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1.
Toxicol Lett ; 325: 25-33, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112875

RESUMO

RATIONALE: Diacetyl (DA; 2,3-butanedione) is a chemical found commonly in foods and e-cigarettes. When inhaled, DA causes epithelial injury, though the mechanism of repair remain poorly understood. The objective of this study was to evaluate airway basal cell repair after DA vapor exposure. METHODS: Primary human bronchial epithelial cells were exposed to DA or PBS for 1 h. Lactate dehydrogenase, cleaved caspase 3/7 and trans-epithelial electrical resistance were measured prior to and following exposure. Exposed cultures were analyzed for the airway basal cell markers keratin 5 and p63 as well as ubiquitin and proteasome activity. Cultures were also treated with a proteasome inhibitor (MG132). RESULTS: DA vapor exposure caused a transient decrease in trans-epithelial electrical resistance in all DA-exposed cultures. Supernatant lactate dehydrogenase and cleaved caspase 3/7 increased significantly at the highest DA concentration but not at lower DA concentrations. Increased keratin 5 ubiquitination occurred after DA exposure but resolved by day 3. Damage to airway basal cells persisted at day 3 in the presence of MG132. CONCLUSIONS: Diacetyl exposure results in airway basal cell injury with keratin 5 ubiquitination and decreased p63 expression. The ubiquitin-proteasome-pathway partially mediates airway basal cell repair after acute DA exposure.


Assuntos
Diacetil/toxicidade , Mucosa Respiratória/patologia , Biomarcadores , Brônquios/citologia , Brônquios/patologia , Caspases/metabolismo , Diacetil/administração & dosagem , Impedância Elétrica , Sistemas Eletrônicos de Liberação de Nicotina , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Exposição por Inalação , Queratina-5/metabolismo , L-Lactato Desidrogenase/metabolismo , Leupeptinas/farmacologia , Proteínas de Membrana , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
2.
PLoS One ; 15(1): e0227727, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940398

RESUMO

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.


Assuntos
Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Compostos de Benzilideno/química , Compostos de Benzilideno/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Estrutura Molecular , Neoplasias/genética , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/uso terapêutico , Ligação Proteica , Relação Estrutura-Atividade , Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nat Commun ; 11(1): 433, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974380

RESUMO

Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Erastin, the ferroptosis activator, binds to voltage-dependent anion channels VDAC2 and VDCA3, but treatment with erastin can result in the degradation of the channels. Here, the authors show that Nedd4 is induced following erastin treatment, which leads to the ubiquitination and subsequent degradation of the channels. Depletion of Nedd4 limits the protein degradation of VDAC2/3, which increases the sensitivity of cancer cells to erastin. By understanding the molecular mechanism of erastin-induced cellular resistance, we can discover how cells adapt to new molecules to maintain homeostasis. Furthermore, erastin-induced resistance mediated by FOXM1-Nedd4-VDAC2/3 negative feedback loop provides an initial framework for creating avenues to overcome the drug resistance of ferroptosis activators.


Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Piperazinas/farmacologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Proteína Forkhead Box M1/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos Nus , Proteínas de Transporte da Membrana Mitocondrial/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitinação/efeitos dos fármacos , Canal de Ânion 2 Dependente de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Adv Exp Med Biol ; 1217: 297-315, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31898235

RESUMO

Neddylation is a posttranslational modification that conjugates a ubiquitin-like protein NEDD8 to substrate proteins. The best-characterized substrates of neddylation are the cullin subunits of cullin-RING E3 ubiquitin ligase complexes (CRLs). CRLs as the largest family of E3 ubiquitin ligases control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. The process of protein neddylation is overactivated in multiple types of human cancers, providing a sound rationale as an attractive anticancer therapeutic strategy, evidenced by the development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Recently, increasing evidence strongly indicates that neddylation inhibition by MLN4924 exerts anticancer effects mainly by triggering cell apoptosis, senescence, and autophagy and causing angiogenesis suppression, inflammatory responses, and chemo-/radiosensitization in a context-dependent manner. Here, we briefly summarize the latest progresses in this field, focusing on the preclinical studies to validate neddylation modification as a promising anticancer target.


Assuntos
Proteína NEDD8/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ciclopentanos/farmacologia , Ciclopentanos/uso terapêutico , Humanos , Proteína NEDD8/metabolismo , Neoplasias/patologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
5.
Nat Commun ; 11(1): 409, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964869

RESUMO

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.


Assuntos
Complexo de Golgi/metabolismo , Ionóforos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteostase/fisiologia , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Ionóforos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Monensin/farmacologia , Monensin/uso terapêutico , Mieloma Múltiplo/patologia , Proteólise/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteína com Valosina/metabolismo
6.
Nat Commun ; 10(1): 5770, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852899

RESUMO

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.


Assuntos
Autofagia/imunologia , Proteína Beclina-1/metabolismo , Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Proteínas Quinases Associadas a Fase S/metabolismo , Animais , Autofagia/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Proteólise/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/imunologia , Células Vero
7.
Nucleic Acids Res ; 47(16): 8502-8520, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616951

RESUMO

Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Reparo do DNA , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Acetilação/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dano ao DNA , Células HEK293 , Humanos , Mutação , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
8.
Mol Carcinog ; 58(12): 2316-2326, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31553086

RESUMO

Primary tumor can induce the formation of premetastatic niche. The hyperpermeability of the vessels in the premetastatic niche is the first step in the development of metastasis. However, the cellular and molecular mechanisms of vascular hyperpermeability remain to be elucidated. In this study, 4T1 breast cells were injected into the breasts of mice to establish a tumor model. Our results showed that primary tumors induced hyperpermeability of the vessels in the premetastatic lung. Subsequent studies showed that the level of vascular endothelial growth factor (VEGF) was elevated in the tumor-bearing mice serum and the levels of tight junction (TJ) proteins occludin and ZO-1 were decreased in the premetastatic lung. In vitro studies demonstrated that VEGF increased the permeability of dextran and decreased the levels of occludin and ZO-1 in human umbilical vein endothelial cells. Moreover, the hyperpermeability of vessels and the degradation of occludin was blocked by bevacizumab. Overexpression of occludin alleviated the VEGF-induced hyperpermeability. Further investigations revealed that VEGF-induced occludin phosphorylation at Ser-490 and ubiquitination. Finally, we showed that VEGF accelerated the process of occludin degradation through the ubiquitin-proteasome system. In conclusion, primary tumor-secrete VEGF induce the occludin phosphorylation/ubiquitination and downregulation, resulting in the disruption of TJs and hyperpermeability of vessels in premetastatic lung. The occludin phosphorylation/ubiquitination pathway may be the mechanism of VEGF-induced vascular hyperpermeability in the lung premetastatic niche.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Experimentais/metabolismo , Ocludina/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Pulmão/irrigação sanguínea , Pulmão/patologia , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ocludina/genética , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Ubiquitinação/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Artif Cells Nanomed Biotechnol ; 47(1): 3720-3728, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31523993

RESUMO

Matrine derivative MASM (M19) is a quinolizine alkaloid with diverse pharmacological effects including preventing postmenopausal osteoporosis. In the current study, we observed that pretreatment with M19 inhibited cell apoptosis of murine osteoblastic MC3T3-E1 and primary osteoblasts induced by 1 µM dexamethasone in a dose-dependent manner. Our study also showed that pretreatment with M19 significantly prevented the upregulation of p53 that is caused by dexamethasone but had no effect on the p53 mRNA expression levels. Further immunoprecipitation (IP) experiments showed that M19 treatment increases the ubiquitination of p53 in dexamethasone-treated MC3T3-E1 cells. The expression of USP14, a deubiquitinating enzyme, increased with dexamethasone and decreased with M19 pretreatment. Co-IP experiments demonstrated the interaction between USP14 and p53, and the induced effect of a USP14 inhibitor (IU1) on p53 ubiquitination, which indicated that USP14 is a potential deubiquitinase for p53. Furthermore, pretreatment with IU1 or a p53 inhibitor (PFT-α) partially blocked dexamethasone-induced apoptosis of MC3T3-E1 cells. The overexpression of USP14 or p53 reversed the antiapoptotic effect of M19 in dexamethasone-treated MC3T3-E1 cells. In addition, PFT-α treatment remarkably blocked the effects of USP14 overexpression. In summary, our findings suggest that M19 exerts protective effects on dexamethasone-treated MC3T3-E1 cells by regulating USP14/p53.


Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Dexametasona/efeitos adversos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Quinolizinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Células 3T3 , Animais , Citoproteção/efeitos dos fármacos , Camundongos , Osteoblastos/metabolismo , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
10.
Food Chem Toxicol ; 133: 110745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31376412

RESUMO

Cadmium (Cd) is a dispensable element for the human body and is usually considered a carcinogen. Occupational and environmental Cd exposure leads to sustained cellular proliferation in some tissues and tumorigenesis via an unclear mechanism. Here, we evaluated the role of Cd in the DNA damage response (DDR). We found that Cd exposure causes extensive DNA double-strand breaks (DSBs) and prevents accumulation of ubiquitination signals at these sites of DNA damage. Cd treatment compromises 53BP1 and BRCA1 recruitment to DSBs induced by itself or DNA damaging agents and partially inactivates the G2/M checkpoint. Mechanistically, Cd directly binds to the E3 ubiquitin ligase RNF168, induces the ubiquitin-proteasome pathway that mediates RNF168 degradation and suppresses RNF168 ubiquitin-ligase activity in vitro. Our study raises the possibility that Cd may target RNF168 to disrupt proper DSB signaling in cultured cells. This pathway may represent a novel mechanism for carcinogenesis induced by Cd.


Assuntos
Compostos de Cádmio/toxicidade , Cádmio/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA/metabolismo , Nitratos/toxicidade , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/metabolismo , Cádmio/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Histonas/metabolismo , Humanos , Ligação Proteica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacos
11.
PLoS One ; 14(7): e0219782, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329620

RESUMO

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 9/metabolismo , Proteínas Culina/metabolismo , Ubiquitinação , Ativação Enzimática/efeitos dos fármacos , Etoposídeo/farmacologia , Células HEK293 , Humanos , Leupeptinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteína bcl-X/metabolismo
12.
Oncol Rep ; 42(4): 1467-1474, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31322269

RESUMO

With the increasing use of poly(ADP­ribose) polymerase (PARP) inhibitors in cancer therapy, understanding their resistance is an urgent research quest. Additionally, CHFR is an E3 ubiquitin ligase, recruited to double­strand breaks (DSBs) by PAR. Furthermore, ALC1 is a new oncogene involved in the invasion and metastasis of breast cancer. Moreover, PARylated PARP1 activates ALC1 at sites of DNA damage, yet the underlying mechanism remains unclear. Mass spectrometric analysis, western blot analysis and immunoprecipitation were performed to confirm the interaction between CHFR and ALC1 in the physiological condition. Deletion mutants of CHFR and ALC1 were generated to map the interaction domain. PARP1/2 inhibitors were added to identify the ubiquitination of ALC1 by CHFR. ALC1 half­life was examined to compare the expression of ALC1 protein in the presence and absence of PARP1/2 inhibitors. The results revealed that the transcriptional level of ALC1 was not upregulated in breast cancer tissues. CHFR interacted with ALC1. The PBZ domain of CHFR, the PMD domain and the MACRO domain of ALC1 domain are the necessary regions for the interaction depending on PAR. Ubiquitination of ALC1 by CHFR was dependent on PARylation and resulted in the degradation of PARylated ALC1. PARP1/2 inhibitors decreased the ubiquitination of PAR­dependent ALC1, and the expression of ALC1 was upregulated by PARP1/2 inhibitors. Ubiquitination mediated by CHFR resulted in the degradation of ALC1. In conclusion, PARP1/2 inhibitors decrease the ubiquitination of ALC1 leading to the accumulation of ALC1, which affects the therapeutic effects of DNA damage response drugs in breast cancer treatment.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Poli(ADP-Ribose) Polimerase-1/metabolismo , Transcrição Genética , Ubiquitinação/efeitos dos fármacos
13.
EMBO J ; 38(13): e101996, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268597

RESUMO

Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.


Assuntos
Antígenos de Bactérias/efeitos adversos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/efeitos adversos , Macrófagos/efeitos dos fármacos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sistemas CRISPR-Cas , Caspase 1/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Inflamassomos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Domínios Proteicos , Proteólise/efeitos dos fármacos , Células RAW 264.7 , RNA Interferente Pequeno/farmacologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
14.
Chemosphere ; 237: 124410, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31362132

RESUMO

The profound influence of environmental chemicals on human health including inducing life-threatening gene mutation has been publicly recognized. Being a substitute for the extensively used endocrine-disrupting chemical BPA, Bisphenol AF (BPAF) has been known as teratogen with developmental toxicities and therefore potentially putting human into the risk of biological hazards. Herein, we deciphered the detrimental effects of BPAF on spermatogenesis and spermiotiliosis in sexual maturity of mice exposing to BPAF (5, 20, 50 mg/kg/d) for consecutive 28 days. BPAF exposure significantly compromises blood-testis barrier integrity and sperm quantity and quality in a dose-dependent manner. Sperms from BPAF exposure mice are featured by severe DNA damage, altered SUMOylation and ubiquitination dynamics and interfered epigenetic inheritance with hypermethylation of H3K27me3 presumably due to the aggregation of cellular reactive oxygen species (ROS). Furthermore, BPAF treatment (50 µM for 24 h) compromises cytoskeleton architecture and tight junction permeability in primary cultured Sertoli cells evidenced by dysfunction of actin regulatory proteins (e.g. Arp3 and Palladin) via activation of ERK signaling, thereby perturbing the privilege microenvironment created by Sertoli cells for spermatogenesis. Overall, our study determines BPAF is deleterious for male fertility, leading to a better appreciation of its toxicological features in our life.


Assuntos
Compostos Benzidrílicos/toxicidade , Barreira Hematotesticular/efeitos dos fármacos , Fenóis/toxicidade , Espermatozoides/efeitos dos fármacos , Animais , Compostos Benzidrílicos/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Disruptores Endócrinos/administração & dosagem , Disruptores Endócrinos/toxicidade , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Fenóis/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/patologia , Transdução de Sinais/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/metabolismo , Espermatozoides/patologia , Sumoilação/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
15.
Cell Prolif ; 52(5): e12665, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31332862

RESUMO

OBJECTIVES: Abnormal activation of NF-κB signalling is a major mechanism of apoptosis resistance in glioblastoma multiforme (GBM). Therefore, better understanding of the regulation of NF-κB signalling has a significant impact for GBM therapy. Here, we uncovered a critical role of the small GTPase RND3 in regulating the p65 subunit of NF-κB and NF-κB signalling in GBM. MATERIALS AND METHODS: Human GBM samples, GBM cells and a human orthotopic GBM-xenografted animal model were used. The mechanisms of RND3 in regulation of NF-κB signalling and GBM cell apoptosis were examined by luciferase assay, quantitative PCR, immunostaining, immunoblotting, immunofluorescence, coimmunoprecipitation, TUNEL staining, JC-1 analysis and flow cytometry. RESULTS: Overexpression of RND3 led to reduced p65 activity in GBM-cultured cells and a GBM animal model, indicating that the NF-κB pathway is negatively regulated by RND3 in GBM. Mechanistically, we found that RND3 bound p65 and promoted p65 ubiquitination, leading to decreased p65 protein levels. Furthermore, RND3 enhanced cleaved caspase 3 levels and promoted apoptosis in GBM cells, and RND3 expression was positively correlated with cleaved caspase 3 and IL-8 in human GBM samples. The effect of RND3 on promoting apoptosis disappeared when p65 ubiquitination was blocked by protease inhibitor carfilzomib or upon co-expression of ectopic p65. CONCLUSIONS: RND3 binds p65 protein and promotes its ubiquitination, resulting in reduced p65 protein expression and inhibition of NF-κB signalling to induce GBM cell apoptosis.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Nus , Oligopeptídeos/farmacologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Transplante Heterólogo , Ubiquitinação/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/genética
16.
Biomed Res Int ; 2019: 6061594, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119177

RESUMO

Aims: Abnormal regulation of autophagy participates in the development of diabetic nephropathy. mTOR is the most common negative regulator of the autophagy signaling pathway. FBW7 constitutes the SCF (Skp1-Cullin1-F-box protein) recognition subunit of E3 ubiquitin ligase, and mTOR is a substrate of FBW7 that can be modified by ubiquitination and be degraded via proteasomes. In this study, we explored the relationship between FBW7 and autophagy and examined the effects of FBW7 on the occurrence of diabetic nephropathy in vitro. Materials and Methods: We cultured mesangial cells induced by high glucose in vitro and used rapamycin as a specific mTOR inhibitor, performed FBW7 gene overexpression, and detected the expression of autophagy signal and inflammatory factors by WB, ELISA, RT-PCR, and immunofluorescence. Results: High glucose can downregulate the expression of FBW7 and activate mTOR signal, which leads to diminished autophagy in renal mesangial cells, as well as renal inflammatory cytokines and fibrotic factors. RAPA, as a specifically inhibitor of mTOR, can decrease inflammatory cytokines and fibrotic factors by inhibiting mTOR. Moreover, FBW7 gene overexpression can increase autophagy by inhibiting mTOR signal; at the same time, the inflammatory cytokines and fibrotic factors were decreased in mesangial cells. Conclusions: FBW7 was decreased in renal mesangial cells induced by high glucose, and FBW7 gene overexpression can increase autophagy by inhibiting mTOR signaling and ameliorate inflammation and fibrosis.


Assuntos
Autofagia/genética , Nefropatias Diabéticas/tratamento farmacológico , Proteína 7 com Repetições F-Box-WD/genética , Serina-Treonina Quinases TOR/genética , Autofagia/efeitos dos fármacos , Linhagem Celular , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/toxicidade , Humanos , Células Mesangiais/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ubiquitinação/efeitos dos fármacos
17.
Cell Mol Biol Lett ; 24: 29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123462

RESUMO

Background: In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex. Methods: An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1ß (IL-1ß). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts. Results: Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1ß treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6. Conclusions: These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.


Assuntos
Lisina/metabolismo , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Células HEK293 , Humanos , Interferon gama/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl/química , Ligante RANK/farmacologia , Domínios RING Finger , Fator 6 Associado a Receptor de TNF/química , Ubiquitinação/efeitos dos fármacos
18.
FEBS Open Bio ; 9(7): 1281-1291, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31125507

RESUMO

Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin- and p62/sequestosome 1 (SQSTM1)-mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m-chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2-associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference-mediated depletion of endogenous BAG6 prevented Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/SQSTM1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.


Assuntos
Mitocôndrias/metabolismo , Chaperonas Moleculares/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/efeitos dos fármacos , Citosol/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
19.
Mar Drugs ; 17(5)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117253

RESUMO

Among malignancies, lung cancer is the major cause of cancer death. Despite the advance in lung cancer therapy, the five-year survival rate is extremely restricted due to therapeutic failure and disease relapse. Targeted therapies selectively inhibiting certain molecules in cancer cells have been accepted as promising ways to control cancer. In lung cancer, evidence has suggested that the myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of the Bcl-2 family, is a target for drug action. Herein, we report the Mcl-1 targeting activity of renieramycin T (RT), a marine-derived tetrahydroisoquinoline alkaloid that was isolated from the Thai blue sponge Xestospongia sp. RT was shown to be dominantly toxic to lung cancer cells compared to the normal cells in the lung. The cytotoxicity of this compound toward lung cancer cells was mainly exerted through apoptosis induction. For the mechanism of action, we found that RT mediated activation of p53 protein and caspase-9 and -3 activations. While others Bcl-2 family proteins (Bcl-2, Bak, and Bax) were minimally changed in response to RT, Mcl-1 protein was dramatically diminished. We further performed the cycloheximide experiment and found that the half-life of Mcl-1 was significantly shortened by RT treatment. When MG132, a potent selective proteasome inhibitor, was utilized, it could restore the Mcl-1 level. Furthermore, immunoprecipitation analysis revealed that RT significantly increased the formation of Mcl-1-ubiquitin complex compared to the non-treated control. In conclusion, we report the potential apoptosis induction of RT with a mechanism of action involving the targeting of Mcl-1 for ubiquitin-proteasomal degradation. As Mcl-1 is critical for cancer cell survival and chemotherapeutic failure, this novel information regarding the Mcl-1-targeted compound would be beneficial for the development of efficient anti-cancer strategies or targeted therapies.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Sistemas de Liberação de Medicamentos , Neoplasias Pulmonares/fisiopatologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Poríferos/química , Proteólise/efeitos dos fármacos , Tetra-Hidroisoquinolinas/uso terapêutico , Tetra-Hidroisoquinolinas/toxicidade , Ubiquitinação/efeitos dos fármacos
20.
Science ; 364(6441)2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31097636

RESUMO

Activation of tumor suppressors for the treatment of human cancer has been a long sought, yet elusive, strategy. PTEN is a critical tumor suppressive phosphatase that is active in its dimer configuration at the plasma membrane. Polyubiquitination by the ubiquitin E3 ligase WWP1 (WW domain-containing ubiquitin E3 ligase 1) suppressed the dimerization, membrane recruitment, and function of PTEN. Either genetic ablation or pharmacological inhibition of WWP1 triggered PTEN reactivation and unleashed tumor suppressive activity. WWP1 appears to be a direct MYC (MYC proto-oncogene) target gene and was critical for MYC-driven tumorigenesis. We identified indole-3-carbinol, a compound found in cruciferous vegetables, as a natural and potent WWP1 inhibitor. Thus, our findings unravel a potential therapeutic strategy for cancer prevention and treatment through PTEN reactivation.


Assuntos
Anticarcinógenos/farmacologia , Indóis/farmacologia , Neoplasias/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Anticarcinógenos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/uso terapêutico , Masculino , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/genética , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos
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