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1.
Nat Commun ; 11(1): 4980, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020477

RESUMO

The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as "Yin and Yang" partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14ARF in human and p19ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53.


Assuntos
Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Carcinogênese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , Sumoilação , Proteína Supressora de Tumor p53/genética , Ubiquitinação
2.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
3.
Int Heart J ; 61(5): 1034-1040, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32999190

RESUMO

Low-density lipoprotein (LDL) particles are known to be atherogenic agents in coronary artery diseases. They adjust to other electronegative forms and can be the subject for the enhancement of inflammatory events in vessel subendothelial spaces. The LDL uptake is related to the membrane scavenger receptors, including LDL receptor (LDLR). The LDLR expression is closely associated with LDL uptake and occurrence of diseases, such as atherosclerotic cardiovascular diseases. Our findings identified USP16 as a novel regulator of LDLR due to its ability to prevent ubiquitylation-dependent LDLR degradation, further promoting the uptake of LDL. The enhancement of USP16-mediated deubiquitination andthe suppressive degradation of the LDLR cause the presentation of a potential strategy to increase LDL cholesterol clearance.


Assuntos
Receptores de LDL/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Células HeLa , Humanos , Lipoproteínas LDL/metabolismo , Processamento de Proteína Pós-Traducional
4.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1484-1492, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924347

RESUMO

Ubiquitination, one type of the most common post-translational modification, mediates the regulation of protein homeostasis in vivo. Since ubiquitin itself contains multiple lysine residues and one N-terminal free amino group, eight types of ubiquitin chains can be formed. The K27 ubiquitin chain is formed through the ubiquitination of the ubiquitin Lys27 (K27), which adopts a compact conformation. In recent years, biological function of the K27 ubiquitin chain in innate immunity, protein homeostasis and DNA damage has been discovered, but the molecular mechanisms of K27 ubiquitin chain assembly, recognition and hydrolysis are still poorly understood. Here we review the structural features and biological functions of K27 ubiquitin chain, to provide a reference for future studies.


Assuntos
Ubiquitina , Imunidade Inata , Lisina , Processamento de Proteína Pós-Traducional , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação
5.
Nat Commun ; 11(1): 4868, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985517

RESUMO

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ubiquitina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição TFIIH/genética , Ubiquitinação
6.
Life Sci ; 259: 118380, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32898524

RESUMO

AIMS: Benign prostatic hyperplasia (BPH) is a progressive disease, which severely affects men's health. Here, we sought to analyze the functions and mechanism of action of the tripartite motif protein 52 (TRIM52), a novel prostate basal cell biomarker in BPH. MATERIALS AND METHODS: Immunohistochemistry assay was performed in sectioned human BPH tissues, BPH-1 cells, and prostate RWPE-1 cells, to detect the expressions of TRIM52 and NF-κB. Western blotting and qRT-PCR analyses were conducted to measure the relative protein and mRNA expression levels, respectively. Further, lentiviral transfection was performed in BPH-1 and RWPE-1 cells to study the overexpression and siRNA knockdown of TRIM52. Dual-luciferase reporter assay was applied to evaluate the relationship between NF-κB and TRIM52. Furthermore, CCK-8 assay and flow cytometry were employed to analyze cell proliferation and apoptosis. KEY FINDINGS: TRIM52 and NF-κB levels were elevated in BPH tissues, and TRIM52 expression positively correlated with NF-κB expression. TRIM52 silencing suppressed the growth of BPH-1 cells and decreased the promoter activity of NF-κB. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed TRIM52-induced proliferation of RWPE-1 cells and inhibited NF-κB promoter activity in oeTRIM52 transfected RWPE-1 cells. Silencing TRIM52 also inhibited TRAF2 ubiquitination in BPH-1 cells. Further, NF-κB promoter activity in siNC transfected cells was enhanced by the recombinant protein TNF-α and inhibited by siTRIM52. SIGNIFICANCE: TRIM52 accelerated the growth of BPH-1 cells by upregulating NF-κB, and TRIM52 could promote TRAF2 ubiquitination. These findings might contribute to the understanding of the biological functions and action mechanisms of TRIM52 in BPH.


Assuntos
NF-kappa B/metabolismo , Hiperplasia Prostática/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Western Blotting , Progressão da Doença , Citometria de Fluxo , Humanos , Masculino , Hiperplasia Prostática/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação
7.
Nat Commun ; 11(1): 4479, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900999

RESUMO

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness 'myosinopathy' patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.


Assuntos
Fibras Musculares Esqueléticas/fisiologia , Proteínas Quinases/fisiologia , Sarcômeros/fisiologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Força Muscular/fisiologia , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Miosinas/metabolismo , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Sarcômeros/patologia , Ubiquitinação
8.
Nat Commun ; 11(1): 3807, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32733036

RESUMO

The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
9.
Nat Commun ; 11(1): 4184, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826889

RESUMO

Oncogenic processes exert their greatest effect by targeting regulators of cell proliferation. Studying the mechanism underlying growth augmentation is expected to improve clinical therapies. The ovarian tumor (OTU) subfamily deubiquitinases have been implicated in the regulation of critical cell-signaling cascades, but most OTUs functions remain to be investigated. Through an unbiased RNAi screen, knockdown of OTUD5 is shown to significantly accelerate cell growth. Further investigation reveals that OTUD5 depletion leads to the enhanced transcriptional activity of TRIM25 and the inhibited expression of PML by altering the ubiquitination level of TRIM25. Importantly, OTUD5 knockdown accelerates tumor growth in a nude mouse model. OTUD5 expression is markedly downregulated in tumor tissues. The reduced OTUD5 level is associated with an aggressive phenotype and a poor clinical outcome for cancers patients. Our findings reveal a mechanism whereby OTUD5 regulates gene transcription and suppresses tumorigenesis by deubiquitinating TRIM25, providing a potential target for oncotherapy.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença/genética , Células HEK293 , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Interferência de RNA , Transdução de Sinais , Transcriptoma , Ubiquitinação
10.
Nat Commun ; 11(1): 3984, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770009

RESUMO

The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aterosclerose/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteólise , Proteínas Adaptadoras de Transporte Vesicular/química , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/patologia , Cálcio/metabolismo , Colesterol/metabolismo , Células Endoteliais/metabolismo , Feminino , Deleção de Genes , Células HEK293 , Homeostase , Humanos , Inflamação/patologia , Masculino , Camundongos Knockout , Ligação Proteica , Domínios Proteicos , Ubiquitinação
11.
Nat Commun ; 11(1): 3965, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770022

RESUMO

Dysregulated Wnt/ß-catenin activation plays a critical role in cancer progression, metastasis, and drug resistance. Genotoxic agents such as radiation and chemotherapeutics have been shown to activate the Wnt/ß-catenin signaling although the underlying mechanism remains incompletely understood. Here, we show that genotoxic agent-activated Wnt/ß-catenin signaling is independent of the FZD/LRP heterodimeric receptors and Wnt ligands. OTULIN, a linear linkage-specific deubiquitinase, is essential for the DNA damage-induced ß-catenin activation. OTULIN inhibits linear ubiquitination of ß-catenin, which attenuates its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association with ß-catenin is enhanced by OTULIN Tyr56 phosphorylation, which depends on genotoxic stress-activated ABL1/c-Abl. Inhibiting OTULIN or Wnt/ß-catenin sensitizes triple-negative breast cancer xenograft tumors to chemotherapeutics and reduces metastasis. Increased OTULIN levels are associated with aggressive molecular subtypes and poor survival in breast cancer patients. Thus, OTULIN-mediated Wnt/ß-catenin activation upon genotoxic treatments promotes drug resistance and metastasis in breast cancers.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Metástase Neoplásica , Fosforilação , Fosfotirosina/metabolismo , Ubiquitinação , beta Catenina/metabolismo
12.
Circulation ; 142(12): 1190-1204, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32755395

RESUMO

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II, a potent vasoconstrictor, to angiotensin-(1-7) and is also a membrane protein that enables coronavirus disease 2019 (COVID-19) infectivity. AMP-activated protein kinase (AMPK) phosphorylation of ACE2 enhances ACE2 stability. This mode of posttranslational modification of ACE2 in vascular endothelial cells is causative of a pulmonary hypertension (PH)-protective phenotype. The oncoprotein MDM2 (murine double minute 2) is an E3 ligase that ubiquitinates its substrates to cause their degradation. In this study, we investigated whether MDM2 is involved in the posttranslational modification of ACE2 through its ubiquitination of ACE2, and whether an AMPK and MDM2 crosstalk regulates the pathogenesis of PH. METHODS: Bioinformatic analyses were used to explore E3 ligase that ubiquitinates ACE2. Cultured endothelial cells, mouse models, and specimens from patients with idiopathic pulmonary arterial hypertension were used to investigate the crosstalk between AMPK and MDM2 in regulating ACE2 phosphorylation and ubiquitination in the context of PH. RESULTS: Levels of MDM2 were increased and those of ACE2 decreased in lung tissues or pulmonary arterial endothelial cells from patients with idiopathic pulmonary arterial hypertension and rodent models of experimental PH. MDM2 inhibition by JNJ-165 reversed the SU5416/hypoxia-induced PH in C57BL/6 mice. ACE2-S680L mice (dephosphorylation at S680) showed PH susceptibility, and ectopic expression of ACE2-S680L/K788R (deubiquitination at K788) reduced experimental PH. Moreover, ACE2-K788R overexpression in mice with endothelial cell-specific AMPKα2 knockout mitigated PH. CONCLUSIONS: Maladapted posttranslational modification (phosphorylation and ubiquitination) of ACE2 at Ser-680 and Lys-788 is involved in the pathogenesis of pulmonary arterial hypertension and experimental PH. Thus, a combined intervention of AMPK and MDM2 in the pulmonary endothelium might be therapeutically effective in PH treatment.


Assuntos
Peptidil Dipeptidase A/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Hipertensão Arterial Pulmonar/patologia , Ubiquitinação , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Animais , Suscetibilidade a Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidil Dipeptidase A/genética , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos
13.
Proc Natl Acad Sci U S A ; 117(34): 20538-20548, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788364

RESUMO

Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) levels are frequently found reduced in human cancers, but how PTEN is down-regulated is not fully understood. In addition, although a compelling connection exists between PRL (phosphatase of regenerating liver) 2 and cancer, how this phosphatase induces oncogenesis has been an enigma. Here, we discovered that PRL2 ablation inhibits PTEN heterozygosity-induced tumorigenesis. PRL2 deficiency elevates PTEN and attenuates AKT signaling, leading to decreased proliferation and increased apoptosis in tumors. We also found that high PRL2 expression is correlated with low PTEN level with reduced overall patient survival. Mechanistically, we identified PTEN as a putative PRL2 substrate and demonstrated that PRL2 down-regulates PTEN by dephosphorylating PTEN at Y336, thereby augmenting NEDD4-mediated PTEN ubiquitination and proteasomal degradation. Given the strong cancer susceptibility to subtle reductions in PTEN, the ability of PRL2 to down-regulate PTEN provides a biochemical basis for its oncogenic propensity. The results also suggest that pharmacological targeting of PRL2 could provide a novel therapeutic strategy to restore PTEN, thereby obliterating PTEN deficiency-induced malignancies.


Assuntos
Carcinogênese , Proteínas Imediatamente Precoces/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Animais , Feminino , Células HEK293 , Humanos , Longevidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação
15.
Kidney Int ; 98(3): 785, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32828241
16.
Proc Natl Acad Sci U S A ; 117(37): 23106-23112, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32848052

RESUMO

Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABAA-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.


Assuntos
Hipnóticos e Sedativos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Teratogênios/metabolismo , Talidomida/farmacologia , Ubiquitinação/efeitos dos fármacos , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
17.
PLoS Genet ; 16(7): e1008933, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692737

RESUMO

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases Flap/genética , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Fase S/genética , Schizosaccharomyces/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
18.
Life Sci ; 257: 118041, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32622945

RESUMO

AIM: Transcription factor CCAAT/Enhancer binding protein alpha (C/EBPα) is a key regulator of myeloid differentiation, granulopoiesis in particular. Although CEBPA mutations are found in more than 10% in AML, functional inhibition of C/EBPα protein is also widely observed in AML. Here, we sought to examine if SKP2, an aberrantly enhanced E3 ubiquitin ligase in primary AMLs inhibits C/EBPα stability to induce differentiation block. MAIN METHODS: Here we employed cell based assays such as transfections, immunoblotting, co-immunoprecipitation, luciferase and gel shift assays along with differentiation assays to investigate SKP2 regulated C/EBPα protein stability in acute myeloid leukemia. KEY FINDINGS: Here we discovered that oncogenic E3 ubiquitin ligase SCFskp2 ubiquitinates and destabilizes C/EBPα in a proteasome-dependent manner. Our data demonstrates that SKP2 physically interacts with C-terminal of C/EBPα and promotes its K48-linked ubiquitination-mediated degradation leading to its reduced transactivation potential, DNA binding ability and cellular functions. We further show that while overexpression of SKP2 inhibits both ectopic as well as endogenous C/EBPα in heterologous (HEK293T) as well as myeloid leukemia cells respectively, SKP2 depletion restores endogenous C/EBPα leading to reduced colony formation and enhanced myeloid differentiation of myeloid leukemia cells. Using Estradiol-inducible K562-C/EBPα-ER cells as yet another model of granulocytic differentiation, we further confirmed that SKP2 overexpression indeed inhibits granulocytic differentiation by mitigating C/EBPα stability. SIGNIFICANCE: Our findings identify SKP2 as a potential negative regulator of C/EBPα stability and function in AML which suggests that SKP2 can be potentially targeted in AML to restore C/EBPα and overcome differentiation block.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Células U937 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
19.
Life Sci ; 257: 118021, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621919

RESUMO

AIMS: Tribbles homolog 3 (TRIB3) is emerging as a multifunctional oncoprotein associated with various cellular events in different tumors. However, the regulatory mechanism of TRIB3 in acute myeloid leukemia (AML) remains unknown. This study aims to investigate the molecular mechanisms and uncover the functions of TRIB3 in AML. METHODS: Western blotting and quantitative real-time PCR were used to analyze the expression levels of TRIB3, peroxisome proliferator-activated receptor α (PPARα), apoptosis markers and autophagy markers in AML cells. Flow cytometry was used to assess cell apoptosis. The interaction of TRIB3 and PPARα was evaluated by immunofluorescence, coimmunoprecipitation, and in vivo ubiquitination assays. KEY FINDINGS: We demonstrated that downregulating TRIB3 in leukemic cells effectively induced apoptosis and autophagy by regulating the degradation of PPARα. Mechanistically, TRIB3 interacted with PPARα and contributed to its destabilization by promoting its ubiquitination. When PPARα was activated by its specific agonist clofibrate, the apoptosis and autophagy of AML cells were significantly enhanced. These results were confirmed by rescue experiments. Blocking PPARα expression using the PPARα inhibitor GW6471 reversed the functional influence of TRIB3 on AML cells. SIGNIFICANCE: The aim of this study is to provide evidence of the degradation of PPARα by TRIB3 via ubiquitin-dependent proteasomal degradation. This process meditates the progression of AML and prolongs the survival of leukemic cells. As a result, these data indicate that TRIB3 is a novel and promising therapeutic target for AML treatment.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Bases de Dados Genéticas , Humanos , Leucemia Mieloide Aguda/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteostase/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Ubiquitinação
20.
Mol Cell ; 79(2): 320-331.e9, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645369

RESUMO

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.


Assuntos
Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Catálise , Citosol/metabolismo , Células HEK293 , Humanos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteólise , Solubilidade , Ubiquitinação
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