RESUMO
DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.
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Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.
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Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.
Assuntos
Proteínas F-Box , Proteínas de Membrana , Proteínas Mitocondriais , Mitofagia , Proteólise , Proteínas Proto-Oncogênicas , Mitofagia/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Ligação Proteica , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Células HeLa , Células HEK293 , Animais , Ubiquitina-Proteína LigasesRESUMO
The transcription factor BACH1 regulates heme homeostasis and oxidative stress responses and promotes cancer metastasis upon aberrant accumulation. Its stability is controlled by two F-box protein ubiquitin ligases, FBXO22 and FBXL17. Here we show that the homodimeric BTB domain of BACH1 functions as a previously undescribed quaternary structure degron, which is deciphered by the two F-box proteins via distinct mechanisms. After BACH1 is released from chromatin by heme, FBXO22 asymmetrically recognizes a cross-protomer interface of the intact BACH1 BTB dimer, which is otherwise masked by the co-repressor NCOR1. If the BACH1 BTB dimer escapes the surveillance by FBXO22 due to oxidative modifications, its quaternary structure integrity is probed by a pair of FBXL17, which simultaneously engage and remodel the two BTB protomers into E3-bound monomers for ubiquitination. By unveiling the multifaceted regulatory mechanisms of BACH1 stability, our studies highlight the abilities of ubiquitin ligases to decode high-order protein assemblies and reveal therapeutic opportunities to block cancer invasion via compound-induced BACH1 destabilization.
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Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.
Assuntos
Ciclinas , Reparo de Erro de Pareamento de DNA , Animais , Ciclinas/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Interfase , Mamíferos/metabolismoRESUMO
The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S.
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Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
Assuntos
Endossomos , ATPases Vacuolares Próton-Translocadoras , Autofagia , Membrana Celular/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , HumanosRESUMO
IMPORTANCE: Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.
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Proteases Semelhantes à Papaína de Coronavírus , Interações entre Hospedeiro e Microrganismos , Glicoproteínas de Membrana , Proteínas de Membrana , Proteínas de Membrana Transportadoras , SARS-CoV-2 , Replicação Viral , Humanos , COVID-19/virologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/metabolismoRESUMO
Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Evasão Tumoral , Humanos , Apresentação de Antígeno , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos HLA , Neoplasias/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.
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Mitofagia , Fagocitose , Autofagia/fisiologia , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Humanos , Animais , CamundongosRESUMO
Immunomodulatory drugs (IMiDs), which include thalidomide and its derivatives, have emerged as the standard of care against multiple myeloma. They function as molecular glues that bind to the E3 ligase cereblon (CRBN) and induce protein interactions with neosubstrates, including the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). The subsequent ubiquitylation and degradation of these transcription factors underlies the antiproliferative activity of IMiDs. Here, we introduce photoswitchable immunomodulatory drugs (PHOIMiDs) that can be used to degrade Ikaros and Aiolos in a light-dependent fashion. Our lead compound shows minimal activity in the dark and becomes an active degrader upon irradiation with violet light. It shows high selectivity over other transcription factors, regardless of its state, and could therefore be used to control the levels of Ikaros and Aiolos with high spatiotemporal precision.
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Cells must avoid licensing of neosynthesized DNA to prevent rereplication. In this issue of Molecular Cell, Ratnayeke et al. (2022)1 reveal how the licensing factor CDT1, prior to its degradation, inhibits DNA elongation by suppressing CMG helicase progression at replication forks.
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Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , DNA , DNA Helicases/genéticaRESUMO
The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFßTrCP E3 ubiquitin ligase and silencing ßTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that ßTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.
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Serina-Treonina Quinase 3 , Ubiquitina-Proteína Ligases , Proteínas Contendo Repetições de beta-Transducina , Humanos , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Matriz Extracelular/metabolismo , Fosforilação , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Serina-Treonina Quinase 3/metabolismoRESUMO
The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Exorribonucleases/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Células A549 , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Antivirais/farmacologia , Humanos , SARS-CoV-2/enzimologia , Replicação Viral/efeitos dos fármacosRESUMO
Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Interferon Tipo I/metabolismo , Neoplasias Pulmonares/imunologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação/genética , Proteínas Repressoras/metabolismo , Evasão Tumoral/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunidade Inata/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
FBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and -2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.
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Fosfatase 6 de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas F-Box/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas Culina/genética , Proteínas Culina/metabolismo , Cicloexilaminas/farmacologia , Fosfatase 6 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 6 de Especificidade Dupla/genética , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Proteínas F-Box/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Indenos/farmacologia , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteólise , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
D-type cyclins (cyclin D1, D2, and D3, together cyclin D) are central drivers of the cell division cycle and well-described proto-oncoproteins. Rapid turnover of cyclin D is critical for its regulation, but the underlying mechanism has remained a matter of debate. Recently, AMBRA1 was identified as the major regulator of the stability of all three D-type cyclins. AMBRA1 serves as the substrate receptor for one of â¼40 CUL4-RING E3 ubiquitin ligase (CRL4) complexes to mediate the polyubiquitylation and subsequent degradation of cyclin D. Consequently, AMBRA1 regulates cell proliferation to impact tumor growth and the cellular response to cell cycle-targeted cancer therapies. Here we discuss the findings that implicate AMBRA1 as a core member of the cell cycle machinery.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciclina D2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Neoplasias/metabolismoAssuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Apresentação de Antígeno , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Ubiquitylation, a highly regulated post-translational modification, controls many cellular pathways that are critical to cell homeostasis. Ubiquitin ligases recruit substrates and promote ubiquitin transfer onto targets, inducing proteasomal degradation or non-degradative signaling. Accumulating evidence highlights the critical role of dysregulated ubiquitin ligases in processes associated with the initiation and progression of cancer. Depending on the substrate specificity and biological context, a ubiquitin ligase can act either as a tumor promoter or as a tumor suppressor. In this review, we focus on the regulatory roles of ubiquitin ligases and how perturbations of their functions contribute to cancer pathogenesis. We also briefly discuss current strategies for targeting or exploiting ubiquitin ligases for cancer therapy.