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1.
Adv Exp Med Biol ; 1164: 63-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576540

RESUMO

Gankyrin (also called PSMD10, p28, or p28GANK) is a crucial oncoprotein that is upregulated in various cancers and assumed to play pivotal roles in the initiation and progression of tumors. Although the in vitro function of gankyrin is relatively well characterized, its role in vivo remains to be elucidated. We have investigated the function of gankyrin in vivo by producing mice with liver parenchymal cell-specific gankyrin ablation (Alb-Cre;gankyrinf/f) and gankyrin deletion both in liver parenchymal and in non-parenchymal cells (Mx1-Cre;gankyrinf/f). Gankyrin deficiency both in non-parenchymal cells and parenchymal cells, but not in parenchymal cells alone, reduced STAT3 activity, interleukin-6 production, and cancer stem cell marker expression, leading to attenuated tumorigenic potential in the diethylnitrosamine hepatocarcinogenesis model. Essentially similar results were obtained by analyzing mice with intestinal epithelial cell-specific gankyrin ablation (Villin-Cre;Gankyrinf/f) and gankyrin deletion both in myeloid and epithelial cells (Mx1-Cre;Gankyrinf/f) in the colitis-associated cancer model. Clinically, gankyrin expression in the tumor microenvironment was negatively correlated with progression-free survival in patients undergoing treatment with Sorafenib for hepatocellular carcinomas. These findings indicate important roles played by gankyrin in non-parenchymal cells as well as parenchymal cells in the pathogenesis of liver cancers and colorectal cancers, and suggest that by acting both on cancer cells and on the tumor microenvironment, anti-gankyrin agents would be promising as therapeutic and preventive strategies against various cancers, and that an in vitro cell culture models that incorporate the effects of non-parenchymal cells and gankyrin would be useful for the study of human cell transformation.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Complexo de Endopeptidases do Proteassoma , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/fisiopatologia , Carcinoma Hepatocelular/terapia , Neoplasias Colorretais/genética , Neoplasias Colorretais/fisiopatologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Deleção de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia , Neoplasias Hepáticas/terapia , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral
2.
Gene ; 717: 144043, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31400407

RESUMO

Genes involved in the repair of DNA damage are emerging as playing important roles during the disease processes caused by pathogenic fungi. However, there are potentially hundreds of genes involved in DNA repair in a fungus and some of those genes can play additional roles within the cell. One such gene is RAD23, required for virulence of the human pathogenic fungus Cryptococcus neoformans, that encodes a protein involved in the nucleotide excision repair (NER) pathway. However, Rad23 is a dual function protein, with a role in either repair of damaged DNA or protein turn over by directing proteins to the proteasome. Here, these two functions of Rad23 were tested by the creation of a series of domain deletion alleles of RAD23 and the assessment of the strains for DNA repair, proteasome functions, and virulence properties. Deletion of the different domains was able to uncouple the two functions of Rad23, and the phenotypes of strains carrying such forms indicated that the role of RAD23 in virulence is due to its function in proteasomal-mediated protein degradation rather than NER.


Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Reparo do DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/microbiologia , Mariposas/microbiologia , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico/genética , Virulência
3.
Mol Biol (Mosk) ; 53(4): 638-647, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397437

RESUMO

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.


Assuntos
Resposta ao Choque Térmico , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteólise , Células U937 , Ubiquitina/metabolismo
4.
Plant Cell Physiol ; 60(8): 1633-1645, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292642

RESUMO

Plants respond to a rise in ambient temperature by increasing the growth of petioles and hypocotyls. In this work, we show that Arabidopsis thaliana class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 are required for optimal petiole and hypocotyl elongation under high ambient temperature. These TCPs influence the levels of the DELLA protein RGA and the expression of growth-related genes, which are induced in response to an increase in temperature. However, the class I TCPs are not required for the induction of the auxin biosynthesis gene YUCCA8 or for auxin-dependent gene expression responses. TCP15 directly targets the gibberellin biosynthesis gene GA20ox1 and the growth regulatory genes HBI1 and PRE6. Several of the genes regulated by TCP15 are also targets of the growth regulator PIF4 and show an enrichment of PIF4- and TCP-binding motifs in their promoters. PIF4 binding to GA20ox1 and HBI1 is enhanced in the presence of the TCPs, indicating that TCP14 and TCP15 directly participate in the induction of genes involved in gibberellin biosynthesis and cell expansion by high temperature functionally interacting with PIF4. In addition, overexpression of HBI1 rescues the growth defects of tcp14 tcp15 double mutants, suggesting that this gene is a major outcome of regulation by both class I TCPs during thermomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Giberelinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Temperatura Ambiente , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
J Enzyme Inhib Med Chem ; 34(1): 1307-1313, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31307247

RESUMO

Proteasome inhibition is a promising strategy for the treatment of multiple myeloma; unfortunately, this disease is often associated with an increasing chemoresistance. One novel approach may be to target the immunoproteasome, a proteasomal isoform mainly present in cells of hematopoietic origin. We investigated the activity of a panel of amides against immunoproteasome core particles as potential agents for the treatment of multiple myeloma (MM). Amide 6 showed an ideal profile since it was able to inhibit both the chymotrypsin-like activities of the immunoproteasome with Ki values of 4.90 µM and 4.39 µM for ß1i and ß5i, respectively, coupled with an EC50 =17.8 µM against MM.1R cells. Compound 6 inhibited also ubiquitinated protein degradation and was able to act on different phases of MM cell cycle reducing the levels of cyclin A/CDK1, cyclin B/CDK1 and cyclin D/CDK4/6 complexes, which turns in cell cycle arrest.


Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Relação Estrutura-Atividade
6.
Chemosphere ; 233: 786-795, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31340409

RESUMO

Microbial volatile organic compounds (mVCs) are formed in the metabolism of microorganisms and widely distributed in nature and pose threats to human health. However, the air pollution by microorganisms is a situation which is poorly understood. In this study, the cytotoxicity of E. aerogenes VCs was evaluated in the model organism Saccharomyces cerevisiae. E. aerogenes VCs inhibited the survival of yeast and triggered the formation of intracellular reactive oxygen species (ROS). The hypersensitive of MAP kinase mpk1/slt2 and 19S regulatory assembly chaperone adc17 mutants to the E. aerogenes VCs indicated cell wall integrity (CWI) pathway together with stress-inducible proteasome assembly regulation are essentially involved in mVCs tolerance mechanism. Furthermore, exposure to the mVCs resulted in the transcriptional upregulation of the CWI pathway, the regulatory particle assembly chaperones, and genes involved in proteasome regulations. Our research suggested that the ROS/MAPK signaling and proteasome regulatory pathway play pivotal roles in the integration and fine-tuning of the mVCs stress response. This study provides a molecular framework for future study of the effects of mVCs on more complex organisms, such as humans.


Assuntos
Enterobacter aerogenes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Compostos Orgânicos Voláteis/farmacologia , Poluentes Atmosféricos/análise , Poluição do Ar/análise , Parede Celular/metabolismo , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional
7.
Nat Commun ; 10(1): 2576, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189900

RESUMO

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.


Assuntos
Dinaminas/metabolismo , Mitocôndrias Musculares/patologia , Dinâmica Mitocondrial/fisiologia , Miopatias Mitocondriais/patologia , Músculo Esquelético/patologia , Animais , Cálcio/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Dinaminas/genética , Homeostase/fisiologia , Humanos , Camundongos , Camundongos Knockout , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/mortalidade , Músculo Esquelético/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitinas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
8.
Virol J ; 16(1): 73, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146743

RESUMO

BACKGROUND: The ubiquitin proteasome system (UPS) regulates the expression levels of cellular proteins by ubiquitination of protein substrates followed by their degradation via the proteasome. As a highly conserved cellular degradation mechanism, the UPS affects a variety of biological processes and participates in viral propagation. MAIN BODY: During hepatitis B virus (HBV) infection, the UPS is shown to act as a double-edged sword in viral pathogenesis. On the one hand, the UPS acts as a host defense mechanism to selectively recognize HBV proteins as well as special cellular proteins that favor the viral life cycle and induces their ubiquitin-dependent proteasomal degradation to limit HBV infection. On the other hand, the HBV has evolved to subvert the UPS function for its own advantage. Moreover, in the infected hepatocytes, certain cellular proteins that are dependent on the UPS are involved in abnormal biological processes which are mediated by HBV. CONCLUSION: The molecular interaction of HBV with the UPS to modulate viral propagation and pathogenesis is summarized in the review. Considering the important role of the UPS in HBV infection, a better understanding of the HBV-UPS interaction could provide novel insight into the mechanisms that are involved in viral replication and pathogenesis and help to develop potential treatment strategies targeting the UPS.


Assuntos
Vírus da Hepatite B/patogenicidade , Interações Hospedeiro-Patógeno , Complexo de Endopeptidases do Proteassoma/metabolismo , Replicação Viral , Animais , Hepatite B/patologia , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Humanos , Camundongos , Ubiquitinação
9.
Nat Chem ; 11(7): 644-652, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182821

RESUMO

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.


Assuntos
Lisina/química , Peptídeos Cíclicos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Células HeLa , Humanos , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinas/síntese química , Ubiquitinas/química
10.
Microb Pathog ; 132: 362-368, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054366

RESUMO

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.


Assuntos
Flavivirus/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Replicação Viral/fisiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Patos , Flavivirus/efeitos dos fármacos , Flavivirus/patogenicidade , Técnicas de Silenciamento de Genes , Leupeptinas/antagonistas & inibidores , Doenças das Aves Domésticas/virologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , RNA Interferente Pequeno , Transfecção , Ubiquitina/efeitos dos fármacos , Ubiquitina/genética , Proteínas do Envelope Viral , Internalização do Vírus
11.
Int J Mol Sci ; 20(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060234

RESUMO

In recent years, functional interconnections emerged between synaptic transmission, inflammatory/immune mediators, and central nervous system (CNS) (patho)-physiology. Such interconnections rose up to a level that involves synaptic plasticity, both concerning its molecular mechanisms and the clinical outcomes related to its behavioral abnormalities. Within this context, synaptic plasticity, apart from being modulated by classic CNS molecules, is strongly affected by the immune system, and vice versa. This is not surprising, given the common molecular pathways that operate at the cross-road between the CNS and immune system. When searching for a common pathway bridging neuro-immune and synaptic dysregulations, the two major cell-clearing cell clearing systems, namely the ubiquitin proteasome system (UPS) and autophagy, take center stage. In fact, just like is happening for the turnover of key proteins involved in neurotransmitter release, antigen processing within both peripheral and CNS-resident antigen presenting cells is carried out by UPS and autophagy. Recent evidence unravelling the functional cross-talk between the cell-clearing pathways challenged the traditional concept of autophagy and UPS as independent systems. In fact, autophagy and UPS are simultaneously affected in a variety of CNS disorders where synaptic and inflammatory/immune alterations concur. In this review, we discuss the role of autophagy and UPS in bridging synaptic plasticity with neuro-immunity, while posing a special emphasis on their interactions, which may be key to defining the role of immunity in synaptic plasticity in health and disease.


Assuntos
Neuroimunomodulação , Plasticidade Neuronal , Animais , Autofagia , Biomarcadores , Suscetibilidade a Doenças , Metabolismo Energético , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Mediadores da Inflamação/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transmissão Sináptica
12.
Genes Dev ; 33(11-12): 684-704, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048545

RESUMO

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
13.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067643

RESUMO

The 26S proteasome is critical for the selective degradation of proteins in eukaryotic cells. This enzyme complex is composed of approximately 70 subunits, including the structurally homologous proteins α1-α7, which combine to form heptameric rings. The correct arrangement of these α subunits is essential for the function of the proteasome, but their assembly does not occur autonomously. Assembly of the α subunit is assisted by several chaperones, including the PAC3-PAC4 heterodimer. In this study we showed that the PAC3-PAC4 heterodimer functions as a molecular matchmaker, stabilizing the α4-α5-α6 subcomplex during the assembly of the α-ring. We solved a 0.96-Å atomic resolution crystal structure for a PAC3 homodimer which, in conjunction with nuclear magnetic resonance (NMR) data, highlighted the mobility of the loop comprised of residues 51 to 61. Based on these structural and dynamic data, we created a three-dimensional model of the PAC3-4/α4/α5/α6 quintet complex, and used this model to investigate the molecular and structural basis of the mechanism of proteasome α subunit assembly, as mediated by the PAC3-PAC4 heterodimeric chaperone. Our results provide a potential basis for the development of selective inhibitors against proteasome biogenesis.


Assuntos
Chaperonas Moleculares/química , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/química , Multimerização Proteica , Humanos , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(4): 387-393, 2019 Apr 30.
Artigo em Chinês | MEDLINE | ID: mdl-31068280

RESUMO

OBJECTIVE: To study the expression of PSMA7 and its effect on proliferation, invasion and migration of gastric cancer and subcutaneous tumorigenesis in nude mice. >and subcutaneous tumorigenesis in nude mice. METHODS: Specimens of tumor tissues and paired adjacent tissues were collected from 60 patients with gastric cancer for detecting the expression levels of PSMA7 using immunohistochemical method. Gastric cancer cell line SGC7901 was transfected with a lentiviral vector to inhibit PSMA7 expression, and the changes in cell proliferation and invasion were observed using cell counting kit-8 (CCK-8), clone formation assay and Transwell assay. A BALB/c mouse model bearing subcutaneous gastric cancer xenograft was established using SGC7901 cells with stable PSMA7 knockdown to assess the effect of low expression of PSMA7 on xenograft growth. RESULTS: Gastric cancer tissues expressed significantly higher levels of PSMA7 than the paired adjacent tissues (P < 0.05). In SGC7901 cells, interference of PSMA7 expression significantly inhibited the cell proliferation and invasion (P < 0.05). In the tumor-bearing BALB/c mice, the xenografts derived from SGC7901 cells with PSMA7 expression interference showed significant growth suppression as compared with the control xenografts (P < 0.05). CONCLUSIONS: PPSMA 7 is overexpressed in gastric cancer tissues, and PSMA7 knockdown inhibits the proliferation, invasion, migration and subcutaneous tumorigenesis of gastric cancer cells in nude mice.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias Gástricas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica
15.
Talanta ; 199: 32-39, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952265

RESUMO

The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.


Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Técnicas Eletroquímicas , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Antineoplásicos/química , Bortezomib/química , Inibidores Enzimáticos/química , Humanos , Microscopia de Força Atômica , Estrutura Molecular , Oligopeptídeos/química , Inibidores de Proteassoma/química , Técnicas de Microbalança de Cristal de Quartzo
16.
Eur J Med Chem ; 174: 159-180, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035238

RESUMO

Inhibitors and nucleic acid based techniques were two main approaches to interfere with protein signaling and respective cascade in the past. Until recently, a new class of small molecules named proteolysis-targeting chimeras (PROTACs) have emerged. Each contains a target warhead, a linker and an E3 ligand. These bifunctional molecules recruit E3 ligases and target specific proteins for degradation via the ubiquitin (Ub) proteasome system (UPS). The degradation provides several advantages over inhibition in potency, selectivity and drug resistance. Thus, a variety of small molecule PROTACs have been discovered so far. In this review, we summarize the biological mechanism, advantages and recent progress of PROTACs, trying to offer an outlook in development of drugs targeting degradation in future.


Assuntos
Oligopeptídeos/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Ligantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas/química , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
17.
Gene ; 702: 66-74, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30930224

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver with high mortality and frequent recurrence. Although various therapies provide potential cure for HCC patients, unfortunately the five-year survival rate of advanced HCC remains dismal. It is critical to explore the pathogenesis of HCC and identify novel biomarkers for early HCC diagnosis. PSMD4 is a major receptor of the 26S proteasome involved in ubiquitindependent and proteasome-mediated protein degradation. In our study, PSMD4 was overexpressed in HCC tissues and cell lines determined by Northern blot, western blot and immunohistochemistry. The silencing of PSMD4 blocked cell proliferation and tumor growth, induced cell apoptosis and inhibited the proteasome activity. Western blot results showed that the knockdown of PSMD4 blocked the expression of cyclooxygenase 2 (COX2), phosphorylated Sarcoma tyrosine kinase (P-SRC) and Bcl-2, but improved the levels of p53 and Bax in HCC, lung cancer, colorectal cancer, breast cancer and endometrial cancer cell lines. Taken together, these findings indicated that the subunit of 26S proteasome PSMD4 exerts as an oncogene in HCC and other cancers via regulating the expression p53, Bcl-2 and Bax. These findings enriched the pathogenesis of HCC, and provided a new biomarker for cancers diagnosis and a new target for cancers therapy.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Animais , Apoptose , Carcinogênese , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA
18.
Chem Biol Interact ; 306: 70-77, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980806

RESUMO

PURPOSE: Skeletal muscle is severely affected in diabetes leading to muscle atrophy. Previously we reported the role of ER stress in muscle atrophy due to hyperglycemia. Hence, in the present study, we investigated the effect of a classical ER stress inhibitor, 4-phenylbutyric acid (PBA), on muscle atrophy in diabetic rats. METHODS: Diabetes was induced in male rats by streptozotocin, and PBA was administered (40 mg/kg/day; intraperitoneal) after two months of diabetes for two more months. Gastrocnemius muscle is collected after four months of experimental period. The cross-sectional area of myocytes was measured on Hematoxylin and Eosin stained muscle sections. Protein levels of ER stress markers, ubiquitin-proteasome system (UPS) components, and apoptosis were analysed by immunoblot. Proteasomal activity and apoptotic cells were measured. RESULTS: ER stress markers (GRP78, ATF6, ATF4 and CHOP) that are elevated in diabetes are decreased with PBA treatment. PBA also averted diabetes-induced alterations in UPS (higher levels of E1, atrogin-1, UCHL1 and UCHL5, accumulation of ubiquitinated proteins and increased proteasomal activity). Apoptosis mediators-p53, BAX, and cleaved caspase-3 protein levels, and TUNEL positive cells were decreased in PBA treated diabetic rats. PBA notably improved the muscle-cross sectional area. CONCLUSIONS: Results highlighted the therapeutic potential of PBA in diabetes muscle wastage.


Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Atrofia Muscular/prevenção & controle , Fenilbutiratos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Injeções Intraperitoneais , Masculino , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologia , Fenilbutiratos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Estreptozocina
19.
J Cancer Res Clin Oncol ; 145(6): 1449-1460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30968255

RESUMO

BACKGROUND: A growing body of evidence suggests that exercise training has beneficial effects in cancer patients. The aim of the present study was to investigate the molecular basis underlying these beneficial effects in skeletal muscle from cancer patients. METHODS: We investigated expression of selected proteins involved in cellular processes known to orchestrate adaptation to exercise training by western blot. Skeletal muscle biopsies were sampled from ten cancer patients before and after 4-7 weeks of ongoing chemotherapy, and subsequently after 10 weeks of continued chemotherapy in combination with exercise training. Biopsies from ten healthy matched subjects served as reference. RESULTS: The expression of the insulin-regulated glucose transporter, GLUT4, increased during chemotherapy and continued to increase during exercise training. A similar trend was observed for ACC, a key enzyme in the biosynthesis and oxidation of fatty acids, but we did not observe any changes in other regulators of substrate metabolism (AMPK and PDH) or mitochondrial proteins (Cyt-C, COX-IV, SDHA, and VDAC). Markers of proteasomal proteolysis (MURF1 and ATROGIN-1) decreased during chemotherapy, but did not change further during chemotherapy combined with exercise training. A similar pattern was observed for autophagy-related proteins such as ATG5, p62, and pULK1 Ser757, but not ULK1 and LC3BII/LC3BI. Phosphorylation of FOXO3a at Ser318/321 did not change during chemotherapy, but decreased during exercise training. This could suggest that FOXO3a-mediated transcriptional regulation of MURF1 and ATROGIN-1 serves as a mechanism by which exercise training maintains proteolytic systems in skeletal muscle in cancer patients. Phosphorylation of proteins that regulate protein synthesis (mTOR at Ser2448 and 4EBP1 at Thr37/46) increased during chemotherapy and leveled off during exercise training. Finally, chemotherapy tended to increase the number of satellite cells in type 1 fibers, without any further change during chemotherapy and exercise training. Conversely, the number of satellite cells in type 2 fibers did not change during chemotherapy, but increased during chemotherapy combined with exercise training. CONCLUSIONS: Molecular signaling cascades involved in exercise training are disturbed during cancer and chemotherapy, and exercise training may prevent further disruption of these pathways. TRIAL REGISTRATION: The study was approved by the local Scientific Ethics Committee of the Central Denmark Region (Project ID: M-2014-15-14; date of approval: 01/27/2014) and the Danish Data Protection Agency (case number 2007-58-0010; date of approval: 01/28/2015). The trial was registered at http//www.clinicaltrials.gov (registration number: NCT02192216; date of registration 07/17-2014).


Assuntos
Exercício , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiopatologia , Neoplasias/fisiopatologia , Adulto , Feminino , Transportador de Glucose Tipo 4/biossíntese , Humanos , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/terapia , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Ubiquitina/metabolismo
20.
Cell Mol Life Sci ; 76(14): 2761-2777, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31030225

RESUMO

Protein silencing is often employed as a means to aid investigations in protein function and is increasingly desired as a therapeutic approach. Several types of protein silencing methodologies have been developed, including targeting the encoding genes, transcripts, the process of translation or the protein directly. Despite these advances, most silencing systems suffer from limitations. Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible. Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Targeted proteolysis has the potential to overcome some of these limitations. The field of targeted proteolysis has witnessed the emergence of many methodologies aimed at targeting specific proteins for degradation in a spatio-temporal manner. In this review, we provide an appraisal of the different targeted proteolytic systems and discuss their applications in understanding protein function, as well as their potential in therapeutics.


Assuntos
Edição de Genes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Ubiquitinação
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