSP, known as SPG54, is caused by biallelic mutations in the DDHD2 gene. The primary pathological feature observed in patients with SPG54 is the massive accumulation of lipid droplets (LDs) in the brain. However, the precise mechanisms and roles of DDHD2 in regulating lipid homeostasis are not yet fully understood. Through Affinity Purification-Mass Spectroscopy (AP-MS) analysis, we identify that DDHD2 interacts with multiple members of the ATG8 family proteins (LC3, GABARAPs), which play crucial roles in lipophagy. Mutational analysis reveals the presence of two authentic LIR motifs in DDHD2 protein that are essential for its binding to LC3/GABARAPs. We show that DDHD2 deficiency leads to LD accumulation, while enhanced DDHD2 expression reduces LD formation. The LC3/GABARAP-binding capacity of DDHD2 and the canonical autophagy pathway both contribute to its LD-eliminating activity. Moreover, DDHD2 enhances the colocalization between LC3B and LDs to promote lipophagy. LD·ATTEC, a small molecule that tethers LC3 to LDs to enhance their autophagic clearance, effectively counteracts DDHD2 deficiency-induced LD accumulation. These findings provide valuable insights into the regulatory roles of DDHD2 in LD catabolism and offer a potential therapeutic approach for treating SPG54 patients.
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Fosfolipasas , Paraplejía Espástica Hereditaria , Humanos , Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia , Mutación/genética , Fosfolipasas/genética , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/patologíaRESUMEN
Mitophagy, the process of removing damaged mitochondria to promote cell survival, plays a crucial role in cellular functionality. However, excessive, or uncontrolled mitophagy can lead to reduced mitochondrial content that burdens the remaining organelles, triggering mitophagy-mediated cell death. FBXL4 mutations, which affect the substrate-binding adaptor of the CUL1 (cullin 1)-RING ubiquitin ligase complex (CRL1), have been linked to mitochondrial DNA depletion syndrome type 13 (MTDPS13) characterized by reduced mtDNA content and impaired energy production in affected organs. However, the mechanism behind FBXL4 mutation-driven MTDPS13 remain poorly understood. In a recent study, we demonstrate that the CRL1-FBXL4 complex promotes the degradation of BNIP3 and BNIP3L, two key mitophagy cargo receptors. Deficiency of FBXL4 results in a strong accumulation of BNIP3 and BNIP3L proteins and triggers high levels of BNIP3- and BNIP3L-dependent mitophagy. Patient-derived FBXL4 mutations do not affect its interaction with BNIP3 and BNIP3L but impair the assembly of an active CRL1-FBXL4 complex. Furthermore, excessive mitophagy is observed in knockin mice carrying a patient-derived FBXL4 mutation, and in cortical neurons generated from human patient induced pluripotent stem cells (hiPSCs). These findings support the model that the CRL1-FBXL4 complex tightly restricts basal mitophagy, and its dysregulation leads to severe symptoms of MTDPS13.
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Autofagia , Enfermedades Mitocondriales , Mitofagia , Animales , Humanos , Ratones , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas F-Box/genética , Ubiquitina-Proteína Ligasas/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismoRESUMEN
Encephalomyopathic mitochondrial DNA (mtDNA) depletion syndrome 13 (MTDPS13) is an autosomal recessive disorder arising from biallelic F-box and leucine-rich repeat (LRR) protein 4 (FBXL4) gene mutations. Recent advances have shown that excessive BCL2 interacting protein 3 (BNIP3)/ BCL2 interacting protein 3 like (BNIP3L)-dependent mitophagy underlies the molecular pathogenesis of MTDPS13. Here, we provide an overview of these groundbreaking findings and discuss potential therapeutic strategies for this fatal disease.
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Encefalomiopatías Mitocondriales , Mitofagia , Humanos , Mitofagia/genética , Mitocondrias/metabolismo , ADN Mitocondrial/genética , Mutación , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/metabolismo , Encefalomiopatías Mitocondriales/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
[This corrects the article on p. 2797 in vol. 9, PMID: 31911863.].
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The mechanistic target of the rapamycin (mTOR) pathway, which participates in the regulation of cellular growth and metabolism, is aberrantly regulated in various cancer types. The mTOR complex 2 (mTORC2), which consists of the core components mTOR, Rictor, mSin1, and mLST8, primarily responds to growth signals. However, the coordination between mTORC2 assembly and activity remains poorly understood. Keap1, a major sensor of oxidative stress in cells, functions as a substrate adaptor for Cullin 3-RING E3 ubiquitin ligase (CRL3) to promote proteasomal degradation of NF-E2-related factor 2 (NRF2), which is a transcription factor that protects cells against oxidative and electrophilic stress. In the present study, we demonstrate that Keap1 binds to mLST8 via a conserved ETGE motif. The CRL3Keap1 ubiquitin ligase complex promotes non-degradative ubiquitination of mLST8, thus reducing mTORC2 complex integrity and mTORC2-AKT activation. However, this effect can be prevented by oxidative/electrophilic stresses and growth factor signaling-induced reactive oxygen species (ROS) burst. Cancer-derived Keap1 or mLST8 mutations disrupt the Keap1-mLST8 interaction and allow mLST8 to evade Keap1-mediated ubiquitination, thereby enhancing mTORC2-AKT activation and promoting cell malignancy and remodeling cell metabolism. Our findings provide new insights into the molecular mechanisms of Keap1/mLST8 mutation-driven tumorigenesis by promoting mTORC2-AKT activation, which is independent of the canonical NRF2 pathway.
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Neoplasias , Proteínas Proto-Oncogénicas c-akt , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias/genética , MutaciónRESUMEN
The KEAP1-NRF2 axis is the principal regulator of cellular responses to oxidative and electrophilic stressors. NRF2 hyperactivation is frequently observed in many types of cancer and promotes cancer initiation, progression, metastasis, and resistance to various therapies. Here, we determined that dipeptidyl peptidase 9 (DPP9) is a regulator of the KEAP1-NRF2 pathway in clear cell renal cell carcinoma (ccRCC). DPP9 was markedly overexpressed at the mRNA and protein levels in ccRCC, and high DPP9 expression levels correlated with advanced tumor stage and poor prognosis in patients with ccRCC. Protein affinity purification to identify functional partners of DPP9 revealed that it bound to KEAP1 via a conserved ESGE motif. DPP9 disrupted KEAP1-NRF2 binding by competing with NRF2 for binding to KEAP1 in an enzyme-independent manner. Upregulation of DPP9 led to stabilization of NRF2, driving NRF2-dependent transcription and thereby decreasing cellular reactive oxygen species levels. Moreover, DPP9 overexpression suppressed ferroptosis and induced resistance to sorafenib in ccRCC cells, which was largely dependent on the NRF2 transcriptional target SLC7A11. Collectively, these findings indicate that the accumulation of DPP9 results in hyperactivation of the NRF2 pathway to promote tumorigenesis and intrinsic drug resistance in ccRCC. SIGNIFICANCE: DPP9 overcomes oxidative stress and suppresses ferroptosis in ccRCC by binding to KEAP1 and promoting NRF2 stability, which drives tumor development and sorafenib resistance.
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Carcinoma de Células Renales , Ferroptosis , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/genética , Sorafenib/farmacologíaRESUMEN
Mitochondria are essential organelles found in eukaryotic cells that play a crucial role in ATP production through oxidative phosphorylation (OXPHOS). Mitochondrial DNA depletion syndrome (MTDPS) is a group of genetic disorders characterized by the reduction of mtDNA copy number, leading to deficiencies in OXPHOS and mitochondrial functions. Mutations in FBXL4, a substrate-binding adaptor of Cullin 1-RING ubiquitin ligase complex (CRL1), are associated with MTDPS, type 13 (MTDPS13). Here, we demonstrate that, FBXL4 directly interacts with the mitophagy cargo receptors BNIP3 and BNIP3L, promoting their degradation through the ubiquitin-proteasome pathway via the assembly of an active CRL1FBXL4 complex. However, MTDPS13-associated FBXL4 mutations impair the assembly of an active CRL1FBXL4 complex. This results in a notable accumulation of BNIP3/3L proteins and robust mitophagy even at basal levels. Excessive mitophagy was observed in Knockin (KI) mice carrying a patient-derived FBXL4 mutation and cortical neurons (CNs)-induced from MTDPS13 patient human induced pluripotent stem cells (hiPSCs). In summary, our findings suggest that abnormal activation of BNIP3/BNIP3L-dependent mitophagy impairs mitochondrial homeostasis and underlies FBXL4-mutated MTDPS13.
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The pathologic significance of the circular RNA DDIT4 (circDDIT4), which is formed by backsplicing at the 3'-untranslated region (UTR) with a 5' splice acceptor site in exon 2 of linear DDIT4 mRNA, has yet to be determined. Our study found that circDDIT4 is downregulated in prostate cancer and functions as a tumor suppressor during prostate cancer progression. By competitively binding to ELAV-like RNA binding protein 1 (ELAVL1/HuR) through its 3'-UTR, circDDIT4 acts as a protein sponge to decrease the expression of prostate cancer-overexpressed anoctamin 7 (ANO7). This promotes prostate cancer cell apoptosis while inhibiting cell proliferation and metastasis. Furthermore, we discovered that N6-methyladenosine (m6A) modification facilitates the biogenesis of circDDIT4. The methyltransferase complex consisting of WTAP/METTL3/METTL14 increases the level of circDDIT4, while the RNA demethylase FTO decreases it. IMPLICATIONS: These findings suggest that abnormal cotranscriptional modification of m6A promotes prostate cancer initiation and progression via a circular RNA-protein-cell signaling network.
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Neoplasias de la Próstata , ARN Circular , Masculino , Humanos , ARN Circular/genética , Metiltransferasas/genética , Neoplasias de la Próstata/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismoRESUMEN
Blockade of programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L1) has evolved into one of the most promising immunotherapy strategies for cancer patients. Tumor cells frequently overexpress PD-L1 to evade T cell-mediated immune surveillance. However, the specific genetic alterations that drive aberrant overexpression of PD-L1 in cancer cells remain poorly understood. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in endometrial cancer (EC). Here, we report that SPOP negatively regulates PD-L1 expression at the transcriptional level. Wild-type SPOP binds to IRF1, a primary transcription factor responsible for the inducible expression of PD-L1, and subsequently triggers its ubiquitin- proteasomal degradation to suppress IRF1-mediated transcriptional upregulation of PD-L1. In contrast, EC-associated SPOP mutants lose their capacity to degrade IRF1 but stabilize IRF1, and upregulate PD-L1 expression. EC-associated SPOP mutations accelerate xenograft tumor growth partially by increasing IRF1 and PD-L1 expression. Together, we identify SPOP as a negative regulator of the IRF1-PD-L1 axis and characterize the critical roles of IRF1 and PD-L1 in SPOP mutation-driven tumor immune evasion in EC.
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Neoplasias Endometriales , Proteínas Nucleares , Escape del Tumor , Femenino , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Neoplasias Endometriales/genética , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Mutación/genética , Proteínas Nucleares/metabolismoRESUMEN
Lysosomes are essential catabolic organelles responsible for the degradation of biomacromolecules into low-molecular-weight materials for subsequent reuse. Neuronal ceroid lipofuscinoses (NCLs) are a group of fatal neurodegenerative lysosomal storage disorders characterized by the intracellular accumulation of lipoprotein aggregates (called ceroid lipofuscin) in neurons and other tissues. Mutations in KCTD7, which encodes a substrate-binding adaptor for the CUL3-RING E3 (CRL3) ubiquitin ligase complex, are categorized as a unique NCL subtype. However, the molecular mechanisms underlying the KCTD7-mutated NCLs remain unclear. In our recent study, we showed that KCTD7 deficiency leads to the accumulation of lysosomal storage deposits owing to lysosomal dysfunction and macroautophagic/autophagic defects. We identified CLN5 as an authentic substrate of CRL3-KCTD7 E3s. Wild-type KCTD7 targets CLN5 for ubiquitination and proteasomal degradation, whereas NCL patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive CLN5 accumulation in the endoplasmic reticulum. Accumulated CLN5 disrupts the interaction between CLN6-CLN8 and lysosomal enzymes, leading to impaired ER-to-Golgi trafficking of lysosomal enzymes. Thus, our findings indicate that KCTD7 is a key player in maintaining lysosomal and autophagic homeostasis and demonstrate that KCTD7 and CLN5, two NCL causative genes, are biochemically linked and function in a common neurodegenerative pathway.
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Lipofuscinosis Ceroideas Neuronales , Humanos , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Autofagia/genética , Tripeptidil Peptidasa 1 , Lisosomas/metabolismo , Mutación/genética , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Canales de Potasio/genéticaRESUMEN
Background: Ubiquitination and deubiquitination modifications play pivotal roles in eukaryotic life processes, regulating protein dynamics via the ubiquitin-proteasome pathway. Dysregulation can impact disease development, including cancer and neurodegenerative disorders. Increasing evidence highlights their role in tumorigenesis, modulating key proteins. OTUD3, a deubiquitinase, stabilizes PTEN, suppressing tumor growth by inhibiting PI3K-AKT signaling. Yet, further OTUD3 substrates remain underexplored. Methods: We employed the In vivo ubiquitination assay to investigate the ubiquitination role of OTUD3 on KPTN within the cellular context. Additionally, CRISPR/Cas9 editing and Immunofluorescence were utilized to study the impact of OTUD3 on the mTOR signaling pathway in cells. Furthermore, Cell proliferation assay and NMR were employed to explore the effects of OTUD3 on cellular growth and proliferation. Results: OTUD3 serves as a deubiquitinase for KPTN. OTUD3 interacts with KPTN, facilitated by the OTU domain within OTUD3. Further investigations confirmed KPTN's ubiquitination modification, primarily at lysine residue 49. Ubiquitination experiments demonstrated OTUD3's ability to mediate KPTN's deubiquitination without affecting its protein levels. This suggests KPTN's ubiquitination is a function-regulated, non-degradable modification. Under various amino acid starvation or stimulation conditions, overexpressing OTUD3 reduces mTORC1 signaling activation, while knocking out OTUD3 further enhances it. Notably, OTUD3's regulation of mTORC1 signaling relies on its deubiquitinase activity, and this effect is observed even in PTEN KO cells, confirming its independence from PTEN, a reported substrate. OTUD3 also promotes GATOR1's lysosomal localization, a process requiring KPTN's involvement. Ultimately, OTUD3 affects cellular metabolic pool products by downregulating the mTORC1 pathway, significantly inhibiting tumor cell growth and proliferation. Discussion: Our experiments shed light on an alternative perspective regarding the intrinsic functions of OTUD3 in inhibiting tumor development. We propose a novel mechanism involving KPTN-mediated regulation of the mTORC1 signaling pathway, offering fresh insights into the occurrence and progression of tumor diseases driven by related genes. This may inspire new approaches for drug screening and cancer treatment, potentially guiding future therapies for relevant tumors.
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BACKGROUND: The gene encoding the E3 ubiquitin ligase substrate-binding adapter Speckle-type BTB/POZ protein (SPOP) is frequently mutated in prostate cancer (PCa) and endometrial cancer (EC); however, the molecular mechanisms underlying the contribution of SPOP mutations to tumorigenesis remain poorly understood. METHODS: BRAF harbors a potential SPOP-binding consensus motif (SBC) motif. Co-immunoprecipitation assays demonstrated that BRAF interacts with SPOP. A series of functional analyses in cell lines were performed to investigate the biological significance of MAPK/ERK activation caused by SPOP mutations. RESULTS: Cytoplasmic SPOP binds to and induces non-degradative ubiquitination of BRAF, thereby reducing the interaction between BRAF and other core components of the MAPK/ERK pathway. SPOP ablation increased MAPK/ERK activation. EC- or PCa-associated SPOP mutants showed a reduced capacity to bind and ubiquitinate BRAF. Moreover, cancer-associated BRAF mutations disrupted the BRAF-SPOP interaction and allowed BRAF to evade SPOP-mediated ubiquitination, thereby upregulating MAPK/ERK signaling and enhancing the neoplastic phenotypes of cancer cells. CONCLUSIONS: Our findings provide new insights into the molecular link between SPOP mutation-driven tumorigenesis and aberrant BRAF-dependent activation of the MAPK/ERK pathway.
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Lysosomes are central organelles for cellular degradation and energy metabolism. Neuronal ceroid lipofuscinoses (NCLs) are a group of the most common neurodegenerative lysosomal storage disorders characterized by intracellular accumulation of ceroid in neurons. Mutations in KCTD7, a gene encoding an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, are categorized as a unique NCL subtype. However, the underlying mechanisms remain elusive. Here, we report various lysosomal and autophagic defects in KCTD7-deficient cells. Mechanistically, the CRL3-KCTD7 complex degrades CLN5, whereas patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive accumulation of CLN5. The accumulated CLN5 disrupts the interaction between CLN6/8 and lysosomal enzymes at the endoplasmic reticulum (ER), subsequently impairing ER-to-Golgi trafficking of lysosomal enzymes. Our findings reveal previously unrecognized roles of KCTD7-mediated CLN5 proteolysis in lysosomal homeostasis and demonstrate that KCTD7 and CLN5 are biochemically linked and function in a common neurodegenerative pathway.
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Lipofuscinosis Ceroideas Neuronales , Aparato de Golgi/metabolismo , Humanos , Hidrolasas , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Canales de Potasio/metabolismoRESUMEN
SQSTM1/p62 is a selective macroautophagy/autophagy receptor that drives ubiquitinated cargos toward the lysosome for degradation, and also a stress-induced scaffold protein that helps cells to cope with oxidative stress through sequestrating KEAP1 and subsequent activation of the NFE2L2/NRF2 antioxidant pathway. Accumulating evidence implicates SQSTM1 dysregulation in the induction of multiple oncogenic transformations in vivo. SPOP (speckle type BTB/POZ protein), an E3 ubiquitin ligase adaptor, is the most frequently mutated gene in prostate cancer (Pca), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis are still largely unknown. In a recent study, we describe a new role for SPOP as a negative regulator of autophagy and NFE2L2 pathway activation. SPOP binds and induces the non-degradative ubiquitination of SQSTM1 at Lys420. This post-translational modification decreases SQSTM1 body formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing SQSTM1-dependent autophagy, KEAP1 sequestration, and NFE2L2 activation. Notably, PCa-associated SPOP mutants lose the capacity to ubiquitinate SQSTM1 and instead enhance autophagy and the antioxidant response in a dominant-negative manner. Thus, our findings indicate the critical roles of autophagy and NFE2L2 pathway activation in PCa tumorigenesis by oncogenic SPOP mutations.
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Factor 2 Relacionado con NF-E2 , Proteínas Nucleares , Neoplasias de la Próstata , Proteínas Represoras , Antioxidantes/metabolismo , Autofagia/genética , Carcinogénesis , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Masculino , Mutación , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Proteínas Represoras/genética , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , UbiquitinaciónRESUMEN
Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival and appear to neutralize the cell death machinery by binding pro-apoptotic caspases. dcaf12 was recently identified as an apoptosis regulator in Drosophila. However, the underlying molecular mechanisms are unknown. Here we revealed that human DCAF12 homolog binds multiple IAPs, including XIAP, cIAP1, cIAP2, and BRUCE, through recognition of BIR domains in IAPs. The pro-apoptotic function of DCAF12 is dependent on its capacity to bind IAPs. In response to apoptotic stimuli, DCAF12 translocates from the nucleus to the cytoplasm, where it blocks the interaction between XIAP and pro-apoptotic caspases to facilitate caspase activation and apoptosis execution. Similarly, DCAF12 suppresses NF-κB activation in an IAP binding-dependent manner. Moreover, DCAF12 acts as a tumor suppressor to restrict the malignant phenotypes of cancer cells. Together, our results suggest that DCAF12 is an evolutionarily conserved IAP antagonist.
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Proteínas Inhibidoras de la Apoptosis , FN-kappa B , Apoptosis , Caspasas/metabolismo , Supervivencia Celular , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/genética , FN-kappa B/metabolismo , Dominios Proteicos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
p62/SQSTM1 is a selective autophagy receptor that drives ubiquitinated cargos towards autophagic degradation. This receptor is also a stress-induced scaffold protein that helps cells to cope with oxidative stress through activation of the Nrf2 pathway. Functional disorders of p62 are closely associated with multiple neurodegenerative diseases and cancers. The gene encoding the E3 ubiquitin ligase substrate-binding adapter SPOP is frequently mutated in prostate cancer (PCa), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis remain poorly understood. Here, we report that cytoplasmic SPOP binds and induces the non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This protein modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing p62-dependent autophagy. Moreover, we show that SPOP relieves p62-mediated Keap1 sequestration, which ultimately decreases Nrf2-mediated transcriptional activation of antioxidant genes. We further show that PCa-associated SPOP mutants lose the capacity to ubiquitinate p62 and instead promote autophagy and the redox response in a dominant-negative manner. Thus, our findings indicate oncogenic roles of autophagy and Nrf2 activation in the tumorigenesis of SPOP-mutated PCa.
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Factor 2 Relacionado con NF-E2 , Proteínas Nucleares , Neoplasias de la Próstata , Proteínas Represoras , Proteína Sequestosoma-1 , Humanos , Masculino , Autofagia/fisiología , Carcinogénesis , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
The cystine/glutamate antiporter SLC7A11 (commonly known as xCT) functions to import cystine for glutathione biosynthesis, thereby protecting cells from oxidative stress and ferroptosis, a regulated form of non-apoptotic cell death driven by the accumulation of lipid-based reactive oxygen species (ROS). p14ARF, a well-established tumor suppressor, promotes ferroptosis by inhibiting NRF2-mediated SLC7A11 transcription. Here, we demonstrate the crucial role of Cullin 2 RING E3 ligase (CRL2)-KLHDC3 E3 ubiquitin ligase complex in regulating p14ARF protein stability. KLHDC3 acts as a CRL2 adaptor that specifically recognizes a C-terminal degron in p14ARF and triggers p14ARF for ubiquitin-proteasomal degradation. This regulation mode is absent in the murine p14ARF homolog, p19arf which lacks the C-terminal degron. We also show that KLHDC3 suppresses ferroptosis in vitro and supports tumor growth in vivo by relieving p14ARF-mediated suppression of SLC7A11 transcription. Overall, these findings reveal that the protein stability and pro-ferroptotic function of p14ARF are controlled by a CRL2 E3 ubiquitin ligase complex, and suggest that suppression of the p14ARF-NRF2-SLC7A11 regulatory pathway by KLHDC3 overexpression likely contributes to cancer progression.