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1.
Theranostics ; 11(16): 7755-7766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335962

RESUMO

Background: Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify in vivo. We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [68Ga]MHLL1 after acute MI. Methods: One-step chemical synthesis and manual as well as module-based radiolabeling yielded [68Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by ex vivo autoradiography and immunostaining for FAP and inflammatory macrophages. Results: [68Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [68Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 vs. 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 vs. 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [68Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution in vivo. Conclusion: The simplified synthesis of [68Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.


Assuntos
Endopeptidases/metabolismo , Radioisótopos de Gálio/farmacologia , Proteínas de Membrana/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Animais , Autorradiografia/métodos , Linhagem Celular Tumoral , Endopeptidases/fisiologia , Fibroblastos/metabolismo , Fibrose/diagnóstico por imagem , Radioisótopos de Gálio/metabolismo , Humanos , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Distribuição Tecidual/fisiologia , Tomografia Computadorizada por Raios X/métodos
2.
Cell Death Dis ; 12(6): 534, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035221

RESUMO

Breast cancer is the most common malignancy in women worldwide. Estrogen receptor α (ERα) is expressed in ∼70% of breast cancer cases and promotes estrogen-dependent cancer progression. In the present study, we identified OTU domain-containing 7B (OTUD7B), a deubiquitylase belonging to A20 subgroup of ovarian tumor protein superfamily, as a bona fide deubiquitylase of ERα in breast cancer. OTUD7B expression was found to be positively correlated with ERα in breast cancer and associated with poor prognosis. OTUD7B could interact with, deubiquitylate, and stabilize ERα in a deubiquitylation activity-dependent manner. Depletion of OTUD7B decreased ERα protein level, the expression of ERα target genes, and the activity of estrogen response element in breast cancer cells. In addition, OTUD7B depletion significantly decreased ERα-positive breast cancer cell proliferation and migration. Finally, overexpression of ERα could rescue the suppressive effect induced by OTUD7B depletion, suggesting that the ERα status was essential to the function of OTUD7B in breast carcinogenesis. In conclusion, our study revealed an interesting post-translational mechanism between ERα and OTUD7B in ERα-positive breast cancer. Targeting the OTUD7B-ERα complex may prove to be a potential approach to treat patients with ERα-positive breast cancer.


Assuntos
Neoplasias da Mama/patologia , Endopeptidases/fisiologia , Receptor alfa de Estrogênio/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Processamento de Proteína Pós-Traducional/genética , Estabilidade Proteica , Ubiquitinação/genética
3.
J Virol ; 95(14): e0032121, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33883227

RESUMO

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a comprehensive view of lysins' structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G-) (higher net charge and amphipathic helices) are responsible for improved interaction with the G- envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecule design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G- surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages' host bacteria, and sequence-predicted physicochemical properties. Based on such classifiers, an investigation of the differential appearance of certain features was conducted. This analysis revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. In particular, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment, and aliphatic index) were associated with G- phage lysins, appearing specifically at their C-terminal end. Information on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts is provided, specifically supporting the nowadays-common hypothesis that lysins from G- usually contain AMP-like regions. IMPORTANCE Phage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The potential of lysins as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysin diversity landscape by examining a set of phage lysin genes. We have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures and, thus, the reach of their specialization regarding cell wall structures. These results provide clarity and evidence to sustain some of the common hypotheses in current literature, as well as making available an updated and characterized database of lysins sequences for further developments.


Assuntos
Antibacterianos , Bacteriófagos/genética , Parede Celular/enzimologia , Endopeptidases/genética , Antibacterianos/farmacologia , Bactérias/enzimologia , Bactérias/genética , Bactérias/virologia , Endopeptidases/química , Endopeptidases/farmacologia , Endopeptidases/fisiologia , Domínios Proteicos , Relação Estrutura-Atividade
4.
Cancer Lett ; 504: 104-115, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33587979

RESUMO

Macrophages, which are highly plastic, can be polarized to M1 or M2 subtypes according to the diverse signals in complex microenvironment. Studies have shown the activation of YAP, an oncogenic transcriptional co-activator, increased macrophage recruitment. However, its role in macrophage polarization remains to be elucidated, especially in triple-negative breast cancer (TNBC) progression. Here we found TNBC cells increased YAP expression in macrophages, which depended on OTUD5-mediated deubiquitination and stabilization of YAP, then the high expression of YAP polarized macrophage to the M2-like phenotype. Moreover, the elevation of YAP in M2-like macrophage promotes the pro-metastatic potential of TNBC cells via MCP-1/CCR2 pathway. We also observed high expression of YAP in M2 macrophage was negatively related to survival. Collectively, our finding suggested the therapeutic strategy that targets YAP+ M2 macrophage could be a novel option for TNBC treatment.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Endopeptidases/fisiologia , Macrófagos/patologia , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Macrófagos/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Ubiquitinação
6.
J Cell Mol Med ; 24(18): 10946-10957, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32798288

RESUMO

Ubiquitin-specific protease 19 (USP19) belongs to USP family and is involved in promoting skeletal muscle atrophy. Although USP19 is expressed in the heart, the role of USP19 in the heart disease remains unknown. The present study provides in vivo and in vitro data to reveal the role of USP19 in preventing pathological cardiac hypertrophy. We generated USP19-knockout mice and isolated neonatal rat cardiomyocytes (NRCMs) that overexpressed or were deficient in USP19 to investigate the effect of USP19 on transverse aortic constriction (TAC) or phenylephrine (PE)-mediated cardiac hypertrophy. Echocardiography, pathological and molecular analysis were used to determine the extent of cardiac hypertrophy, fibrosis, dysfunction and inflammation. USP19 expression was markedly increased in rodent hypertrophic heart or cardiomyocytes underwent TAC or PE culturing, the increase was mediated by the reduction of Seven In Absentia Homolog-2. The extent of TAC-induced cardiac hypertrophy, fibrosis, dysfunction and inflammation in USP19-knockout mice was exacerbated. Consistently, gain-of-function and loss-of-function approaches that involved USP19 in cardiomyocytes suggested that the down-regulation of USP19 promoted the hypertrophic phenotype, while the up-regulation of USP19 improved the worsened phenotype. Mechanistically, the USP19-elicited cardiac hypertrophy improvement was attributed to the abrogation of the transforming growth factor beta-activated kinase 1 (TAK1)-p38/JNK1/2 transduction. Furthermore, the inhibition of TAK1 abolished the aggravated hypertrophy induced by the loss of USP19. In conclusion, the present study revealed that USP19 and the downstream of TAK1-p38/JNK1/2 signalling pathway might be a potential target to attenuate pathological cardiac hypertrophy.


Assuntos
Cardiomegalia/fisiopatologia , Endopeptidases/fisiologia , MAP Quinase Quinase Quinases/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Miócitos Cardíacos/enzimologia , Angiotensina II/toxicidade , Animais , Animais Recém-Nascidos , Estenose da Valva Aórtica , Sistemas CRISPR-Cas , Cardiomegalia/induzido quimicamente , Cardiomegalia/diagnóstico por imagem , Modelos Animais de Doenças , Endopeptidases/biossíntese , Endopeptidases/deficiência , Endopeptidases/genética , Fibrose , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fenilefrina/farmacologia , Pressão , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Remodelação Ventricular/fisiologia
7.
J Virol ; 94(13)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295921

RESUMO

Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD.IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.


Assuntos
Endopeptidases/genética , Endopeptidases/metabolismo , Vírus da Febre Aftosa/genética , Animais , Antivirais/metabolismo , Linhagem Celular , Citocinas/metabolismo , Endopeptidases/fisiologia , Feminino , Febre Aftosa/virologia , Vírus da Febre Aftosa/metabolismo , Vírus da Febre Aftosa/patogenicidade , Células HEK293 , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Serina Endopeptidases/metabolismo , Suínos , Ubiquitinas/metabolismo , Vacinas Atenuadas/imunologia
8.
J Biosci Bioeng ; 129(4): 423-427, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31640922

RESUMO

In the yeast Saccharomyces cerevisiae, the transcriptional factor Msn2 plays an essential role in response to a variety of environmental stresses by activating the transcription of many genes that contain the stress-responsive elements in the promoters. We previously reported that overexpression of the MSN2 gene confers tolerance to various stresses in industrial yeast strains. Recently, the overexpression of MSN2 was shown to increase the amount of the amino acid permease Gnp1 on the plasma membrane, leading to the increased uptake of proline into the cell, suggesting a novel link between the Msn2-mediated stress response and amino acid homeostasis in yeast. Here, we found that overexpression of MSN2 increased ubiquitinated protein levels with reduced free ubiquitin. Among deubiquitinating enzymes (DUBs), it was revealed that the loss of Ubp6 depleted the free ubiquitin level and decreased tolerance to the toxic amino acid analogues. The overexpression of UBP6 in MSN2-overexpressing cells clearly complemented the impaired tolerance towards the toxic amino acid analogues. Both the protein level and the plasma-membrane localization of Gnp1 were increased in ubp6-deleted cells, as shown in MSN2-overexpressing cells. These results suggest that an excess level of Msn2 impairs endocytic degradation of Gnp1 through dysfunction of Ubp6 and other DUBs.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Endopeptidases/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae , Estresse Fisiológico/fisiologia , Fatores de Transcrição/fisiologia , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Organismos Geneticamente Modificados , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Ubiquitina/metabolismo
9.
Biochem Soc Trans ; 47(6): 1867-1879, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31845722

RESUMO

Protein modification by ubiquitin is one of the most versatile posttranslational regulations and counteracted by almost 100 deubiquitinating enzymes (DUBs). USP8 was originally identified as a growth regulated ubiquitin-specific protease and is like many other DUBs characterized by its multidomain architecture. Besides the catalytic domain, specific protein-protein interaction modules were characterized which contribute to USP8 substrate recruitment, regulation and targeting to distinct protein complexes. Studies in mice and humans impressively showed the physiological relevance and non-redundant function of USP8 within the context of the whole organism. USP8 knockout (KO) mice exhibit early embryonic lethality while induced deletion in adult animals rapidly causes lethal liver failure. Furthermore, T-cell specific ablation disturbs T-cell development and function resulting in fatal autoimmune inflammatory bowel disease. In human patients, somatic mutations in USP8 were identified as the underlying cause of adrenocorticotropic hormone (ACTH) releasing pituitary adenomas causing Cushing's disease (CD). Here we provide an overview of the versatile molecular, cellular and pathology associated function and regulation of USP8 which appears to depend on specific protein binding partners, substrates and the cellular context.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Cílios/metabolismo , Endopeptidases/genética , Endopeptidases/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitofagia/fisiologia , Mutação , Hipersecreção Hipofisária de ACTH/genética , Ligação Proteica , Transdução de Sinais , Linfócitos T/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/fisiologia
10.
Science ; 366(6469): 1150-1156, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31780561

RESUMO

To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human ß2-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Endopeptidases/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/fisiologia , Dobramento de Proteína , Receptores Adrenérgicos beta 2/fisiologia , Evolução Biológica , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Modificação Traducional de Proteínas , Imagem Individual de Molécula
11.
Endocrinology ; 160(8): 1982-1998, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199479

RESUMO

Leptin has neurotrophic actions in the hippocampus to increase synapse formation and stimulate neuronal plasticity. Leptin also enhances cognition and has antidepressive and anxiolytic-like effects, two hippocampal-dependent behaviors. In contrast, mice lacking leptin or the long form of the leptin receptor (LepRb) have lower cortical volume and decreased memory and exhibit depressive-like behaviors. A number of the signaling pathways regulated by LepRb are known, but how membrane LepRb levels are regulated in the central nervous system is not well understood. Here, we show that the lysosomal inhibitor chloroquine increases LepRb expression in hippocampal cultures, suggesting that LepRb is degraded in the lysosome. Furthermore, we show that leptin increases surface expression of its own receptor by decreasing the level of ubiquitinated LepRbs. This decrease is mediated by the deubiquitinase ubiquitin-specific protease 8 (USP8), which we show is in complex with LepRb. Acute leptin stimulation increases USP8 activity. Moreover, leptin stimulates USP8 gene expression through cAMP response element-binding protein (CREB)-dependent transcription, an effect blocked by expression of a dominant-negative CREB or with short hairpin RNA knockdown of CREB. Increased expression of USP8 causes increased surface localization of LepRb, which in turn enhances leptin-mediated activation of the MAPK kinase/extracellular signal-regulated kinase pathway and CREB activation. Lastly, increased USP8 expression increases glutamatergic synapse formation in hippocampal cultures, an effect dependent on expression of LepRbs. Leptin-stimulated synapse formation also requires USP8. In conclusion, we show that USP8 deubiquitinates LepRb, thus inhibiting lysosomal degradation and enhancing surface localization of LepRb, which are essential for leptin-stimulated synaptogenesis in the hippocampus.


Assuntos
Endopeptidases/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Leptina/farmacologia , Receptores para Leptina/metabolismo , Sinapses/fisiologia , Ubiquitina Tiolesterase/fisiologia , Ubiquitinação , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Ubiquitina Tiolesterase/genética
12.
Nucleic Acids Res ; 47(13): 7035-7048, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31114929

RESUMO

The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5'-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). Several experimental evidences suggest that GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. However, the underlying molecular mechanism is unknown. Here, we investigated the role of the uncharacterized Drosophila melanogaster (Dm) GIGYF protein in post-transcriptional mRNA regulation. We show that, when in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. We identified the RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4-NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP-GIGYF complex. Our findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Fator de Iniciação 4E em Eucariotos/fisiologia , Regulação da Expressão Gênica , Proteínas de Ligação ao Cap de RNA/fisiologia , RNA Mensageiro/genética , Proteínas Repressoras/fisiologia , Sequência de Aminoácidos , Animais , Sequência Conservada , RNA Helicases DEAD-box/fisiologia , Regulação para Baixo , Endopeptidases/fisiologia , Genes Reporter , Complexos Multiproteicos , Biossíntese de Proteínas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ribonucleases/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
13.
Med Sci Monit ; 25: 3469-3475, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31075090

RESUMO

BACKGROUND The aim of this study was to investigate the role of deubiquitinase [ovarian tumor domain-containing protein 5 (OTUD5)] in regulating Akt ubiquitination and its effect on the radiosensitivity of cervical cancer. MATERIAL AND METHODS Cervical cancer C33A cells were cultured, and then 2 groups of cells (overexpressed cells and silenced cells) were established by overexpressing and silencing OTUD5 gene. Next, quantitative polymerase chain reaction (qPCR) was employed to detect the expression level of OTUD5 in cells in each group. Co-immunoprecipitation and Western blot (WB) analysis were applied to measure the expression level of phosphorylated protein kinase B (Akt) and the level of ubiquitination. The sensitivity of cells to radiotherapy in each group was detected via clone-forming efficiency assay. After that, Statistical Product and Service Solutions (SPSS) 17.0 software was employed for analyses. The t test, one-way analysis of variance (ANOVA), and p test were used. P<0.05 suggested that a difference was statistically significant. RESULTS The levels of phosphorylated Akt and ubiquitination in OTUD5-overexpressed C33A cells were lower than those in the OTUD5-silenced group and control group. The sensitivity of OTUD5-overexpressed C33A cells to radiotherapy was higher than that of the OTUD5-silenced group and control group. CONCLUSIONS OTUD5 affects the radiosensitivity of cervical cancer through the regulation of Akt deubiquitination.


Assuntos
Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias do Colo do Útero/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/fisiologia , Feminino , Inativação Gênica , Humanos , Neoplasias Ovarianas/genética , RNA Interferente Pequeno , Tolerância a Radiação/genética , Tolerância a Radiação/fisiologia , Transdução de Sinais , Ubiquitinação , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/radioterapia
14.
Brain Res ; 1719: 40-48, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31075263

RESUMO

Sepsis-associated encephalopathy (SAE) is a common and serious complication of sepsis, which is thought to be caused by neuroinflammation. In our previous study, ubiquitin-specific protease 8 (USP8), was reported to regulate inflammation in vitro. In the current study, we investigated whether increased USP8 expression would ameliorate the cognitive and motor impairments induced by cecal ligation and puncture (CLP) in mice, a model of SAE. Male adult mice were randomly divided into four groups: control, sham, CLP, and CLP + USP8 groups. The CLP + USP8 mice showed reduced weight loss on day 4 post-CLP, with a slight increase noted on day 7. The mortality rate in the CLP group was 70% 48 h after CLP; however, USP8 significantly improved survival after CLP. USP8 modulated the neurobehavioral scores in CLP mice. Our results also indicate that USP8 attenuated the CLP-induced cognitive and motor impairments, based on the performance of mice in the Morris water maze (MWM), pole-climbing, and wire suspension tests. USP8 suppressed the release of pro-inflammatory mediators, including prostaglandin E2(PGE2) in the serum and nitric oxide (NO) in brain tissue, as well as levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in brain tissue. Immunofluorescence experiments revealed that USP8 inhibited CLP-induced increases in microglial size and density in the hippocampus, and protected hippocampal neurons. Our findings indicate that neuroinflammation occurs in the brains of CLP mice, and that USP8 exerts protective effects against CLP-induced neuroinflammation and cognitive and motor impairments, which may aid in the development of novel therapeutic strategies for SAE.


Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Encefalopatia Associada a Sepse/fisiopatologia , Ubiquitina Tiolesterase/metabolismo , Animais , Encéfalo/metabolismo , Ceco , Cognição/efeitos dos fármacos , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Endopeptidases/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Hipocampo/metabolismo , Inflamação/metabolismo , Inibição Psicológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Atividade Motora/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Sepse/complicações , Ubiquitina Tiolesterase/fisiologia
15.
Neurophysiol Clin ; 49(1): 69-80, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30327170

RESUMO

OBJECTIVES: The aim of our study was to verify the effectiveness of single fiber potential (SFP) criteria in cases when the potential recorded using single fiber electrode (SFE) or concentric needle electrode (CNE) is contaminated by distant fibers. METHODS: Morphological counterparts of SFP were studied using computer simulations. In this study, we examined triphasic potentials using a model of a linear source of SFP. The criteria defining SFP in the case of SFP contaminated by distant fibers were analyzed, and the effect of second fiber contamination on jitter and fiber diameter determination evaluated. RESULTS: We found that SFP criteria prevent detection of SFP from fibers smaller than about 30µm in diameter, but do not prevent classification of a potential as an SFP even though it is formed by two or more fibers. This suggests that the presently used criteria may lead to incorrect interpretation of SFP potentials. SFPs contaminated by fibers of diameters differing by a few percent fulfill the criteria but a negative peak may be shifted in time and therefore impact jitter and diameter measurements. This contamination generally tends to decrease both the jitter and the determined diameter. A new approach to the identification of SFP is presented, determining fiber diameter and distance from the electrode to enable maximum sensitivity to potential contamination by the effect of a second fiber. CONCLUSION: A new parameter characterizing SFP shape changes is introduced. This parameter is used in the method by which additional fibers affecting the SFP may be detected.


Assuntos
Eletromiografia , Endopeptidases/fisiologia , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Potenciais de Ação/fisiologia , Simulação por Computador , Eletrodos , Eletromiografia/métodos , Humanos
16.
Mol Biol Rep ; 45(6): 2393-2401, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30386973

RESUMO

Macrophages play pivotal roles in innate and adaptive immune response, tissue homeostasis and cancer development. Their development and heterogeneity are tightly controlled by epigenetic program and transcription factors. Deubiquitinase Mysm1 plays crucial roles in regulating stem cell maintenance and immune cell development. Here we show that Mysm1 expression is up regulated during bone marrow macrophage development. Mysm1 deficient cells exhibit accelerating proliferation with more cells going to S phase and higher cyclin D1, cyclin D2 and c-Myc expression. However, compared to WT counterparts, more cell death is also detected in Mysm1 deficient cells no matter M-CSF deprived or not. In LPS-condition medium, Mysm1-/- macrophages show more pro-inflammatory factors IL-1ß, TNFα and iNOS production. In addition, much higher expression of surface marker CD86 is detected in Mysm1-/- macrophages. In vivo tumor model data demonstrate that in contrast to WT macrophages promoting tumor growth, Mysm1-/- macrophages inhibit tumor growth, showing the properties of M1 macrophages. Collectively, these data indicate that Mysm1 is essential for macrophage survival and plays an important role in macrophage polarization and might be a target for cell therapy.


Assuntos
Endopeptidases/metabolismo , Macrófagos/metabolismo , Animais , Apoptose , Ciclo Celular/fisiologia , Diferenciação Celular , Células Cultivadas , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/fisiologia , Regulação da Expressão Gênica/genética , Camundongos Knockout , Células-Tronco , Transativadores , Fatores de Transcrição , Proteases Específicas de Ubiquitina , Ubiquitinação/fisiologia
17.
Blood ; 132(4): 423-434, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29844011

RESUMO

Ras mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic KrasG12D/+ USP22 deficiency in KrasG12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient KrasG12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient KrasG12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient KrasG12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis.


Assuntos
Transformação Celular Neoplásica/patologia , Endopeptidases/fisiologia , Leucemia Mieloide/patologia , Leucemia Mielomonocítica Juvenil/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prognóstico , Proteínas Proto-Oncogênicas/genética , Taxa de Sobrevida , Transativadores/genética , Ubiquitina Tiolesterase
18.
Curr Rheumatol Rep ; 20(7): 39, 2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29846841

RESUMO

PURPOSE OF REVIEW: The nuclear factor κB (NF-κB) pathway is tightly regulated through multiple posttranslational mechanisms including ubiquitination. Mutations in these regulatory pathways can cause disease and are the focus of this review. RECENT FINDINGS: The linear ubiquitin chain assembly complex (LUBAC) is a trimer made up of HOIL-1L, SHARPIN, and the catalytic subunit HOIP. Loss of function mutations in HOIL-1L and HOIP result in largely overlapping phenotypes, characterized by multi-organ autoinflammation, immunodeficiency, and amylopectinosis. Interestingly, patient fibroblasts exhibited diminished IL-1ß- and TNF-induced NF-κB activation, yet monocytes were hyper-responsive to IL-1ß, hinting at cell type or target specific roles of LUBAC-mediated ubiquitination. Ubiquitin-driven signaling is counterbalanced by deubiquitinase enzymes (DUBs), such as OTULIN and A20. Hypomorphic mutations in OTULIN result in elevated NF-κB signaling causing an autoinflammatory syndrome. Similarly, patients with high-penetrance heterozygous mutations in the gene encoding A20 (haploinsufficiency of A20 (HA20)) display excessive ubiquitination and increased activity of NF-κB and of NLRP3 inflammasome activation. HA20 patients present with Behçet-like characteristics or an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype, indicating diverse protein functions. This review summarizes recent discoveries in the field of NF-kB-related autoinflammatory diseases (relopathies) within the past 3 years and points to several questions that still remain unanswered.


Assuntos
Doenças Hereditárias Autoinflamatórias/genética , Endopeptidases/genética , Endopeptidases/fisiologia , Doenças Hereditárias Autoinflamatórias/metabolismo , Humanos , Mutação , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologia
19.
Nat Commun ; 9(1): 688, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29449677

RESUMO

Although approximately 100 deubiquitinating enzymes (DUBs) are encoded in the human genome, very little is known about the DUBs that function in mitosis. Here, we demonstrate that DUB USP35 functions as a mitotic regulator by controlling the protein levels and downstream signaling of Aurora B and the depletion of USP35 eventually leads to several mitotic defects including cytokinesis failures. USP35 binds to and deubiquitinates Aurora B, and inhibits the APCCDH1-mediated proteasomal degradation of Aurora B, thus maintaining its steady-state levels during mitosis. In addition, the loss of USP35 decreases the phosphorylation of histone H3-Ser10, an Aurora B substrate. Finally, the transcription factor FoxM1 promotes the expression of USP35, as well as that of Aurora B, during the cell cycle. Our findings suggest that USP35 regulates the stability and function of Aurora B by blocking APCCDH1-induced proteasomal degradation, thereby controlling mitotic progression.


Assuntos
Aurora Quinase B/metabolismo , Endopeptidases/fisiologia , Mitose/fisiologia , Endopeptidases/genética , Endopeptidases/metabolismo , Estabilidade Enzimática , Proteína Forkhead Box M1/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes APC , Células HeLa , Histonas/metabolismo , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica
20.
Nat Commun ; 8(1): 481, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28883390

RESUMO

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.It is unclear how Gram-positive bacteria, with a thick cell wall, can release membrane vesicles. Here, Toyofuku et al. show that a prophage-encoded endolysin can generate holes in the cell wall through which cytoplasmic membrane material protrudes and is released as vesicles.


Assuntos
Bacillus subtilis/ultraestrutura , Peptidoglicano/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Parede Celular/ultraestrutura , Tomografia com Microscopia Eletrônica , Endopeptidases/metabolismo , Endopeptidases/fisiologia
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