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1.
Nat Commun ; 12(1): 5263, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489457

RESUMO

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Evolução Molecular , Células HEK293 , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Lenalidomida/química , Lenalidomida/farmacologia , Camundongos , Organoides , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Nihon Yakurigaku Zasshi ; 156(5): 271-274, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-34470930

RESUMO

Vertebral bone and limb bone are formed by endochondral ossification, which is replaced with bone tissue by osteoblasts after cartilage formation. Bone growth is regulated by the balance between epiphyseal chondrocyte proliferation and ossification. We attempted to elucidate the mechanism of chondrocyte differentiation and maturation regulated by the Extracellular-signal-regulated kinase 5 (Erk5) signal. Erk5 is a serine/threonine kinase belonging to the mitogen-activated protein kinase (MAPK) family, which includes Erk1/2, JNK, and p38. Mesenchymal stem cell-specific Erk5-deficient mice exhibited the phenotype of deformities of the metatarsal bones, enlargement of the long bones in limbs, and overgrowth of cartilage tissue. Based on this result, we searched for factors that directly phosphorylate Erk5, and We demonstrated that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249 to activate its function and promotes ubiquitination-mediated degradation. The TGF-ß-Smad signal suppresses the proliferation of many cells and regulates the production of extracellular matrix. Our findings may lead to the development of novel drugs targeting TGF-ß associated diseases. In this paper, we investigated the function of Smurf2Thr249 phosphorylation and the possibility as new therapeutic target for various diseases.


Assuntos
Proteína Quinase 7 Ativada por Mitógeno , Fator de Crescimento Transformador beta , Ubiquitina-Proteína Ligases , Animais , Diferenciação Celular , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fosforilação , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
3.
Biol Aujourdhui ; 215(1-2): 25-43, 2021.
Artigo em Francês | MEDLINE | ID: mdl-34397373

RESUMO

Targeted protein degradation (TPD), discovered twenty years ago through the PROTAC technology, is rapidly developing thanks to the implication of many scientists from industry and academia. PROTAC chimeras are heterobifunctional molecules able to link simultaneously a protein to be degraded and an E3 ubiquitin ligase. This allows the protein ubiquitination and its degradation by 26S proteasome. PROTACs have evolved from small peptide molecules to small non-peptide and orally available molecules. It was shown that PROTACs are capable to degrade proteins considered as "undruggable" i.e. devoid of well-defined pockets and deep grooves possibly occupied by small molecules. Among these "hard to drug" proteins, several can be degraded by PROTACs: scaffold proteins, BAF complex, transcription factors, Ras family proteins. Two PROTACs are clinically tested for breast (ARV471) and prostate (ARV110) cancers. The protein degradation by proteasome is also induced by other types of molecules: molecular glues, hydrophobic tagging (HyT), HaloPROTACs and homo-PROTACs. Other cellular constituents are eligible to induced degradation: RNA-PROTACs for RNA binding proteins and RIBOTACs for degradation of RNA itself (SARS-CoV-2 RNA). TPD has recently moved beyond the proteasome with LYTACs (lysosome targeting chimeras) and MADTACs (macroautophagy degradation targeting chimeras). Several techniques such as screening platforms together with mathematical modeling and computational design are now used to improve the discovery of new efficient PROTACs.


Assuntos
COVID-19/tratamento farmacológico , Desenho de Fármacos , Terapia de Alvo Molecular/métodos , Proteólise , Proteínas Recombinantes de Fusão/farmacologia , SARS-CoV-2/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Autofagia , Catálise , Humanos , Lisossomos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica , Proteólise/efeitos dos fármacos , RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Nat Commun ; 12(1): 4794, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373456

RESUMO

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.


Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sumoilação/fisiologia , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Proteína 28 com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
5.
FASEB J ; 35(9): e21825, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383978

RESUMO

Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.


Assuntos
Glicólise/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteômica/métodos , Especificidade por Substrato , Ubiquitina/metabolismo
6.
J Plant Physiol ; 264: 153484, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34343729

RESUMO

High concentrations of As in contaminated environments pose a serious threat to plant, human, and animal health. In this study, we characterized an As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase gene under arsenate (AsV) stress, named as Oryza sativa As-Induced RING E3 ligase 3 (OsAIR3). AsV treatment highly induced the expression of OsAIR3. OsAIR3-EYFP was localized to the nucleus in rice protoplasts and exhibited E3 ligase activity. Yeast two-hybrid screening and bimolecular fluorescence complementation and pull-down assays revealed the interaction of OsAIR3 with an O. sativa molybdate transporter (OsMOT1;3) in the plasma membrane and cytoplasm. In addition, an in vitro cell-free degradation assay was performed to demonstrate the degradation of OsMOT1;3 by OsAIR3 via the 26S proteasome system. Heterogeneous overexpression of OsAIR3 in Arabidopsis yielded AsV-tolerant phenotypes, as indicated by the comparison of cotyledon expansion, root elongation, shoot fresh weight, and As accumulation between the OsAIR3-overexpressing and control plants. Collectively, these findings suggest that OsAIR3 positively regulates plant response to AsV stress.


Assuntos
Proteínas de Transporte/metabolismo , Molibdênio/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arsênio/toxicidade , Oryza/enzimologia , Transcriptoma , Técnicas do Sistema de Duplo-Híbrido
7.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34452991

RESUMO

COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.


Assuntos
Proteases 3C de Coronavírus/metabolismo , Estabilidade Proteica , SARS-CoV-2/patogenicidade , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Regulação da Expressão Gênica , Interferons/fisiologia , SARS-CoV-2/imunologia , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Células Vero
8.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360883

RESUMO

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.


Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Suplementos Nutricionais , Mitofagia/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Valeratos/farmacologia , Adenocarcinoma/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo
9.
Nat Commun ; 12(1): 5016, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408138

RESUMO

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína BRCA1/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445478

RESUMO

Peptide-based target protein degradation inducers called PROTACs/SNIPERs have low cell penetrability and poor intracellular stability as drawbacks. These shortcomings can be overcome by easily modifying these peptides by conjugation with cell penetrating peptides and side-chain stapling. In this study, we succeeded in developing the stapled peptide stPERML-R7, which is based on the estrogen receptor alpha (ERα)-binding peptide PERML and composed of natural amino acids. stPERML-R7, which includes a hepta-arginine motif and a hydrocarbon stapling moiety, showed increased α-helicity and similar binding affinity toward ERα when compared with those of the parent peptide PERML. Furthermore, we used stPERML-R7 to develop a peptide-based degrader LCL-stPERML-R7 targeting ERα by conjugating stPERML-R7 with a small molecule LCL161 (LCL) that recruits the E3 ligase IAPs to induce proteasomal degradation via ubiquitylation. The chimeric peptide LCL-stPERML-R7 induced sustained degradation of ERα and potently inhibited ERα-mediated transcription more effectively than the unstapled chimera LCL-PERML-R7. These results suggest that a stapled structure is effective in maintaining the intracellular activity of peptide-based degraders.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Receptor alfa de Estrogênio/metabolismo , Tiazóis/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Receptor alfa de Estrogênio/genética , Humanos , Células MCF-7 , Ligação Proteica , Ubiquitinação
11.
Theriogenology ; 174: 160-168, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34455243

RESUMO

Vitrification is an effective technique for fertility preservation, but is known to lead to mitochondrial dysfunction in porcine oocytes. Mitophagy is induced to rebalance mitochondrial function, a process in which reactive oxygen species (ROS) plays a role. In this study, vitrified-warmed porcine oocytes were incubated for 4 h with the oxidant AAPH or antioxidant α-tocopherol to alter ROS levels. A series of tests suggested that vitrification damaged mitochondrial structure and caused dysfunction, including blurred mitochondrial cristae, decreased mitochondrial membrane potential, decreased mtDNA copy number and increased ROS generation. This dysfunction resulted in mitophagy and the loss of embryonic developmental potential. Incubation with AAPH or α-tocopherol altered mitochondrial function and mitophagy flux status in vitrified oocytes. The PINK1/Parkin pathway was involved in oxidative stress regulation in vitrified oocytes. Under AAPH-induced oxidative stress, increased fluorescence intensity of Parkin, increased expression of PINK1, Parkin, and LC3B-II, and decreased expression of MFN2 and p62 were observed, whereas the opposite effects were induced under α-tocopherol treatment. The inhibition of ROS by α-tocopherol benefitted mitochondrial homeostasis and alleviated PINK1/Parkin-mediated mitophagy, resulting in the recovery of embryonic developmental potential in vitrified porcine oocytes. Therefore, this study provides a new mechanism for the application of antioxidants to aid the cryopreservation of porcine oocytes.


Assuntos
Mitocôndrias , Mitofagia , Animais , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Oócitos/metabolismo , Estresse Oxidativo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Suínos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
12.
Science ; 373(6558): 998-1004, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446601

RESUMO

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361089

RESUMO

UBR box E3 ligases, also called N-recognins, are integral components of the N-degron pathway. Representative N-recognins include UBR1, UBR2, UBR4, and UBR5, and they bind destabilizing N-terminal residues, termed N-degrons. Understanding the molecular bases of their substrate recognition and the biological impact of the clearance of their substrates on cellular signaling pathways can provide valuable insights into the regulation of these pathways. This review provides an overview of the current knowledge of the binding mechanism of UBR box N-recognin/N-degron interactions and their roles in signaling pathways linked to G-protein-coupled receptors, apoptosis, mitochondrial quality control, inflammation, and DNA damage. The targeting of these UBR box N-recognins can provide potential therapies to treat diseases such as cancer and neurodegenerative diseases.


Assuntos
Apoptose , Dano ao DNA , Inflamação/patologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Inflamação/metabolismo
14.
Theranostics ; 11(16): 8112-8128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335983

RESUMO

The coiled-coil domain containing protein members have been well documented for their roles in many diseases including cancers. However, the function of the coiled-coil domain containing 65 (CCDC65) remains unknown in tumorigenesis including gastric cancer. Methods: CCDC65 expression and its correlation with clinical features and prognosis of gastric cancer were analyzed in tissue. The biological role and molecular basis of CCDC65 were performed via in vitro and in vivo assays and a various of experimental methods including co-immunoprecipitation (Co-IP), GST-pull down and ubiquitination analysis et al. Finally, whether metformin affects the pathogenesis of gastric cancer by regulating CCDC65 and its-mediated signaling was investigated. Results: Here, we found that downregulated CCDC65 level was showed as an unfavourable factor in gastric cancer patients. Subsequently, CCDC65 or its domain (a.a. 130-484) was identified as a significant suppressor in GC growth and metastasis in vitro and in vivo. Molecular basis showed that CCDC65 bound to ENO1, an oncogenic factor has been widely reported to promote the tumor pathogenesis, by its domain (a.a. 130-484) and further promoted ubiquitylation and degradation of ENO1 by recruiting E3 ubiquitin ligase FBXW7. The downregulated ENO1 decreased the binding with AKT1 and further inactivated AKT1, which led to the loss of cell proliferation and EMT signal. Finally, we observed that metformin, a new anti-cancer drug, can significantly induce CCDC65 to suppress ENO1-AKT1 complex-mediated cell proliferation and EMT signals and finally suppresses the malignant phenotypes of gastric cancer cells. Conclusion: These results firstly highlight a critical role of CCDC65 in suppressing ENO1-AKT1 pathway to reduce the progression of gastric cancer and reveals a new molecular mechanism for metformin in suppressing gastric cancer. Our present study provides a new insight into the mechanism and therapy for gastric cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , China , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Glicoproteínas/genética , Humanos , Masculino , Metformina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oncogenes , Prognóstico , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
15.
Life Sci ; 282: 119817, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34273374

RESUMO

R-spondins 2 (RSPO2) protein is a member of RSPO family which plays an essential role in stem cell survival, development and tumorigenicity. There has several evidence suggested that RSPO2 involved in breast, gastric, liver and colorectal cancer. However, the specific function and mechanism of RSPO2 in nasopharyngeal carcinoma (NPC) remain unknown. In the present study, we first observed that RSPO2 expression was elevated in NPC cell lines SUNE-6-10B, SUNE-5-8F, and CNE-1 compared with the normal laryngeal epithelia cell line NP69. Knockdown of RSPO2 significantly inhibits SUNE-6-10B and CNE-1 cell survival and proliferation by using CCK-8 assay and Edu assay, respectively. Further studies verified that RSPO2 silence suppressed migration and invasion of SUNE-6-10B and CNE-1 cells. Further studies suggested that RSPO2 silence suppressed epithelial-to-mesenchymal transition (EMT) related protein E-cadherin expression and promoted Vimentin and N-cadherin expression both in SUNE-6-10B and CNE-1 cells. Molecular mechanism explorations showed that RSPO2 deletion increased ZNRF3 expression and inhibited Gli1 expression. Additionally, knockdown ZNRF3 expression or overexpression Gli1 both reversed the effects of RSPO2 silence on NPC growth and metastasis. Finally, RSPO2 depletion was impaired NPC tumor growth in vivo animal experiments. In conclusion, the present study confirmed that RSPO2 silence inhibits the tumorigenesis of NPC via ZNRF3/Hedgehog-Gli1 signal pathway.


Assuntos
Carcinogênese/metabolismo , Inativação Gênica , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proteínas Hedgehog/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Ubiquitina-Proteína Ligases/genética , Proteína GLI1 em Dedos de Zinco/genética
16.
Redox Biol ; 45: 102058, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34218200

RESUMO

Tripartite motif (TRIM) 31 has been implicated in diverse biological and pathological conditions. However, whether TRIM31 plays a role in ischemic stroke progression is not clarified. Here we demonstrated that TRIM31 was significantly downregulated in the ischemic brain and the deficiency of TRIM31 alleviated brain injury induced by middle cerebral artery occlusion by reducing reactive oxygen species production and maintaining mitochondrial homeostasis. Mechanistically, we found that TRIM31 is an E3 ubiquitin ligase for TP53-induced glycolysis and apoptosis regulator (TIGAR), which confers protection against brain ischemia by increasing the pentose phosphate pathway flux and preserving mitochondria function. TRIM31 interacted with TIGAR and promoted the polyubiquitination of TIGAR, consequently facilitated its degradation in a proteasome-dependent pathway. Furthermore, TIGAR knockdown effectively abolished the protective effect of TRIM31 deficiency after cerebral ischemia. In conclusion, we identified that TRIM31 was a novel E3 ubiquitin ligase for TIGAR, played a critical role in regulating its protein level, and subsequently involved in the ischemic brain injury, suggesting TRIM31 as a potential therapeutic target for ischemic stroke.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Lesões Encefálicas , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Animais , Apoptose , Glicólise , Masculino , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
17.
Cell Death Dis ; 12(7): 708, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267179

RESUMO

The dysregulated microRNAs (miRNAs) are involved in diabetic retinopathy progression. Epithelial mesenchymal transition (EMT) and cell permeability are important events in diabetic retinopathy. However, the function and mechanism of miR-195 in EMT and cell permeability in diabetic retinopathy remain largely unclear. Diabetic retinopathy models were established using streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated ARPE-19 cells. Retina injury was investigated by hematoxylin-eosin (HE) staining. EMT and cell permeability were analyzed by western blotting, immunofluorescence, wound healing, and FITC-dextran assays. MiR-195 expression was detected via qRT-PCR. YY1, VEGFA, Snail1, and Smurf2 levels were detected via western blotting. The interaction relationship was analyzed via ChIP, Co-IP, or dual-luciferase reporter assay. The retina injury, EMT, and cell permeability were induced in STZ-induced diabetic mice. HG induced EMT and cell permeability in ARPE-19 cells. MiR-195, YY1, VEGFA, and Snail1 levels were enhanced, but Smurf2 abundance was reduced in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells. VEGFA knockdown decreased Snail1 expression and attenuated HG-induced EMT and cell permeability. YY1 silence reduced VEGFA and Snail1 expression, and mitigated HG-induced EMT and cell permeability. YY1 could bind with VEGFA and Snail1, and it was degraded via Smurf2-mediated ubiquitination. MiR-195 knockdown upregulated Smurf2 to decrease YY1 expression and inhibited HG-induced EMT and cell permeability. MiR-195 targeted Smurf2, increased expression of YY1, VEGFA, and Snail1, and promoted HG-induced EMT and cell permeability. MiR-195 promotes EMT and cell permeability of HG-stimulated ARPE-19 cells by increasing VEGFA/Snail1 via inhibiting the Smurf2-mediated ubiquitination of YY1.


Assuntos
Retinopatia Diabética/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , MicroRNAs/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Glucose/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Permeabilidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de Transcrição YY1/genética
18.
Molecules ; 26(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34299435

RESUMO

KRIBB11, an HSF1 inhibitor, was shown to sensitize various types of cancer cells to treatment with several anticancer drugs. However, the exclusive effects of KRIBB11 in preventing the growth of glioblastoma cells and the related mechanisms have not been elucidated yet. Herein, we aimed to examine the potential of KRIBB11 as an anticancer agent for glioblastoma. Using MTT and colony formation assays and Western blotting for c-PARP, we demonstrated that KRIBB11 substantially inhibits the growth of A172 glioma cells by inducing apoptosis. At the molecular level, KRIBB11 decreased anti-apoptotic protein MCL-1 levels, which was attributable to the increase in MULE ubiquitin ligase levels. However, the constitutive activity of HSF1 in A172 cells was not influenced by the exclusive treatment with KRIBB11. Additionally, based on cycloheximide chase assay, we found that KRIBB11 markedly retarded the degradation of MULE. In conclusion, stabilization of MULE upon KRIBB11 treatment is apparently an essential step for degradation of MCL-1 and the subsequent induction of apoptosis in A172 cells. Our results have expanded the knowledge on molecular pathways controlled by KRIBB11 and could be potentially effective for developing an inhibitory therapeutic strategy for glioblastoma.


Assuntos
Aminopiridinas/farmacologia , Glioblastoma/tratamento farmacológico , Indazóis/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Aminopiridinas/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Indazóis/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Supressoras de Tumor/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo
19.
Biomolecules ; 11(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209255

RESUMO

Various environmental stimuli, including oxidative stress, could lead to granulosa cell (GC) death through mitophagy. Recently, it was reported that melatonin (MEL) has a significant effect on GC survival during oxidative damage. Here, we found that MEL inhibited oxidative stress-induced mitophagy to promote GC survival. The loss of cell viability upon H2O2 exposure was significantly restored after MEL treatment. Concomitantly, MEL inhibited the activation of mitophagy during oxidative stress. Notably, blocking mitophagy repressed GC death caused by oxidative stress. However, MEL cannot further restore viability of cells treated with mitophagy inhibitor. Moreover, PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase, was inhibited by MEL during oxidative stress. As a result, the E3 ligase Parkin failed to translocate to mitochondria, leading to impaired mitochondria clearance. Using RNAi to knock down PINK1 expression, we further verified the role of the MEL-PINK1-Parkin (MPP) pathway in maintaining GC survival by suppressing mitophagy. Our findings not only clarify the protective mechanisms of MEL against oxidative damage in GCs, but also extend the understanding about how circadian rhythms might influence follicles development in the ovary. These findings reveal a new mechanism of melatonin in defense against oxidative damage to GCs by repressing mitophagy, which may be a potential therapeutic target for anovulatory disorders.


Assuntos
Células da Granulosa/metabolismo , Melatonina/farmacologia , Mitofagia/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células da Granulosa/fisiologia , Peróxido de Hidrogênio/farmacologia , Masculino , Melatonina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Substâncias Protetoras/farmacologia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
20.
Cell Death Dis ; 12(7): 664, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215725

RESUMO

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3-/- retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Fotorreceptoras de Vertebrados/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Degeneração Retiniana/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/metabolismo , Autofagossomos/patologia , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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