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1.
Cell ; 173(7): 1622-1635.e14, 2018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29779948

RESUMO

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.


Assuntos
Proteoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Leupeptinas/farmacologia , Fases de Leitura Aberta/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteólise , Proteoma/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo
2.
Genes Dev ; 33(19-20): 1441-1455, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31467088

RESUMO

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in nonplant systems. However, tagging proteins at their endogenous loci results in chronic auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the auxin response transcription factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems but is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin, and we found that expression of the ARF PB1 (Phox and Bem1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transcriptional profiling indicates that ZNF143 activates transcription in cis and regulates promoter-proximal paused RNA polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.


Assuntos
Técnicas Genéticas , Proteínas/metabolismo , Proteólise , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Ácidos Indolacéticos/farmacologia , Leupeptinas/farmacologia , Células MCF-7 , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Transativadores/genética , Transativadores/metabolismo
3.
Nat Immunol ; 15(6): 562-70, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24777531

RESUMO

Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses.


Assuntos
Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Células Th1/imunologia , Proteases Específicas de Ubiquitina/imunologia , Transferência Adotiva , Animais , Apoptose/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HCT116 , Humanos , Leupeptinas/farmacologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-mdm2/genética , Evasão Tumoral , Proteína Supressora de Tumor p53/imunologia , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/imunologia
4.
Cell ; 142(4): 637-46, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20723762

RESUMO

Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.


Assuntos
Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Engenharia de Proteínas , Caspase 6/metabolismo , Linhagem Celular , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma
5.
PLoS Genet ; 18(1): e1010015, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35025870

RESUMO

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.


Assuntos
Atrofia Muscular/patologia , Distrofia Muscular Oculofaríngea/patologia , Proteína I de Ligação a Poli(A)/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Testes Genéticos , Humanos , Leupeptinas/farmacologia , Leupeptinas/uso terapêutico , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/tratamento farmacológico , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutação , Proteína I de Ligação a Poli(A)/química , Estudo de Prova de Conceito , Agregados Proteicos/efeitos dos fármacos
6.
Plant Cell Environ ; 47(11): 4449-4463, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39007522

RESUMO

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estabilidade Proteica , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ligação Proteica , Temperatura , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Leupeptinas/farmacologia , Vernalização
7.
J Muscle Res Cell Motil ; 45(3): 155-169, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39080182

RESUMO

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.


Assuntos
Fibras Musculares Esqueléticas , Piruvato Desidrogenase Quinase de Transferência de Acetil , Animais , Humanos , Ratos , Células Cultivadas , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leupeptinas/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Ubiquitina/metabolismo
8.
J Pharmacol Sci ; 155(2): 52-62, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38677786

RESUMO

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.


Assuntos
Apoptose , Leupeptinas , NADPH Oxidase 5 , NADPH Oxidases , Neuroblastoma , Inibidores de Proteassoma , Superóxidos , Humanos , Apoptose/efeitos dos fármacos , Apoptose/genética , Inibidores de Proteassoma/farmacologia , Superóxidos/metabolismo , Linhagem Celular Tumoral , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Leupeptinas/farmacologia , NADPH Oxidases/metabolismo , NADPH Oxidases/genética , NADPH Oxidase 5/genética , NADPH Oxidase 5/metabolismo , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Acetilcisteína/farmacologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos
9.
Mol Biol Rep ; 51(1): 770, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38896168

RESUMO

BACKGROUND: MG132, a proteasome inhibitor, is widely used to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity by proteasome-mediated degradation of IκB. It has been marketed as a specific, reversible, cell-permeable and low-cost inhibitor. However, adverse effects of the compound have been reported in the literature. We recently discovered and characterised a point mutation in the acute phase protein serum amyloid A (SAA) in chickens, by overexpressing the protein in chicken hepatocellular carcinoma (LMH) cells. This serine to arginine exchange at amino acid position 90 (SAA.R90S) leads to intra- and extracellular accumulation of SAA, which is surprisingly counteracted by MG132 treatment, independent of SAA's intrinsic promoter. METHODS AND RESULTS: To test, whether low proteasomal degradation of SAA.R90S is responsible for the observed intra- and extracellular SAA accumulation, we intended to inhibit the proteasome in SAA wild type (SAA.WT) overexpressing cells with MG132. However, we observed an unexpected drastic decrease in SAA protein expression at the transcript level. NF-κB gene expression was unchanged by MG132 at the measured time point. CONCLUSIONS: The observed results demonstrate that MG132 inhibits SAA expression at the transcript level, independent of its endogenous promoter. Further, the data might indicate that NF-κB is not involved in the observed MG132-induced inhibition of SAA expression. We, consequently, question in this brief report whether MG132 should truly be categorised as a specific ubiquitin proteasome inhibitor and recommend the usage of alternative compounds.


Assuntos
Carcinoma Hepatocelular , Galinhas , Leupeptinas , Neoplasias Hepáticas , NF-kappa B , Regiões Promotoras Genéticas , Proteína Amiloide A Sérica , Animais , Leupeptinas/farmacologia , Galinhas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Regiões Promotoras Genéticas/genética , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos
10.
Mol Cell ; 63(5): 796-810, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27570075

RESUMO

Stress granules (SGs) are ribonucleoprotein complexes induced by stress. They sequester mRNAs and disassemble when the stress subsides, allowing translation restoration. In amyotrophic lateral sclerosis (ALS), aberrant SGs cannot disassemble and therefore accumulate and are degraded by autophagy. However, the molecular events causing aberrant SG formation and the molecular players regulating this transition are largely unknown. We report that defective ribosomal products (DRiPs) accumulate in SGs and promote a transition into an aberrant state that renders SGs resistant to RNase. We show that only a minor fraction of aberrant SGs is targeted by autophagy, whereas the majority disassembles in a process that requires assistance by the HSPB8-BAG3-HSP70 chaperone complex. We further demonstrate that HSPB8-BAG3-HSP70 ensures the functionality of SGs and restores proteostasis by targeting DRiPs for degradation. We propose a system of chaperone-mediated SG surveillance, or granulostasis, which regulates SG composition and dynamics and thus may play an important role in ALS.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ribossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Arsenitos/farmacologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/efeitos dos fármacos , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Proteínas de Choque Térmico/genética , Homeostase , Humanos , Leupeptinas/farmacologia , Chaperonas Moleculares , Estresse Oxidativo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Ribossomos/genética
11.
Cryobiology ; 115: 104882, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38452847

RESUMO

Oocyte cryopreservation is useful for human fertility treatment and strain preservation in both experimental and domestic animals. However, the embryonic development of vitrified rat oocytes was lower than that of vitrified embryos. To increase the viability of vitrified oocytes, intracellular ice formation during cooling and warming must be prevented. Rapid warming is important to prevent ice formation. Furthermore, suppressing the spontaneous activation of oocytes is also important because vitrification promotes the spontaneous activation of rat oocytes, and thus compromise developmental competence of the gametes. MG132, a proteasome inhibitor, suppresses the spontaneous activation of rat oocytes. Here, we examined the effects of rapid warming and MG132 treatment on the survival and embryonic development of vitrified rat oocytes. The warming rate was adjusted by changing the vitrification solution volume and warming solution temperature. The survival rate of oocytes vitrified in 10 µL solution and warmed at 50 °C (94%) was significantly higher than that of oocytes vitrified in 100 µL and 10 µL solution and warmed at 37 °C (49% and 81%, respectively). Furthermore, the rate of embryonic development of vitrified oocytes treated with MG132 during vitrification, warming, and intracytoplasmic sperm injection (ICSI) (44%) was significantly higher than that of untreated gametes (10%). Offspring were obtained after transferring embryos derived from MG132-treated vitrified oocytes (14%). Altogether, the survivability of vitrified rat oocytes increased by rapid warming, and MG132 improved embryonic development after ICSI.


Assuntos
Criopreservação , Desenvolvimento Embrionário , Leupeptinas , Oócitos , Injeções de Esperma Intracitoplásmicas , Vitrificação , Animais , Oócitos/efeitos dos fármacos , Oócitos/citologia , Ratos , Feminino , Leupeptinas/farmacologia , Criopreservação/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Injeções de Esperma Intracitoplásmicas/métodos , Sobrevivência Celular/efeitos dos fármacos , Masculino , Crioprotetores/farmacologia
12.
Lipids Health Dis ; 23(1): 282, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39232759

RESUMO

OBJECTIVE: This study aimed to reveal the role and mechanism of MG-132 in delaying hyperlipidemia-induced senescence of vascular smooth muscle cells (VSMCs). METHODS: Immunohistochemistry and hematoxylin-eosin staining confirmed the therapeutic effect of MG-132 on arterial senescence in vivo and its possible mechanism. Subsequently, VSMCs were treated with sodium palmitate (PA), an activator (Recilisib) or an inhibitor (Pictilisib) to activate or inhibit PI3K, and CCK-8 and EdU staining, wound healing assays, Transwell cell migration assays, autophagy staining assays, reactive oxygen species assays, senescence-associated ß-galactosidase staining, and Western blotting were performed to determine the molecular mechanism by which MG-132 inhibits VSMC senescence. Validation of the interaction between MG-132 and PI3K using molecular docking. RESULTS: Increased expression of p-PI3K, a key protein of the autophagy regulatory system, and decreased expression of the autophagy-associated proteins Beclin 1 and ULK1 were observed in the aortas of C57BL/6J mice fed a high-fat diet (HFD), and autophagy was inhibited in aortic smooth muscle. MG-132 inhibits atherosclerosis by activating autophagy in VSMCs to counteract PA-induced cell proliferation, migration, oxidative stress, and senescence, thereby inhibiting VSMC senescence in the aorta. This process is achieved through the PI3K/AKT/mTOR signaling pathway. CONCLUSION: MG-132 activates autophagy by inhibiting the PI3K/AKT/mTOR pathway, thereby inhibiting palmitate-induced proliferation, migration, and oxidative stress in vascular smooth muscle cells and suppressing their senescence.


Assuntos
Autofagia , Senescência Celular , Leupeptinas , Músculo Liso Vascular , Miócitos de Músculo Liso , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Autofagia/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Senescência Celular/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Leupeptinas/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Ácido Palmítico/farmacologia , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos
13.
COPD ; 21(1): 2342797, 2024 12.
Artigo em Inglês | MEDLINE | ID: mdl-38712759

RESUMO

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.


Assuntos
Proteínas Quinases Ativadas por AMP , Células Epiteliais , Proteínas F-Box , Proteínas Serina-Treonina Quinases , Fumaça , Animais , Humanos , Masculino , Camundongos , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular , Fumar Cigarros/efeitos adversos , Cicloeximida/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Leupeptinas/farmacologia , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Mucosa Respiratória/metabolismo , Mucosa Respiratória/efeitos dos fármacos , RNA Interferente Pequeno , Fumaça/efeitos adversos
14.
Int J Mol Sci ; 25(10)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38791473

RESUMO

Reduced graphene oxide (rGO) and a proteasome inhibitor (MG-132) are some of the most commonly used compounds in various biomedical applications. However, the mechanisms of rGO- and MG-132-induced cytotoxicity remain unclear. The aim of this study was to investigate the anticancer effect of rGO and MG-132 against ZR-75-1 and MDA-MB-231 breast cancer cell lines. The results demonstrated that rGO, MG-132 or a mix (rGO + MG-132) induced time- and dose-dependent cytotoxicity in ZR-75-1 and MDA-MB-231 cells. Apart from that, we found that treatment with rGO and MG-132 or the mix increased apoptosis, necrosis and induction of caspase-8 and caspase-9 activity in both breast cancer cell lines. Apoptosis and caspase activation were accompanied by changes in the ultrastructure of mitochondria in ZR-75-1 and MDA-MB-231 cells incubated with rGO. Additionally, in the analyzed cells, we observed the induction of oxidative stress, accompanied by increased apoptosis and cell necrosis. In conclusion, oxidative stress induces apoptosis in the tested cells. At the same time, both mitochondrial and receptor apoptosis pathways are activated. These studies provided new information on the molecular mechanisms of apoptosis in the ZR-75-1 and MDA-MB-231 breast cancer cell lines.


Assuntos
Apoptose , Neoplasias da Mama , Grafite , Estresse Oxidativo , Inibidores de Proteassoma , Humanos , Grafite/farmacologia , Grafite/química , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Inibidores de Proteassoma/farmacologia , Feminino , Leupeptinas/farmacologia , Sinergismo Farmacológico , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
15.
Blood ; 137(21): 2947-2957, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33259592

RESUMO

BH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic-resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Compostos Macrocíclicos/farmacologia , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/fisiologia , Cicloeximida/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Compostos Macrocíclicos/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Antígenos de Histocompatibilidade Menor/biossíntese , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/antagonistas & inibidores , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Piridinas/farmacologia , Sulfonamidas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cell ; 134(5): 769-81, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18775310

RESUMO

Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.


Assuntos
Doenças por Armazenamento dos Lisossomos/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/metabolismo , Humanos , Leupeptinas/farmacologia , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Chaperonas Moleculares/farmacologia , Triterpenos Pentacíclicos , Doença de Tay-Sachs/tratamento farmacológico , Doença de Tay-Sachs/metabolismo , Triterpenos/farmacologia
17.
Mol Cell ; 58(6): 1053-66, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26004230

RESUMO

Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Família da Proteína 8 Relacionada à Autofagia , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Leupeptinas/farmacologia , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação/efeitos dos fármacos
18.
J Bacteriol ; 204(1): e0045621, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34633870

RESUMO

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections. IMPORTANCE The blood-brain barrier (BBB) represents a unique cellular barrier that provides structural integrity and protection to the CNS from pathogen invasion. Recently, ubiquitination, which is key for cellular homeostasis, was shown to be involved in pathogen clearance. In this study, we deciphered that the BBB deploys differential ubiquitination as an effective strategy to prevent S. pneumoniae trafficking into the brain. The different ubiquitin chain topologies formed on S. pneumoniae dictated the selection of downstream degradative pathways, namely, autophagy and proteasomes, among which the contribution of the proteasomal system in S. pneumoniae killing is more pronounced. Overall our study revealed how the BBB deploys differential ubiquitination as a strategy for synchronization of various intracellular defense pathways, which work in tandem to ensure the brain's identity as an immunologically privileged site.


Assuntos
Barreira Hematoencefálica/fisiologia , Células Endoteliais/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Streptococcus pneumoniae/fisiologia , Ubiquitinas/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Autofagia/efeitos dos fármacos , Biomarcadores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Gentamicinas/administração & dosagem , Gentamicinas/farmacologia , Humanos , Leupeptinas/farmacologia , Imagem Óptica/métodos , Penicilinas/administração & dosagem , Penicilinas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , Ubiquitinas/química
19.
PLoS Pathog ; 16(2): e1008105, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092124

RESUMO

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.


Assuntos
Morte Celular Autofágica/efeitos dos fármacos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Leupeptinas/farmacologia , Lisossomos/metabolismo , Proteínas Oncogênicas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Animais , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Lisossomos/genética , Camundongos , Proteínas Oncogênicas/genética , Complexo de Endopeptidases do Proteassoma/genética
20.
Hepatology ; 73(2): 776-794, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32380568

RESUMO

BACKGROUND AND AIMS: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier. APPROACH AND RESULTS: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain ß-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of ß-catenin, independent of HCC-associated ß-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/ß-catenin through APC stabilization and ß-catenin cytosolic retention. CONCLUSIONS: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Carcinogênese/patologia , Carcinoma Hepatocelular/genética , Núcleo Celular/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HEK293 , Células Hep G2 , Humanos , Leupeptinas/farmacologia , Neoplasias Hepáticas/genética , Regeneração Hepática , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteólise/efeitos dos fármacos , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismo
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