Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.747
Filtrar
1.
Chem Pharm Bull (Tokyo) ; 68(1): 34-45, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902900

RESUMO

Enzymatic and post-translational modifications (PTMs) such as ubiquitination, acetylation, and methylation occur at lysine residues. The PTMs play critical roles in the regulation of the protein functions, and thus, various cellular processes. In addition, aberrations of the PTMs are associated with various diseases, such as cancer and neurodegenerative disorders. Therefore, we hypothesized that modulation of the PTMs and normalization of the PTM abnormalities could be useful as methods to control various cellular mechanisms and as a therapeutic strategy, respectively. To modulate the PTMs, we have focused on lysine-modifying enzymes and have pursued drug discovery researches on ubiquitination inducers, lysine deacetylase (KDAC) inhibitors, and lysine demethylase (KDM) inhibitors. For the identification of the modulators, we have used not only conventional drug design, such as structure-based drug design (SBDD) and ligand-based drug design (LBDD), but also "strategic chemistry approaches," such as drug design based on enzyme catalytic mechanism. As a result, we have identified several modulators which have pharmacological effects in animal models or in cellular studies. In this review, focusing on the drug design based on enzyme catalytic mechanism, our drug discovery researches have been discussed.


Assuntos
Carboxiliases/metabolismo , Inibidores Enzimáticos/química , Histona Desmetilases/metabolismo , Lisina/química , Carboxiliases/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/síntese química , Histona Desmetilases/antagonistas & inibidores , Humanos , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo
2.
Parasitol Res ; 118(12): 3387-3398, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31728719

RESUMO

Leucine aminopeptidase of Taenia pisiformis (TpLAP) belonging to the M17 peptidase family has been implicated as a stage-differentially expressed protein in the adult stage of T. pisiformis. In order to further dissect the biological functions of TpLAP in the growth and development of adult worms, TpLAP-interacting partners were investigated. In this study, a yeast two-hybrid (Y2H) cDNA library from adult T. pisiformis was constructed. Using pGBKT7-TpLAP as bait, proteins interacting with TpLAP were screened by Y2H system and positive preys were sequenced and analyzed using the Basic Local Alignment Search Tool (BLAST). Our results showed that six genuine TpLAP-interacting proteins, including LAP, dynein light chain (DLC), SUMO-conjugating enzyme (UBC9), histone-lysine n-methyltransferase, trans-acting transcriptional, and one unknown protein, were identified via Y2H assay. Furthermore, the interaction between TpLAP and UBC9 of T. pisiformis (TpUBC9), an important protein involved in SUMOylation pathway, was further validated by one-to-one Y2H assay, co-immunoprecipitation, and confocal analysis. These findings provide a deeper understanding of the biological functions of TpLAP and offer the first clue that TpLAP may act as a novel SUMOylated substrate, suggesting that the SUMO modification pathway plays an important role in regulation of adult worm growth and development.


Assuntos
Leucil Aminopeptidase/metabolismo , Proteínas de Protozoários/metabolismo , Taenia/metabolismo , Animais , Sequência de Bases , Dineínas/metabolismo , Biblioteca Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imunoprecipitação , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/metabolismo
3.
World J Microbiol Biotechnol ; 35(11): 168, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654206

RESUMO

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P < 0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.


Assuntos
Metilação de DNA , Epigênese Genética , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/genética , Tuberculose/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Tuberculose Extensivamente Resistente a Medicamentos , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/microbiologia , Metiltransferases/genética , Mycobacterium tuberculosis/patogenicidade , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Células THP-1 , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 2 Toll-Like/genética , Tuberculose/microbiologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
4.
Nat Cell Biol ; 21(9): 1152-1163, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481791

RESUMO

Ca2+/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase family, and its δ isoform is predominant in the heart. Excessive CaMKII activation plays a pivotal role in the pathogenesis of severe heart conditions, including myocardial infarction, cardiomyopathy and heart failure. However, the identity of CaMKII splice variants and the mechanism(s) underlying CaMKII-mediated cardiac pathology remain elusive. Here, we show that CaMKII-δ9, the most abundant CaMKII-δ splice variant in human heart, potently promotes cardiomyocyte death, cardiomyopathy and heart failure by disrupting cardiomyocyte genome stability. Mechanistically, CaMKII-δ9, but not the previously well-studied CaMKII-δ2 and CaMKII-δ3, targets the ubiquitin-conjugating enzyme E2T (UBE2T) for phosphorylation and degradation, disrupting UBE2T-dependent DNA repair and leading to the accumulation of DNA damage and genome instability. These findings not only reveal a crucial role of CaMKII in the regulation of DNA repair, but also mark the CaMKII-δ9-UBE2T-DNA damage pathway as an important therapeutic target for cardiomyopathy and heart failure.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Reparo do DNA/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/genética
5.
Nat Commun ; 10(1): 3668, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31413316

RESUMO

Breast cancer stem cells (BCSCs) are unique in their ability to undergo unlimited self-renewal, an essential process in breast cancer recurrence following metastatic dormancy. Emergent metastatic lesions were subjected to microarray analysis, which identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) as a differentially expressed gene coupled to metastatic recurrence. Here, we report that elevated Pfkfb3 expression correlates with the appearance of aggressive breast cancers and reduces relapse-free survival, as well as enhances BCSC self-renewal and metastatic outgrowth. We observe an inverse relationship between Pfkfb3 expression and autophagy, which reduces Pfkfb3 expression and elicits cellular dormancy. Targeted depletion of Atg3, Atg7, or p62/sequestosome-1 to inactivate autophagy restores aberrant Pfkfb3 expression in dormant BCSCs, leading to their reactivation of proliferative programs and outgrowth. Moreover, Pfkfb3 interacts physically with autophagy machinery, specifically the UBA domain of p62/sequestosome-1. Importantly, disrupting autophagy and this event enables Pfkfb3 to drive dormant BCSCs and metastatic lesions to recur.


Assuntos
Autofagia/genética , Neoplasias da Mama/genética , Células-Tronco Neoplásicas/metabolismo , Fosfofrutoquinase-2/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Autorrenovação Celular , Feminino , Humanos , Camundongos , Metástase Neoplásica , Proteína Sequestossoma-1/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
6.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 8): 552-560, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31397327

RESUMO

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2-E2BR complexes from both species have been determined. In addition to sequence and structural differences between the human and S. cerevisiae E2BRs, the binding of E2BRs also elicits different biochemical outcomes with respect to E2 charging by E1 and E2 discharge. Here, the Schizosaccharomyces pombe E2BR was identified and purified with Ubc7 to resolve a 1.7 Šresolution co-crystal structure of the E2BR in complex with Ubc7. The S. pombe E2BR binds to the back side of the E2 as an α-helix and, while differences exist, it exhibits greater similarity to the human E2BR. Structure-based sequence alignments reveal differences and conserved elements among these species. Structural comparisons and biochemistry reveal that the S. pombe E2BR presents a steric impediment to E1 binding and inhibits E1-mediated charging, respectively.


Assuntos
Cristalografia por Raios X/métodos , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Retículo Endoplasmático/metabolismo , Modelos Moleculares , Conformação Proteica , Ubiquitina/química
7.
Nat Commun ; 10(1): 3600, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399562

RESUMO

Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12-Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3's catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3's E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy.


Assuntos
Regulação Alostérica/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Domínio Catalítico , Cristalografia por Raios X , Ligases , Modelos Moleculares , Mutação , Conformação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/metabolismo
8.
Mol Cell Biochem ; 462(1-2): 51-59, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31428903

RESUMO

Interferon-stimulated gene 15 (ISG15) is a member of the family of ubiquitin-like proteins. Similar to ubiquitin, conjugation of ISG15 to cellular proteins requires cascade reactions catalyzed by at least 2 enzymes, UbE1L and UbcH8. Expression of ISG15 and its conjugates is up-regulated in many cancer cells, yet the underlying mechanism of up-regulation is still unclear. In this study, we showed that TNF-α, similar to the response by IFN-ß, could directly induce expression of ISG15 and its conjugation machinery, UbE1L and UbcH8, in human lung carcinoma, A549. The early response of their expression was effectively blocked by specific inhibitors of p38 MAPK (SB202190) and JNK (SP600125), but not by B18R, a soluble type-I IFN receptor. In addition, luciferase reporter assay together with serial deletions and site-directed mutagenesis identified a putative C/EBPß binding element in the ISG15 promoter, which is necessary to the response by TNF-α. Taken together, expression of ISG15 and ISG15 conjugation machinery in cancer cells is directly up-regulated by TNF-α via p38 MAPK and JNK pathways through the activation of C/EBPß binding element in the ISG15 promoter. This study provides a new insight toward understanding the molecular mechanism of ISG15 system and inflammatory response in cancer progression.


Assuntos
Citocinas/genética , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , Fator de Necrose Tumoral alfa/farmacologia , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/metabolismo
9.
DNA Cell Biol ; 38(9): 996-1004, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31393166

RESUMO

Osteosarcoma (OS), a highly aggressive bone tumor, mainly occurs in young patients and always presents abnormalities in molecular biology, such as microRNAs (miRNAs). However, the characteristic and underlying mechanism of miR-671-5p in OS are still unclear. In this study, we certify that miR-671-5p is remarkably downregulated in OS tissues and cells. Overexpressed miR-671-5p can suppress OS cell proliferation in vivo and in vitro, by the way of arresting cell-cycle progression. The overexpression of cyclin D1 (CCND1) and CDC34 promotes cell proliferation and cell-cycle promotion, whose functions are contrary to miR-671-5p. miR-671-5p directly binds to CCND1 and CDC34, which are thought as the key factors in regulating cell cycle. Taken together, our results suggest that by targeting CCND1 and CDC34, miR-671-5p plays a tumor suppressor in OS to inhibit the development of OS, implicating it as a novel target for therapeutic intervention in OS.


Assuntos
Ciclo Celular , Proliferação de Células , MicroRNAs/genética , Osteossarcoma/genética , Animais , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Osteossarcoma/patologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
10.
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
11.
BMC Cancer ; 19(1): 710, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319803

RESUMO

BACKGROUND: One major hallmark of colorectal cancers (CRC) is genomic instability with its contribution to tumor heterogeneity and therapy resistance. To facilitate the investigation of intra-sample phenotypes and the de novo identification of tumor sub-populations, imaging mass spectrometry (IMS) provides a powerful technique to elucidate the spatial distribution patterns of peptides and proteins in tissue sections. METHODS: In the present study, we analyzed an in-house compiled tissue microarray (n = 60) comprising CRCs and control tissues by IMS. After obtaining protein profiles through direct analysis of tissue sections, two validation sets were used for immunohistochemical evaluation. RESULTS: A total of 28 m/z values in the mass range 800-3500 Da distinguished euploid from aneuploid CRCs (p < 0.001, ROC AUC values < 0.385 or > 0.635). After liquid chromatograph-mass spectrometry identification, UBE2N could be successfully validated by immunohistochemistry in the initial sample cohort (p = 0.0274, ROC AUC = 0.7937) and in an independent sample set of 90 clinical specimens (p = 0.0070, ROC AUC = 0.6957). CONCLUSIONS: The results showed that FFPE protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value for improved molecular classification. Particularly, the protein expression of UBE2N was validated in an independent clinical cohort to distinguish euploid from aneuploid CRCs.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Instabilidade Genômica , Enzimas de Conjugação de Ubiquitina/metabolismo , Idoso , Aneuploidia , Área Sob a Curva , Biomarcadores Tumorais/metabolismo , Cromatografia Líquida , Estudos de Coortes , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Curva ROC , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Distribuição Tecidual
12.
EMBO J ; 38(13): e101996, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268597

RESUMO

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


Assuntos
Antígenos de Bactérias/efeitos adversos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/efeitos adversos , Macrófagos/efeitos dos fármacos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sistemas CRISPR-Cas , Caspase 1/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Inflamassomos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Domínios Proteicos , Proteólise/efeitos dos fármacos , Células RAW 264.7 , RNA Interferente Pequeno/farmacologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
13.
Nat Commun ; 10(1): 3020, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289263

RESUMO

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with ß2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune , Imunidade Humoral , Proteínas de Ligação a RNA/metabolismo , Receptores Fc/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/virologia , Degradação Associada com o Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Fc/imunologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia
14.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261719

RESUMO

Ubiquitin is required under both normal and stress conditions. Under stress conditions, upregulation of the polyubiquitin gene UBC is essential to meet the requirement of increased ubiquitin levels to confer stress resistance. However, UBC upregulation is usually observed only under stress conditions and not under normal conditions. Therefore, it has not been possible to upregulate UBC under normal conditions to study the effect of excess ubiquitin on cellular machinery. Recently, the CRISPR/Cas9 system has been widely used in biological research as a useful tool to study gene disruption effects. In this study, using an inducible CRISPR/Cas9 variant, a dCas9-VP64 fusion protein, combined with a single guide RNA (sgRNA) containing MS2 aptamer loops and MS2-p65-HSF1, we developed a system to increase the ubiquitin pool via upregulation of UBC. Although it is challenging to upregulate the expression of a gene that is already expressed at high levels, the significance of our system is that UBC upregulation can be induced in an efficient, reversible manner that is compatible with cellular processes, even under normal conditions. This system can be used to study ubiquitin pool dynamics and it will be a useful tool in identifying the role of ubiquitin under normal and stress conditions.


Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Enzimas de Conjugação de Ubiquitina/genética , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima
15.
Int J Mol Sci ; 20(13)2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31262043

RESUMO

Interleukin-13 (IL-13) drives symptoms in asthma with high levels of T-helper type 2 cells (Th2-cells). Since tight junctions (TJ) constitute the epithelial diffusion barrier, we investigated the effect of IL-13 on TJ in human tracheal epithelial cells. We observed that IL-13 increases paracellular permeability, changes claudin expression pattern and induces intracellular aggregation of the TJ proteins zonlua occludens protein 1, as well as claudins. Furthermore, IL-13 treatment increases expression of ubiquitin conjugating E2 enzyme UBE2Z. Co-localization and proximity ligation assays further showed that ubiquitin and the proteasomal marker PSMA5 co-localize with TJ proteins in IL-13 treated cells, showing that TJ proteins are ubiquitinated following IL-13 exposure. UBE2Z upregulation occurs within the first day after IL-13 exposure. Proteasomal aggregation of ubiquitinated TJ proteins starts three days after IL-13 exposure and transepithelial electrical resistance (TEER) decrease follows the time course of TJ-protein aggregation. Inhibition of JAK/STAT signaling abolishes IL-13 induced effects. Our data suggest that that IL-13 induces ubiquitination and proteasomal aggregation of TJ proteins via JAK/STAT dependent expression of UBE2Z, resulting in opening of TJs. This may contribute to barrier disturbances in pulmonary epithelia and lung damage of patients with inflammatory lung diseases.


Assuntos
Células Epiteliais/metabolismo , Interleucina-13/farmacologia , Junções Íntimas/metabolismo , Traqueia/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Janus Quinases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição STAT/metabolismo , Junções Íntimas/efeitos dos fármacos , Traqueia/citologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
16.
Mol Med Rep ; 20(2): 1212-1220, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173226

RESUMO

Renal cell carcinoma (RCC) is a common malignant tumor globally. The overall survival of patients with RCC is poor; one important factor is tumor heterogeneity. Ubiquitin­conjugating enzyme E2T (UBE2T) has been reported to act as an oncogene in various types of cancer; however, its role in RCC has yet to be investigated. In the present study, UBE2T was demonstrated via reverse transcription­quantitative PCR analysis to be significantly upregulated in RCC samples and cell lines compared with in normal tissue and cells. Additionally, UBE2T expression was significantly associated with late tumor stage and high grade in patients with RCC, and patients with high UBE2T expression exhibited poor prognosis compared with patients with low expression. Following knockdown of UBE2T in 786­O cells using RNA interference technology, the proliferation and colony formation of cells were inhibited as determined by an MTT assay and crystal violet staining, respectively; however, the migration and invasion of 786­O cells were not affected, as determined by wound­healing assay and Transwell assays, respectively. Xenograft RCC tumor growth in vivo was also significantly suppressed. The expression levels of two mesenchymal cell markers, N­cadherin and vimentin, were reduced following UBE2T knockdown, whereas E­cadherin and fibronectin levels were increased as determined by western blotting, indicating that epithelial­mesenchymal transition was suppressed. In addition, the phosphorylation levels of PI3K, Akt and mTOR were notably decreased following UBE2T knockdown, but were increased when UBE2T was overexpressed. Wortmannin, an Akt inhibitor, reversed the UBE2T overexpression­induced increase in the phosphorylation of PI3K, Akt and mTOR. Similarly, the UBE2T overexpression­induced promotion of 786­O cell proliferation was also attenuated by wortmannin. In conclusion, UBE2T promoted the proliferation of RCC cells by regulating PI3K/Akt signaling, suggesting it may be a novel target for the treatment of patients with RCC.


Assuntos
Carcinoma de Células Renais/metabolismo , Proliferação de Células , Neoplasias Renais/metabolismo , Transdução de Sinais , Enzimas de Conjugação de Ubiquitina/metabolismo , Adolescente , Adulto , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/fisiopatologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
17.
Life Sci ; 229: 157-165, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31077719

RESUMO

AIM: This study investigates the insulin sensitizer effect of carbenoxolone (CBX) and potentially involved peripheral mechanisms. MAIN METHODS: Taking glucose transporter 4 (GLUT4) as a marker of glucose disposal, we investigated the CBX effects on whole-body insulin sensitivity and solute carrier 2a4 (Slc2a4)/GLUT4 expression in visceral (VAT) and subcutaneous (SAT) adipose tissues and soleus muscle of monosodium glutamate (MSG)-induced obese rats. Sterol regulatory element binding protein (SREBP1), an enhancer of Slc2a4 expression was analyzed through mRNA content and SREBP1-binding to Slc2a4 promoter. Finally, the small ubiquitin-modifier conjugating enzyme 9 (UBC9), whose low content indicates accelerated GLUT4 degradation was analyzed in soleus. KEY FINDINGS: Hypercorticosteronemia, hyperinsulinemia and low glucose decay rate in the insulin tolerance test of obese rats were restored by CBX (P < 0.05). Slc2a4/GLUT4 increased in SAT (P < 0.05) and decreased in VAT (P < 0.01) of obese rats. In soleus, obesity increased Slc2a4 but decreased GLUT4 (P < 0.01), possibly by accelerating GLUT4 degradation, as suggested by decreased UBC9 (P < 0.01). CBX restored both UBC9 and GLUT4 contents. SREBP1 did not participate in the Slc2a4 transcriptional regulation. SIGNIFICANCE: The insulin sensitizer effect of CBX involves the increase of GLUT4 expression in soleus, indicating an increased glucose disposal in skeletal muscle. This observation reinforces the skeletal muscle as the main site of insulin-induced glucose uptake and sheds new light on the metabolic effects of 11ßHSD1 inhibitors, since most of the studies so far have focused on its effects on liver and adipose tissues.


Assuntos
Carbenoxolona/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Hiperinsulinismo/tratamento farmacológico , Resistência à Insulina , Músculo Esquelético/metabolismo , Obesidade/fisiopatologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Antiulcerosos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Masculino , Músculo Esquelético/patologia , Ratos , Ratos Wistar , Enzimas de Conjugação de Ubiquitina/genética
18.
Biosci Biotechnol Biochem ; 83(6): 1011-1026, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31074699

RESUMO

TRAF6 is highly expressed in many tumors and plays an important role in the immune system. The aim of this study is to confirm anti-tumor activities of all naturally occurring Cinchona alkaloids that have been screened using computational docking program, and to validate the accuracy and specificity of the RING domain of TRAF6 as a potential anti-tumor target, and to explore their effect on the immune system. Results reported herein would demonstrate that Cinchona alkaloids could induce apoptosis in HeLa cells, inhibit the ubiquitination and phosphorylation of both AKT and TAK1, and up-regulate the ratio of Bax/Bcl-2. In addition, these compounds could induce apoptosis in vivo, and increase the secretion of TNF-α, IFN-γ, and IgG, while not significantly impacting the ratio of CD4+T/CD8+T. These investigations suggest that the RING domain of TRAF6 could serve as a de novo biological target for therapeutic treatment in cancers.


Assuntos
Apoptose/efeitos dos fármacos , Alcaloides de Cinchona/metabolismo , Alcaloides de Cinchona/farmacologia , Domínios Proteicos , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , Células HeLa , Humanos , Imunoglobulina G/sangue , Marcação In Situ das Extremidades Cortadas , Interferon gama/sangue , Contagem de Linfócitos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/química , Fator de Necrose Tumoral alfa/sangue , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
19.
Medicine (Baltimore) ; 98(16): e15232, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31008954

RESUMO

Ubiquitin-conjugating enzyme E2C (UBE2C), a crucial part of the ubiquitin-conjugating enzyme complex, is reported to promote progression of various cancers. Leucine-rich repeated-containing G protein-coupled receptor (LGR5), a biomarker of cancer stem cells, is reported to be responsible for the initiation and progression of cancers. WW domain-containing oxidoreductase (WWOX), a suppressor of tumor, is reported to inhibit initiation and progression of cancers. Vasculogenic mimicry (VM), a new blood supply pattern, is associated with progression of cancers. However, the clinicopathological significance of UBE2C, LGR5, WWOX, and VM in invasive breast carcinoma (IBC) remains elusive. The aim of this study is to investigate the positive rate of UBE2C, LGR5, WWOX, and VM in IBC and their clinical significance.Positive rates of UBE2C, LGR5, WWOX, and VM in 247 whole IBC samples were detected through immunohistochemistry. Patients data (including clinical, demography, follow-up) were collected.Levels of UBE2C, LGR5, VM, and microvessel density (MVD) were significantly higher, and level of WWOX was significantly lower in IBC specimens when compared with normal mammary gland tissues. Levels of UBE2C, LGR5, VM, and MVD were all positively associated with tumor stages, lymph node metastasis (LNM) stages, tumor grades, and tumor-node-metastasis (TNM) stages, and unfavorably with patients' overall survival (OS) and disease-free survival (DFS). Level of WWOX was negatively associated with tumor stages, LNM stages, grades, and TNM stages, and favorably with patients' OS and DFS. Multivariate analysis indicated that levels of UBE2C, LGR5, VM, MVD, and WWOX, as well as TNM stages were independently prognostic factors for OS and DFS in patients with IBC.UBE2C, LGR5, VM, MVD, and WWOX may be considered as promising indicator of IBC prognosis.


Assuntos
Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Oxidorredutase com Domínios WW/metabolismo , Adulto , Idoso , Mama/enzimologia , Mama/patologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/irrigação sanguínea , Carcinoma/mortalidade , Carcinoma/patologia , China/epidemiologia , Feminino , Humanos , Pessoa de Meia-Idade
20.
Genes Dev ; 33(11-12): 705-717, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30948432

RESUMO

The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.


Assuntos
RNA Polimerase II/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Mutantes/metabolismo , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA