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1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203850

RESUMO

Steroid receptor coactivator-1 (SRC-1) is a transcription coactivator playing a pivotal role in mediating a wide range of signaling pathways by interacting with related transcription factors and nuclear receptors. Aberrantly elevated SRC-1 activity is associated with cancer metastasis and progression, and therefore, suppression of SRC-1 is emerging as a promising therapeutic strategy. In this study, we developed a novel SRC-1 degrader for targeted degradation of cellular SRC-1. This molecule consists of a selective ligand for SRC-1 and a bulky hydrophobic group. Since the hydrophobic moiety on the protein surface could mimic a partially denatured hydrophobic region of a protein, SRC-1 could be recognized as an unfolded protein and experience the chaperone-mediated degradation in the cells through the ubiquitin-proteasome system (UPS). Our results demonstrate that a hydrophobic-tagged chimeric molecule is shown to significantly reduce cellular levels of SRC-1 and suppress cancer cell migration and invasion. Together, these results highlight that our SRC-1 degrader represents a novel class of therapeutic candidates for targeting cancer metastasis. Moreover, we believe that the hydrophobic tagging strategy would be widely applicable to develop peptide-based protein degraders with enhanced cellular activity.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Coativador 1 de Receptor Nuclear/metabolismo , Proteólise , Transativadores/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Chaperonas Moleculares/metabolismo , Invasividade Neoplásica , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
Sci Signal ; 14(690)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230209

RESUMO

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Polifosfatos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Administração por Inalação , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/administração & dosagem , Antivirais/química , COVID-19/metabolismo , COVID-19/virologia , Células CACO-2 , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Citocinas/metabolismo , Células HEK293 , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Técnicas In Vitro , Modelos Biológicos , Simulação de Acoplamento Molecular , Nebulizadores e Vaporizadores , Polifosfatos/administração & dosagem , Polifosfatos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteólise/efeitos dos fármacos , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
3.
Anticancer Res ; 41(7): 3271-3279, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230121

RESUMO

BACKGROUND/AIM: Androgen receptor (AR) degradation is the primary regulator of androgen receptor activity. This study was designed to investigate the influence of the proteasome on AR protein stability after enzalutamide (Enz) treatment. MATERIALS AND METHODS: Cell counting after treatment was utilized to assess the effect of Enz on cell proliferation. Changes in mRNA levels were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Proteasome activity was assessed by measurement of the chymotrypsin-like activity of the beta-5 subunit of the proteasome. Changes in protein levels after treatment with Enz, MG132 (MG), bortezomib (Bor), or their combination were assessed using western blot analysis. RESULTS: Treatment with Enz led to a significant reduction of cell proliferation and AR protein levels. However, AR mRNA levels were unchanged. Inhibition of proteasome activity by MG counteracts the Enz-mediated AR degradation transiently, whereas Bor showed no inhibition of the Enz-mediated AR degradation. CONCLUSION: Enz-mediated change in AR stability as an early and essential event after treatment was shown. However, investigations of the ubiquitin/proteasome system indicate involvement of several proteases in the Enz-mediated AR degradation process.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Nitrilas/farmacologia , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Masculino , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo
4.
Nat Commun ; 12(1): 4227, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244482

RESUMO

Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.


Assuntos
Carcinogênese/genética , Glioma/genética , Glicina Desidrogenase (Descarboxilante)/metabolismo , Glicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Acetilação , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Pirimidinas/biossíntese , Proteínas Repressoras/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Ativação Transcricional , Ubiquitinação/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065512

RESUMO

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.


Assuntos
Antígenos de Neoplasias/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células T Matadoras Naturais/metabolismo , RNA Helicases/metabolismo , Espermatogênese/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Ativação Linfocitária/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo
6.
Nat Commun ; 12(1): 3720, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140524

RESUMO

Low levels of reactive oxygen species (ROS) are crucial for maintaining cancer stem cells (CSCs) and their ability to resist therapy, but the ROS regulatory mechanisms in CSCs remains to be explored. Here, we discover that prohibitin (PHB) specifically regulates mitochondrial ROS production in glioma stem-like cells (GSCs) and facilitates GSC radiotherapeutic resistance. We find that PHB is upregulated in GSCs and is associated with malignant gliomas progression and poor prognosis. PHB binds to peroxiredoxin3 (PRDX3), a mitochondrion-specific peroxidase, and stabilizes PRDX3 protein through the ubiquitin-proteasome pathway. Knockout of PHB dramatically elevates ROS levels, thereby inhibiting GSC self-renewal. Importantly, deletion or pharmacological inhibition of PHB potently slows tumor growth and sensitizes tumors to radiotherapy, thus providing significant survival benefits in GSC-derived orthotopic tumors and glioblastoma patient-derived xenografts. These results reveal a selective role of PHB in mitochondrial ROS regulation in GSCs and suggest that targeting PHB improves radiotherapeutic efficacy in glioblastoma.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Adulto , Idoso , Animais , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Gradação de Tumores , Peroxirredoxinas/metabolismo , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 12(1): 3291, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078905

RESUMO

The formation of hyperphosphorylated intracellular Tau tangles in the brain is a hallmark of Alzheimer's disease (AD). Tau hyperphosphorylation destabilizes microtubules, promoting neurodegeneration in AD patients. To identify suppressors of tau-mediated AD, we perform a screen using a microRNA (miR) library in Drosophila and identify the miR-9 family as suppressors of human tau overexpression phenotypes. CG11070, a miR-9a target gene, and its mammalian orthologue UBE4B, an E3/E4 ubiquitin ligase, alleviate eye neurodegeneration, synaptic bouton defects, and crawling phenotypes in Drosophila human tau overexpression models. Total and phosphorylated Tau levels also decrease upon CG11070 or UBE4B overexpression. In mammalian neuroblastoma cells, overexpression of UBE4B and STUB1, which encodes the E3 ligase CHIP, increases the ubiquitination and degradation of Tau. In the Tau-BiFC mouse model, UBE4B and STUB1 overexpression also increase oligomeric Tau degradation. Inhibitor assays of the autophagy and proteasome systems reveal that the autophagy-lysosome system is the major pathway for Tau degradation in this context. These results demonstrate that UBE4B, a miR-9 target gene, promotes autophagy-mediated Tau degradation together with STUB1, and is thus an innovative therapeutic approach for AD.


Assuntos
Doença de Alzheimer/genética , Proteínas de Drosophila/genética , MicroRNAs/genética , Ubiquitina-Proteína Ligases/genética , Proteínas tau/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Autofagia/genética , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/metabolismo , Olho/patologia , Humanos , Lisossomos/metabolismo , Camundongos , MicroRNAs/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas tau/metabolismo
8.
Mol Cell ; 81(12): 2507-2519, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34107306

RESUMO

Protein homeostasis mechanisms are fundamentally important to match cellular needs and to counteract stress conditions. A fundamental challenge is to understand how defective proteins are recognized and extracted from cellular organelles to be degraded in the cytoplasm. The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway is the best-understood organellar protein quality control system. Here, we review new insights into the mechanism of recognition and retrotranslocation of client proteins in ERAD. In addition to the membrane-integral ERAD E3 ubiquitin ligases, we highlight one protein family that is remarkably often involved in various aspects of membrane protein quality control and protein dislocation: the rhomboid superfamily, which includes derlins and intramembrane serine proteases. Rhomboid-like proteins have been found to control protein homeostasis in the ER, but also in other eukaryotic organelles and in bacteria, pointing toward conserved principles of membrane protein quality control across organelles and evolution.


Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteostase/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Células Eucarióticas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo
9.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34070986

RESUMO

Oral health is an integral part of the general health and well-being of individuals. The presence of oral disease is potentially indicative of a number of systemic diseases and may contribute to their early diagnosis and treatment. The ubiquitin (Ub) system has been shown to play a role in cellular immune response, cellular development, and programmed cell death. Ubiquitination is a post-translational modification that occurs in eukaryotes. Its mechanism involves a number of factors, including Ub-activating enzymes, Ub-conjugating enzymes, and Ub protein ligases. Deubiquitinating enzymes, which are proteases that reversely modify proteins by removing Ub or Ub-like molecules or remodeling Ub chains on target proteins, have recently been regarded as crucial regulators of ubiquitination-mediated degradation and are known to significantly affect cellular pathways, a number of biological processes, DNA damage response, and DNA repair pathways. Research has increasingly shown evidence of the relationship between ubiquitination, deubiquitination, and oral disease. This review investigates recent progress in discoveries in diseased oral sites and discusses the roles of ubiquitination and deubiquitination in oral disease.


Assuntos
Doenças da Boca/metabolismo , Processamento de Proteína Pós-Traducional , Doenças Dentárias/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Síndrome de Dente Quebrado/metabolismo , Cárie Dentária/metabolismo , Sensibilidade da Dentina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Previsões , Gengivite/metabolismo , Humanos , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/metabolismo , Doenças Periodontais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
10.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071136

RESUMO

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfoide/patologia , Proteínas de Neoplasias/metabolismo , Inibidores de Proteases/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular Tumoral , Enzimas Desubiquitinantes , Fluoresceínas/metabolismo , Genes cdc/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Leucemia Linfoide/genética , Leucemia Linfoide/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteínas Ubiquitinadas/metabolismo , Vincristina/farmacologia
11.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072267

RESUMO

The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell-cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), the activities of which are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins and CDK inhibitors (CKIs). Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell-cycle process via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor proteins and oncoproteins or, inversely, abnormally high accumulation results in cell proliferation deregulation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the UPS machinery regulation of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell-cycle transition, cancer treatment, and the development of anti-cancer drugs.


Assuntos
Ciclo Celular , Neoplasias/metabolismo , Animais , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Progressão da Doença , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Neoplasias/etiologia , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
12.
Anticancer Res ; 41(6): 2901-2912, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083281

RESUMO

BACKGROUND/AIM: Activation of the ubiquitin-proteasome system (UPS) has been shown to be associated with drug resistance in cancer. Using bladder cancer cells, we investigated the association between UPS activation and cisplatin resistance and also the efficacy of UPS-targeting drugs. MATERIALS AND METHODS: We established cisplatin-resistant bladder cancer cells (J82-cisR, T24-cisR) and examined the activation status of the UPS and the efficacy of MLN7243, oprozomib, ixazomib, and RTS-V5. RESULTS: The UPS in cisplatin-resistant bladder cancer cells was activated compared to that in their parental controls. All the UPS-targeting drugs induced apoptosis and inhibited growth more effectively in the cisplatin-resistant bladder cancer cells than they did in the parental controls. Furthermore, these UPS-targeting drugs induced endoplasmic reticulum stress by causing unfolded protein accumulation at lower concentrations in the cisplatin-resistant bladder cancer cells. CONCLUSION: Targeting the UPS could be an effective strategy for treating cisplatin-resistant bladder cancer.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Neoplasias da Bexiga Urinária/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos
13.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072647

RESUMO

Steroid-induced glaucoma is a severe pathological condition, sustained by a rapidly progressive increase in intraocular pressure (IOP), which is diagnosed in a subset of subjects who adhere to a glucocorticoid (GC)-based therapy. Molecular and clinical studies suggest that either natural or synthetic GCs induce a severe metabolic dysregulation of Trabecular Meshwork Cells (TMCs), an endothelial-derived histotype with phagocytic and secretive functions which lay at the iridocorneal angle in the anterior segment of the eye. Since TMCs physiologically regulate the composition and architecture of trabecular meshwork (TM), which is the main outflow pathway of aqueous humor, a fluid which shapes the eye globe and nourishes the lining cell types, GCs are supposed to trigger a pathological remodeling of the TM, inducing an IOP increase and retina mechanical compression. The metabolic dysregulation of TMCs induced by GCs exposure has never been characterized at the molecular detail. Herein, we report that, upon dexamethasone exposure, a TMCs strain develops a marked inhibition of the autophagosome biogenesis pathway through an enhanced turnover of two members of the Ulk-1 complex, the main platform for autophagy induction, through the Ubiquitin Proteasome System (UPS).


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Dexametasona/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proliferação de Células/efeitos dos fármacos , Dexametasona/efeitos adversos , Suscetibilidade a Doenças , Glaucoma/etiologia , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexo de Endopeptidases do Proteassoma/metabolismo
14.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070855

RESUMO

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA/genética , Regulação da Expressão Gênica , Reparo de DNA por Recombinação , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Antineoplásicos Fitogênicos/farmacologia , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Etoposídeo/farmacologia , Técnicas de Inativação de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Fatores de Transcrição/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
15.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063751

RESUMO

The role of immunoproteasome (iP) in astroglia, the cellular component of innate immunity, has not been clarified. The results so far indicate that neuroinflammation, a prominent hallmark of Alzheimer's disease, strongly activates the iP subunits expression. Since omega-3 PUFAs possess anti-inflammatory and pro-resolving activity in the brain, we investigated the effect of DHA and EPA on the gene expression of constitutive (ß1 and ß5) and inducible (iß1/LMP2 and iß5/LMP7) proteasome subunits and proteasomal activity in IL-1ß-stimulated astrocytes. We found that both PUFAs downregulated the expression of IL-1ß-induced the iP subunits, but not the constitutive proteasome subunits. The chymotrypsin-like activity was inhibited in a dose-dependent manner by DHA, and much strongly in the lower concentration by EPA. Furthermore, we established that C/EBPα and C/EBPß transcription factors, being the cis-regulatory element of the transcription complex, frequently activated by inflammatory mediators, participate in a reduction in the iP subunits' expression. Moreover, the expression of connexin 43 the major gap junction protein in astrocytes, negatively regulated by IL-1ß was markedly increased in PUFA-treated cells. These findings indicate that omega-3 PUFAs attenuate inflammation-induced hyperactivity of iPs in astrocytes and have a beneficial effect on preservation of interastrocytic communication by gap junctions.


Assuntos
Astrócitos/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Interleucina-1beta/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Astrócitos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Conexina 43/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Insaturados/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
16.
Mol Biol (Mosk) ; 55(3): 355-361, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34097672

RESUMO

The ubiquitin-proteasome system (UPS) is an important regulator of the main cellular processes. The components of the UPS are involved in the regulation of the cell cycle, signal transduction, the cell response to DNA damage, metabolism, and transcription control. E3 ubiquitin ligases (the enzymes that covalently attaches ubiquitin to target proteins) play a key role in the functioning of the UPS. The Drosophila tumor suppressor Hyd (hyperplastic discs) is one of the most interesting E3 ligases; it is required for the regulation of proliferation, growth, and cell differentiation. The study of hyd mutations in different tissues of Drosophila demonstrated that depending on the cellular context, Hyd can not only perform proteolytic functions associated with protein degradation, but can also, interacting with other proteins and/or nucleic acids, act as an important regulator of cellular processes.


Assuntos
Drosophila , Ubiquitina-Proteína Ligases , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
17.
Nat Commun ; 12(1): 3481, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108482

RESUMO

TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.


Assuntos
Proteínas de Escherichia coli/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Ubiquitinação , Fatores de Virulência/metabolismo , Animais , Cromatina/metabolismo , Citrulinação , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/genética , Histonas/metabolismo , Evasão da Resposta Imune , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína-Arginina Desiminase do Tipo 4/genética , Pielonefrite/imunologia , Pielonefrite/patologia , Transcrição Genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/genética
18.
J Med Chem ; 64(11): 7839-7852, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34038131

RESUMO

Inspired by the success of dual-targeting drugs, especially bispecific antibodies, we propose to combine the concept of proteolysis targeting chimera (PROTAC) and dual targeting to design and synthesize dual PROTAC molecules with the function of degrading two completely different types of targets simultaneously. A library of novel dual-targeting PROTAC molecules has been rationally designed and prepared. A convergent synthetic strategy has been utilized to achieve high synthetic efficiency. These dual PROTAC structures are characterized using trifunctional natural amino acids as star-type core linkers to connect two independent inhibitors and E3 ligands together. In this study, gefitinib, olaparib, and CRBN or VHL E3 ligands were used as substrates to synthesize novel dual PROTACs. They successfully degraded both the epidermal growth factor receptor (EGFR) and poly(ADP-ribose) polymerase (PARP) simultaneously in cancer cells. Being the first successful example of dual PROTACs, this technique will greatly widen the range of application of the PROTAC method and open up a new field for drug discovery.


Assuntos
Desenho de Fármacos , Receptores ErbB/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gefitinibe/química , Humanos , Ligantes , Ftalazinas/química , Piperazinas/química , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
19.
Biol Psychiatry ; 89(12): 1176-1187, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33934885

RESUMO

BACKGROUND: Posttranslational histone modifications play a critical role in the regulation of gene transcription underlying synaptic plasticity and memory formation. One such epigenetic change is histone ubiquitination, a process that is mediated by the ubiquitin-proteasome system in a manner similar to that by which proteins are normally targeted for degradation. However, histone ubiquitination mechanisms are poorly understood in the brain and in learning. In this article, we describe a new role for the ubiquitin-proteasome system in histone crosstalk, showing that learning-induced monoubiquitination of histone H2B (H2Bubi) is required for increases in the transcriptionally active H3 lysine 4 trimethylation (H3K4me3) mark at learning-related genes in the hippocampus. METHODS: Using a series of molecular, biochemical, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA-mediated knockdown and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated upregulation of ubiquitin ligases, deubiquitinating enzymes and histone methyltransferases in the rat dorsal hippocampus during memory consolidation. RESULTS: We show that H2Bubi recruits H3K4me3 through a process that is dependent on the 19S proteasome subunit RPT6 and that a loss of H2Bubi in the hippocampus prevents learning-induced increases in H3K4me3, gene transcription, synaptic plasticity, and memory formation. Furthermore, we show that CRISPR-dCas9-mediated increases in H2Bubi promote H3K4me3 and memory formation under weak training conditions and that promoting histone methylation does not rescue memory impairments resulting from loss of H2Bubi. CONCLUSIONS: These results suggest that H2B ubiquitination regulates histone crosstalk in learning by way of nonproteolytic proteasome function, demonstrating a novel mechanism by which histone modifications are coordinated in response to learning.


Assuntos
Histonas , Complexo de Endopeptidases do Proteassoma , Animais , Cromatina , Histonas/metabolismo , Metilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ubiquitinação
20.
J Med Chem ; 64(9): 6262-6272, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33949190

RESUMO

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.


Assuntos
Mycobacterium tuberculosis/enzimologia , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Desenho de Fármacos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos Cíclicos/química , Relação Estrutura-Atividade
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