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1.
Cancer Sci ; 110(10): 3275-3287, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368616

RESUMO

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.


Assuntos
Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição CHOP/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismo
2.
Biomed Khim ; 65(4): 306-310, 2019 Jun.
Artigo em Russo | MEDLINE | ID: mdl-31436171

RESUMO

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.


Assuntos
Técnicas Biossensoriais , Complexo de Endopeptidases do Proteassoma/química , Proteínas Ubiquitinadas/química , Humanos , Ubiquitina , Ubiquitinação
3.
Adv Exp Med Biol ; 1152: 365-375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456194

RESUMO

Massively parallel sequencing, genomic and proteomic technologies have provided near complete resolution of signaling landscape of breast cancer (BCa). NEDD4 family of E3-ubiquitin ligases comprises a large family of proteins particularly, SMURFs (SMURF1, SMURF2), WWPs and NEDD4 which are ideal candidates for targeted therapy. However, it is becoming progressively more understandable that SMURFs and NEDD4 have "split-personalities". These molecules behave dualistically in breast cancer and future studies must converge on detailed identification of context specific role of these proteins in BCa. Finally, we provide scattered clues of regulation of SMURF2 by oncogenic miRNAs, specifically considering longstanding questions related to regulation of SMURF1 and WWPs by miRNAs in BCa. SMURFS, WWPs and NEDD4 are versatile regulators and represent a fast-growing field in cancer research and better understanding of the underlying mechanisms will be helpful in transition of our knowledge from a segmented view to a more conceptual continuum.


Assuntos
Neoplasias da Mama/enzimologia , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases/genética , Neoplasias da Mama/genética , Feminino , Humanos , MicroRNAs/genética , Oncogenes , Proteômica , Transdução de Sinais , Ubiquitinação
4.
Cancer Sci ; 110(10): 3145-3156, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31393050

RESUMO

Yes-associated protein (YAP) is a component of the canonical Hippo signaling pathway that is known to play essential roles in modulating organ size, development, and tumorigenesis. Activation or upregulation of YAP1, which contributes to cancer cell survival and chemoresistance, has been verified in different types of human cancers. However, the molecular mechanism of YAP1 upregulation in cancer is still unclear. Here we report that the E3 ubiquitin ligase STUB1 ubiquitinates and destabilizes YAP1, thereby inhibiting cancer cell survival. Low levels of STUB1 expression were correlated with increased protein levels of YAP1 in human gastric cancer cell lines and patient samples. Moreover, we revealed that STUB1 ubiquitinates YAP1 at the K280 site by K48-linked polyubiquitination, which in turn increases YAP1 turnover and promotes cellular chemosensitivity. Overall, our study establishes YAP1 ubiquitination and degradation mediated by the E3 ligase STUB1 as an important regulatory mechanism in gastric cancer, and provides a rationale for potential therapeutic interventions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Neoplasias Gástricas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Lisina/metabolismo , Camundongos , Transplante de Neoplasias , Estabilidade Proteica , Proteólise , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Ubiquitinação
5.
DNA Cell Biol ; 38(10): 1030-1039, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368785

RESUMO

Both endogenous and exogenous factors can cause DNA damage that compromises genomic integrity and cell viability. A proper DNA damage response (DDR) plays a role in maintaining genome stability and preventing tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion, whose response is dominated by the ataxia-telangiectasia mutated (ATM) protein kinase. After being activated by the sensor Mre11-Rad50-Nbs1 (MRN) complex or acetyltransferase Tip60, ATM rapidly phosphorylates downstream targets to launch DDR signaling when DNA is damaged. However, the exact mechanism of DDR is complex and ambiguous. Ufmylation, one type of ubiquitin-like modification, proceeds mainly through a three-step enzymatic reaction to help ubiquitin-fold modifier 1 (Ufm1), attach to substrates with ubiquitin-like modifier-activating enzyme 5 (Uba5), Ufm1-conjugating enzyme 1 (Ufc1) and Ufm1-specific ligase 1 (Ufl1). Although ubiquitination is essential to the DSBs response, the potential function of ufmylation in DDR is largely unknown. Herein, we review the relationship between ufmylation and DDR to elucidate the function and mechanism of ufmylation in DDR, which would reveal the pathogenesis of some diseases and provide new guidance to create a therapeutic method.


Assuntos
Doenças Cardiovasculares/metabolismo , Quebras de DNA de Cadeia Dupla , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Esquizofrenia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Reparo do DNA , Genoma Humano , Instabilidade Genômica , Humanos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Proteínas/genética , Esquizofrenia/genética , Esquizofrenia/patologia , Transdução de Sinais , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
6.
Braz J Med Biol Res ; 52(9): e8525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31411316

RESUMO

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1ß, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1ß by suppressing the increase in IL-1ß, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1ß-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1ß to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.


Assuntos
Condrócitos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1beta/efeitos dos fármacos , Degeneração do Disco Intervertebral/metabolismo , Adulto , Idoso , Agrecanas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Ginsenosídeos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Dor Lombar/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
7.
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
8.
Genes Dev ; 33(15-16): 1083-1094, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31296559

RESUMO

The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Células Acinares/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo , Pancreatite/genética , Processamento de Proteína , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinação/genética
9.
Nat Commun ; 10(1): 2998, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278365

RESUMO

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we design a forward genetic screen to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, we find that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, we show that Peflin, a Ca2+-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment. Our study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses.


Assuntos
Proteínas Culina/metabolismo , Proteínas de Drosophila/metabolismo , Somação de Potenciais Pós-Sinápticos/fisiologia , Terminações Pré-Sinápticas/metabolismo , Sono/fisiologia , Animais , Drosophila melanogaster , Feminino , Homeostase/fisiologia , Masculino , Modelos Animais , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Ubiquitinação/fisiologia
10.
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
11.
Nat Methods ; 16(8): 771-777, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308549

RESUMO

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.


Assuntos
Técnicas Biossensoriais , Homeostase , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Células HeLa , Humanos , Ligação Proteica , Conformação Proteica , Proteínas/química
12.
Rinsho Ketsueki ; 60(6): 680-690, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31281161

RESUMO

Recent progress in whole genome sequencing has identified recurrent somatic mutations in the additional sex combs like 1 (ASXL1) gene in a variety of hematological disorders and even in premalignant conditions. However, the molecular mechanisms regarding the contribution of ASXL1 mutation to the pathogenesis of premalignant conditions remain largely unelucidated. Thus, we investigated the biological effects of mutant Asxl1 using newly-generated knock-in (KI) mice. Heterozygous mutant KI mice developed phenotypes resembling human low-risk myelodysplastic syndromes (MDS), and some of them developed an MDS/myeloproliferative neoplasm-like disease after a long latency. The H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in KI hematopoietic stem cells (HSCs), suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutant Asxl1. The mutant Asxl1 failed to interact with Bmi1, although wild type ASXL1 protein did not. When p16Ink4a expression was depleted in Asxl1 KI mice, the HSC pool was restored, and apoptosis was ameliorated in HSCs. These findings demonstrate that the loss of protein interaction between mutant Asxl1 and Bmi1 affected the activity of Prc1. The subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in low-risk MDS-like phenotypes in heterozygous Asxl1 KI mice.


Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Proteínas Repressoras/genética , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células-Tronco Hematopoéticas , Histonas/metabolismo , Camundongos , Fenótipo , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Ubiquitinação
13.
Gene ; 712: 143963, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279706

RESUMO

BACKGROUND: The aim of this study was to identify the expression of LIM and calponin-homology domains 1 (LIMCH1) in lung cancer and normal tissues, to determine the interaction between LIMCH1 and HUWE1 in regulating p53 stability. METHODS: The expression of LIMCH1 was detected by the Oncomine and Cancer Genome Atlas databases. Expression of LIMCH1 mRNA was identified using qRT-PCR. In transfected human lung cancer cells, co-immunoprecipitation experiments were performed. The mechanism that HUWE1 sustained lung cancer malignancy was verified by western blotting. The proliferation of tranfected cells was assessed by CCK-8 assay and colony formation. RESULTS: Bioinformatic data and e TCGA database suggested LIMCH1 mRNA levels in tumor tissues were down-regulated compared to tumor adjacent tissues. We found low expression of LIMCH1 mRNA in tumor sites and tumor cell line. Exogenous expression of LIMCH1 interacts with HUWE1 promotes expression of p53. Use of siRNA or shRNA against LIMCH1 resulted in decreased p53 protein levels. LIMCH1 deletion lead to enhance of p53 ubiquitination and protein expression of p53 and substrate p21, puma. Growth curve showed that LIMCH1 deletion significantly promoted the proliferation of A549 cells. CONCLUSIONS: LIMCH1 was a negative regulator and indicated a new molecular mechanism for the pathogenesis of lung cancer via modulating HUWE1 and p53.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Proteínas com Domínio LIM/genética , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
14.
Mol Cell ; 74(5): 862-863, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173720

RESUMO

Cryo-EM structures of Dot1L in complex with a ubiquitinated nucleosome provide the long-sought-after molecular mechanism of Dot1L-mediated methylation of lysine 79 in histone H3 and explain crosstalk with histone H2B ubiquitination.


Assuntos
Histonas , Nucleossomos , Lisina , Metilação , Metiltransferases/genética , Ubiquitinação
15.
Genes Dev ; 33(15-16): 1069-1082, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31221664

RESUMO

Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Células-Tronco Embrionárias , Células HEK293 , Humanos , Camundongos , Mutação/genética , Domínios Proteicos , Estabilidade Proteica , Proteólise , RNA Polimerase II/metabolismo , Transdução de Sinais , Ubiquitinação
16.
Nat Commun ; 10(1): 2849, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253762

RESUMO

Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Proteínas Nucleares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
17.
Nat Commun ; 10(1): 2429, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160553

RESUMO

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.


Assuntos
Proteína ADAMTS5/metabolismo , Cartilagem Articular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoartrite/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Cartilagem Articular/diagnóstico por imagem , Modelos Animais de Doenças , Humanos , Articulação do Joelho/diagnóstico por imagem , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/metabolismo , RNA Mensageiro/farmacologia , Transdução de Sinais , Crânio/diagnóstico por imagem , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Microtomografia por Raio-X , Adulto Jovem
18.
Nat Commun ; 10(1): 2534, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182716

RESUMO

The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. Here, we report co-crystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 Å and 2.69 Å, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, but capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop is fully defined in the electron density, and does not contact the substrate degron directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design.


Assuntos
Proteínas Supressoras da Sinalização de Citocina/química , Ubiquitina-Proteína Ligases/química , Cristalografia por Raios X , Humanos , Fosfotirosina/química , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Receptores da Eritropoetina/química , Receptores da Somatotropina/química , Alinhamento de Sequência , Especificidade por Substrato , Proteínas Supressoras da Sinalização de Citocina/genética , Ubiquitinação
19.
Nat Commun ; 10(1): 2625, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201299

RESUMO

Enormous efforts have been made to target metabolic dependencies of cancer cells for developing new therapies. However, the therapeutic efficacy of glycolysis inhibitors is limited due to their inability to elicit cell death. Hexokinase 2 (HK2), via its mitochondrial localization, functions as a central nexus integrating glycolysis activation and apoptosis resilience. Here we identify that K63-linked ubiquitination by HectH9 regulates the mitochondrial localization and function of HK2. Through stable isotope tracer approach and functional metabolic analyses, we show that HectH9 deficiency impedes tumor glucose metabolism and growth by HK2 inhibition. The HectH9/HK2 pathway regulates cancer stem cell (CSC) expansion and CSC-associated chemoresistance. Histological analyses show that HectH9 expression is upregulated and correlated with disease progression in prostate cancer. This work uncovers that HectH9 is a novel regulator of HK2 and cancer metabolism. Targeting HectH9 represents an effective strategy to achieve long-term tumor remission by concomitantly disrupting glycolysis and inducing apoptosis.


Assuntos
Hexoquinase/metabolismo , Células-Tronco Neoplásicas/fisiologia , Neoplasias da Próstata/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Glicólise , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Nus , Próstata/patologia , RNA Interferente Pequeno , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nat Immunol ; 20(7): 835-851, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160797

RESUMO

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.


Assuntos
Apresentação do Antígeno/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/imunologia , Oncogenes , RNA Longo não Codificante/genética , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Adenoma/genética , Adenoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
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