RESUMEN
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.
Asunto(s)
Proteoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos de Neoplasias/metabolismo , Sistemas CRISPR-Cas/genética , Biología Computacional/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Leupeptinas/farmacología , Sistemas de Lectura Abierta/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Proteolisis , Proteoma/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismoRESUMEN
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.
Asunto(s)
Técnicas Genéticas , Proteínas/metabolismo , Proteolisis , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ácidos Indolacéticos/farmacología , Leupeptinas/farmacología , Células MCF-7 , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
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.
Asunto(s)
Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Células TH1/inmunología , Proteasas Ubiquitina-Específicas/inmunología , Traslado Adoptivo , Animales , Apoptosis/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular , Células HCT116 , Humanos , Leupeptinas/farmacología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/genética , Escape del Tumor , Proteína p53 Supresora de Tumor/inmunología , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/genética , Ubiquitinación/inmunologíaRESUMEN
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.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Ingeniería de Proteínas , Caspasa 6/metabolismo , Línea Celular , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal , Inhibidores de ProteasomaRESUMEN
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.
Asunto(s)
Atrofia Muscular/patología , Distrofia Muscular Oculofaríngea/patología , Proteína I de Unión a Poli(A)/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , Regulación de la Expresión Génica , Pruebas Genéticas , Humanos , Leupeptinas/farmacología , Leupeptinas/uso terapéutico , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/tratamiento farmacológico , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutación , Proteína I de Unión a Poli(A)/química , Prueba de Estudio Conceptual , Agregado de Proteínas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Fibras Musculares Esqueléticas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Animales , Humanos , Ratas , Células Cultivadas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Leupeptinas/farmacología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Ubiquitina/metabolismoRESUMEN
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.
Asunto(s)
Apoptosis , Leupeptinas , NADPH Oxidasa 5 , NADPH Oxidasas , Neuroblastoma , Inhibidores de Proteasoma , Superóxidos , Humanos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Inhibidores de Proteasoma/farmacología , Superóxidos/metabolismo , Línea Celular Tumoral , Neuroblastoma/patología , Neuroblastoma/metabolismo , Leupeptinas/farmacología , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasa 5/genética , NADPH Oxidasa 5/metabolismo , Antioxidantes/farmacología , Relación Dosis-Respuesta a Droga , Acetilcisteína/farmacología , Neuronas/metabolismo , Neuronas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular , Pollos , Leupeptinas , Neoplasias Hepáticas , FN-kappa B , Regiones Promotoras Genéticas , Proteína Amiloide A Sérica , Animales , Leupeptinas/farmacología , Pollos/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Regiones Promotoras Genéticas/genética , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/genética , Gránulos Citoplasmáticos/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Arsenitos/farmacología , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/efectos de los fármacos , Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Proteínas de Choque Térmico/genética , Homeostasis , Humanos , Leupeptinas/farmacología , Chaperonas Moleculares , Estrés Oxidativo , Inhibidores de Proteasoma/farmacología , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Ribosomas/genéticaRESUMEN
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.
Asunto(s)
Criopreservación , Desarrollo Embrionario , Leupeptinas , Oocitos , Inyecciones de Esperma Intracitoplasmáticas , Vitrificación , Animales , Oocitos/efectos de los fármacos , Oocitos/citología , Ratas , Femenino , Leupeptinas/farmacología , Criopreservación/métodos , Desarrollo Embrionario/efectos de los fármacos , Inyecciones de Esperma Intracitoplasmáticas/métodos , Supervivencia Celular/efectos de los fármacos , Masculino , Crioprotectores/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Células Epiteliales , Proteínas F-Box , Proteínas Serina-Treonina Quinasas , Humo , Animales , Humanos , Masculino , Ratones , Quinasas de la Proteína-Quinasa Activada por el AMP , Línea Celular , Fumar Cigarrillos/efectos adversos , Cicloheximida/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Leupeptinas/farmacología , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteolisis/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/efectos de los fármacos , ARN Interferente Pequeño , Humo/efectos adversosRESUMEN
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.
Asunto(s)
Apoptosis , Neoplasias de la Mama , Grafito , Estrés Oxidativo , Inhibidores de Proteasoma , Humanos , Grafito/farmacología , Grafito/química , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inhibidores de Proteasoma/farmacología , Femenino , Leupeptinas/farmacología , Sinergismo Farmacológico , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
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.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Compuestos Macrocíclicos/farmacología , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/fisiología , Cicloheximida/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leupeptinas/farmacología , Compuestos Macrocíclicos/uso terapéutico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Antígenos de Histocompatibilidad Menor/biosíntesis , Antígenos de Histocompatibilidad Menor/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Piridinas/farmacología , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal/metabolismo , Pliegue de Proteína , Proteínas/metabolismo , Línea Celular , Fibroblastos/metabolismo , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/metabolismo , Humanos , Leupeptinas/farmacología , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Chaperonas Moleculares/farmacología , Triterpenos Pentacíclicos , Enfermedad de Tay-Sachs/tratamiento farmacológico , Enfermedad de Tay-Sachs/metabolismo , Triterpenos/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Leupeptinas/farmacología , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica/efectos de los fármacos , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Ubiquitinación/efectos de los fármacosRESUMEN
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.
Asunto(s)
Barrera Hematoencefálica/fisiología , Células Endoteliales/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Streptococcus pneumoniae/fisiología , Ubiquitinas/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Autofagia/efectos de los fármacos , Biomarcadores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Gentamicinas/administración & dosificación , Gentamicinas/farmacología , Humanos , Leupeptinas/farmacología , Imagen Óptica/métodos , Penicilinas/administración & dosificación , Penicilinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Ubiquitinas/químicaRESUMEN
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.
Asunto(s)
Muerte Celular Autofágica/efectos de los fármacos , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Leupeptinas/farmacología , Lisosomas/metabolismo , Proteínas Oncogénicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteolisis/efectos de los fármacos , Animales , Antígenos Nucleares del Virus de Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Lisosomas/genética , Ratones , Proteínas Oncogénicas/genética , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
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.
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Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Tetracloruro de Carbono/administración & dosificación , Tetracloruro de Carbono/toxicidad , Carcinogénesis/patología , Carcinoma Hepatocelular/genética , Núcleo Celular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Células HEK293 , Células Hep G2 , Humanos , Leupeptinas/farmacología , Neoplasias Hepáticas/genética , Regeneración Hepática , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteolisis/efectos de los fármacos , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
PURPOSE: We previously reported that a calpain inhibitor (CAI) prevents the development of atherosclerosis in rats. This study aimed to investigate the effects of CAI (1 mg/kg) on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (HFD) and explore the underlying mechanism by analyzing the expression of genes related to the uptake and efflux of cholesterol. METHODS: Atherosclerotic plaques were evaluated. The activity of calpain in the aorta and that of superoxide dismutase (SOD) in the serum were assessed. Lipid profiles in the serum and liver were examined. Serum oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels were measured. The mRNA expressions of CD68, TNF-α, IL-6, CD36, scavenger receptor (SR-A), peroxisome proliferator-activated receptor gamma (PPAR-γ), liver-x-receptor alpha (LXR-α), and ATP-binding cassette transporter class A1 (ABCA1) in the aorta and peritoneal macrophages were also evaluated. RESULTS: CAI reduced calpain activity in the aorta. CAI also impeded atherosclerotic lesion formation and mRNA expression of CD68 in the aorta and peritoneal macrophages of ApoE KO mice compared with those of mice receiving HFD. However, CAI had no effect on body weight and lipid levels in both the serum and liver. CAI significantly decreased MDA, oxLDL, TNF-α, and IL-6 levels and increased SOD activity in the serum. Moreover, CAI significantly inhibited the mRNA expression of TNF-α and IL-6 genes in the aorta and peritoneal macrophages. In addition, CAI significantly downregulated the mRNA expression of scavenger receptors CD36 and SR-A and upregulated the expression of genes involved in the cholesterol efflux pathway, i.e., PPAR-γ, LXR-α, and ABCA1 in the aorta and peritoneal macrophages. CONCLUSIONS: CAI inhibited the development of atherosclerotic lesions in ApoE KO mice, and this effect might be related to the reduction of oxidative stress and inflammation and the improvement of cholesterol intake and efflux pathways.
Asunto(s)
Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Calpaína/antagonistas & inhibidores , Colesterol/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Leupeptinas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , ARN Mensajero/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aorta/enzimología , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Calpaína/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Metabolismo de los Lípidos/genética , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , PPAR gamma/genética , PPAR gamma/metabolismo , Placa Aterosclerótica , ARN Mensajero/genética , Receptores Depuradores de Clase A/genética , Receptores Depuradores de Clase A/metabolismoRESUMEN
Viruses confiscate cellular components of the ubiquitin-proteasome system (UPS) to facilitate many aspects of the infectious cycle. The 26S proteasome is an ATP-dependent, multisubunit proteolytic machine present in all eukaryotic cells. The proteasome executes the controlled degradation of functional proteins, as well as the hydrolysis of aberrantly folded polypeptides. There is growing evidence for the role of the UPS in viral entry. The UPS assists in several steps of the initiation of infection, including endosomal escape of the entering virion, intracellular transport of incoming nucleocapsids and uncoating of the viral genome. Inhibitors of proteasome activity, including MG132, epoxomicin, lactacystin and bortezomib have been integral to developments in this area. Here, we review the mechanistic details of UPS involvement in the entry process of viruses from a multitude of families. The possibility of proteasome inhibitors as therapeutic antiviral agents is highlighted.