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1.
Cell Rep ; 43(2): 113752, 2024 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-38341852

RESUMEN

We here demonstrate that SERTAD1 is an adaptor protein responsible for the regulation of lysine 63 (K63)-linked NLRP3 polyubiquitination by the Cullin1 E3 ubiquitin ligase upon inflammasome activation. SERTAD1 specifically binds to NLRP3 but not to other inflammasome sensors. This endogenous interaction increases after inflammasome activation, interfering with the interaction between NLRP3 and Cullin1. Interleukin (IL)-1ß and IL-18 secretion, as well as the cleavage of gasdermin D, are decreased in SERTAD1 knockout bone-marrow-derived macrophages, together with reduced formation of the NLRP3 inflammasome complex. Additionally, SERTAD1-deficient mice show attenuated severity of monosodium-uric-acid-induced peritonitis and experimental autoimmune encephalomyelitis. Analysis of public datasets indicates that expression of SERTAD1 mRNA is significantly increased in the patients of autoimmune diseases. Thus, our findings uncover a function of SERTAD1 that specifically reduces Cullin1-mediated NLRP3 polyubiquitination via direct binding to NLRP3, eventually acting as a crucial factor to regulate the initiation of NLRP3-mediated inflammasome activation.


Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Humanos , Ratones , Inflamasomas/metabolismo , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
2.
Exp Mol Med ; 55(6): 1218-1231, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37258579

RESUMEN

The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3-/- knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3ß were decreased in primary hepatocytes obtained from Peli3-/- KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3ß compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3ß. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3-/- KO hepatocytes restored the mitochondrial translocation of GSK3ß, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3ß polyubiquitination.


Asunto(s)
Acetaminofén , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Animales , Ratones , Acetaminofén/efectos adversos , Fosforilación , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ratones Endogámicos C57BL
3.
Int J Mol Sci ; 21(9)2020 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-32354117

RESUMEN

p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.


Asunto(s)
Lisina/metabolismo , Proteína Sequestosoma-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Benzamidas/farmacología , Supervivencia Celular/efectos de los fármacos , Cicloheximida/farmacología , Regulación de la Expresión Génica , Células HEK293 , Células HT29 , Células HeLa , Humanos , FN-kappa B/metabolismo , Piperazinas/farmacología , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Piridinas/farmacología , Pirroles/farmacología , Proteína Sequestosoma-1/química , Transducción de Señal , Ubiquitina Tiolesterasa/genética
4.
EBioMedicine ; 49: 55-71, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31685442

RESUMEN

BACKGROUND: Although bone morphogenetic protein 6 (BMP6) signaling pathway has been implicated in many types of cancer, its role of tumorigenesis seems to be controversial and its ubiquitin-modifying mechanisms have not been fully addressed. Our study was designed to investigate how BMP6 signaling pathway is regulated by ubiquitin-modifying systems and to address molecular and clinical significance in colorectal cancers. METHODS: Human deubiquitnase (DUB) siRNA library was used to screen the specific DUB, named PSMD14, involved in BMP6 signaling pathway. Immunoblot, immunoprecipitation and ubiquitination assays were used to analyze targets of the PSMD14. A role of PSMD14-mediated BMP6 signaling pathway for malignant cancer progression was investigated using in vitro and in vivo model of colorectal cancers as well as clinical samples of colorectal cancer patients. FINDINGS: The deubiquitinase PSMD14 acts as a positive regulator for the initiation of the BMP6 signaling pathway through deubiquitinating K48-linked ALK2 type I receptor ubiquitination mediated by Smurf1 E3 ligase, resulting in increased stability of the ALK2. This role of PSMD14 is independent of its intrinsic role in the 26S proteasome system. Furthermore, either PSMD14 or ALK2 depletion significantly decreases tumorigenesis of HCT116 colorectal cancer cells in a xenograft model as well as cancer stemness/chemoresistance, and expression of the PSMD14 and ALK2 gene are correlated with malignant progression and the survival of colorectal cancer patients. INTERPRETATION: These findings suggest that the PSMD14-ALK2 axis plays an essential role in initiation of the BMP6 signaling pathway and contributes to tumorigenesis and chemoresistance of colorectal cancers.


Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Proteína Morfogenética Ósea 6/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , Lisina/metabolismo , Modelos Biológicos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Poliubiquitina/metabolismo , Pronóstico , Unión Proteica , Estabilidad Proteica , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
5.
EMBO Rep ; 19(4)2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29487085

RESUMEN

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin-specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation-induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.


Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Ubiquitina Tiolesterasa/metabolismo , Animales , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Línea Celular , Supervivencia Celular , Expresión Génica , Células HEK293 , Humanos , Lisosomas/metabolismo , Ratones , Unión Proteica , Estabilidad Proteica , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Ubiquitina Tiolesterasa/genética , Ubiquitinación
6.
Nat Cell Biol ; 19(10): 1260-1273, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28892081

RESUMEN

Although the ubiquitin-editing enzyme A20 is a key player in inflammation and autoimmunity, its role in cancer metastasis remains unknown. Here we show that A20 monoubiquitylates Snail1 at three lysine residues and thereby promotes metastasis of aggressive basal-like breast cancers. A20 is significantly upregulated in human basal-like breast cancers and its expression level is inversely correlated with metastasis-free patient survival. A20 facilitates TGF-ß1-induced epithelial-mesenchymal transition (EMT) of breast cancer cells through multi-monoubiquitylation of Snail1. Monoubiquitylated Snail1 has reduced affinity for glycogen synthase kinase 3ß (GSK3ß), and is thus stabilized in the nucleus through decreased phosphorylation. Knockdown of A20 or overexpression of Snail1 with mutation of the monoubiquitylated lysine residues into arginine abolishes lung metastasis in mouse xenograft and orthotopic breast cancer models, indicating that A20 and monoubiquitylated Snail1 are required for metastasis. Our findings uncover an essential role of the A20-Snail1 axis in TGF-ß1-induced EMT and metastasis of basal-like breast cancers.


Asunto(s)
Neoplasias de la Mama/enzimología , Movimiento Celular , Neoplasias Pulmonares/enzimología , Factores de Transcripción de la Familia Snail/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Lisina , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Fosforilación , Estabilidad Proteica , Interferencia de ARN , Transducción de Señal , Factores de Transcripción de la Familia Snail/genética , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta1/farmacología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacos
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