RESUMEN
Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.
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Interferón Tipo I , Ubiquitina , Humanos , Ubiquitina/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Beclina-1 , Ubiquitinación , Inmunidad Innata , Interferón Tipo I/metabolismo , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Endopeptidasas/genética , Endopeptidasas/metabolismoRESUMEN
Exploring the connection between ubiquitin-like modifiers (ULMs) and the DNA damage response (DDR), we employed several advanced DNA damage and repair assay techniques and identified a crucial role for LC3B. Notably, its RNA recognition motif (RRM) plays a pivotal role in the context of transcription-associated homologous recombination (HR) repair (TA-HRR), a particular subset of HRR pathways. Surprisingly, independent of autophagy flux, LC3B interacts directly with R-loops at DNA lesions within transcriptionally active sites via its RRM, promoting TA-HRR. Using native RNA immunoprecipitation (nRIP) coupled with high-throughput sequencing (nRIP-seq), we discovered that LC3B also directly interacts with the 3'UTR AU-rich elements (AREs) of BRCA1 via its RRM, influencing its stability. This suggests that LC3B regulates TA-HRR both proximal to and distal from DNA lesions. Data from our LC3B depletion experiments showed that LC3B knockdown disrupts end-resection for TA-HRR, redirecting it towards the non-homologous end joining (NHEJ) pathway and leading to chromosomal instability, as evidenced by alterations in sister chromatid exchange (SCE) and interchromosomal fusion (ICF). Thus, our findings unveil autophagy-independent functions of LC3B in DNA damage and repair pathways, highlighting its importance. This could reshape our understanding of TA-HRR and the interaction between autophagy and DDR.
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Proteína BRCA1 , Proteínas Asociadas a Microtúbulos , Estructuras R-Loop , Reparación del ADN por Recombinación , Transcripción Genética , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Daño del ADN , Reparación del ADN por Unión de Extremidades , Regiones no Traducidas 3' , Recombinación Homóloga , Línea Celular Tumoral , Intercambio de Cromátides HermanasRESUMEN
Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.
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Proteínas de Unión al ADN , Neoplasias , Humanos , Proteínas de Unión al ADN/genética , Neoplasias/genética , Mutaciones Letales Sintéticas , Medicina de Precisión , Factores de Transcripción/genética , Péptidos , Hidrolasas Diéster Fosfóricas/genéticaRESUMEN
The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria-associated E3 ligase, MARCH5. Myeloid cell-specific March5 conditional knockout (March5 cKO) mice failed to secrete IL-1ß and IL-18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL-1ß and IL-18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27-linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination-defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL-1ß production. Thus, MARCH5-dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3-NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Interleucina-18/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Caspasa 1/metabolismoRESUMEN
BACKGROUND: Anti-heat shock protein (HSP) autoantibodies are detected in autoimmune diseases. We sought to ascertain whether anti-HSP10 IgG is present in patients with CSU and to elucidate the role of HSP10 in CSU pathogenesis. METHOD: Using a human proteome microarray, six potential autoantibodies had higher expression in 10 CSU samples compared with 10 normal controls (NCs). Among them, HSP10 IgG autoantibody was quantified by immune dot-blot assay in sera from 86 CSU patients and 44 NCs. The serum levels of HSP10 and microRNA-101-5p were measured in CSU patients and NCs. The effects of HSP10 and miR-101-5p on mast cell degranulation in response to IgE, compound 48/80, and platelet-activating factor (PAF) were investigated. RESULTS: CSU patients had higher IgG positivity to HSP10 (40.7% vs. 11.4%, p = .001), lower serum HSP10 levels (5.8 ± 3.6 vs. 12.2 ± 6.6 pg/mL, p < .001) than in NCs, and their urticaria severity was associated with anti-HSP10 IgG positivity, while HSP10 levels were related to urticaria control status. MiR-101-5p was increased in CSU patients. PAF enhanced IL4 production in PBMCs from CSU patients. IL-4 upregulated miR-101-5p and reduced HSP10 expression in keratinocytes. Transfection of miR-101-5p reduced HSP10 expression in keratinocytes. MiR-101-5p promoted PAF-induced mast cell degranulation, while HSP10 specifically prevented it. CONCLUSION: A new autoantibody, anti-HSP10 IgG was detected in CSU patients, which showed a significant correlation with UAS7 scores. A decreased serum HSP10 level was associated with upregulation of miR-101-5p due to increased IL-4 and PAF in CSU patients. Modulation of miR-101-5p and HSP10 may be a novel therapeutic approach for CSU.
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Urticaria Crónica , MicroARNs , Urticaria , Humanos , MicroARNs/genética , Factor de Activación Plaquetaria , Interleucina-4 , Enfermedad Crónica , Autoanticuerpos , Inmunoglobulina GRESUMEN
Osteoarthritis (OA) is a degenerative joint disorder associated with inflammation, functional disability, and high socioeconomic costs. The development of effective therapies against inflammatory OA has been limited owing to its complex and multifactorial nature. The efficacy of Prussian blue nanozymes coated with Pluronic (PPBzymes), US Food and Drug Administration-approved components, and their mechanisms of action have been described in this study, and PPBzymes have been characterized as a new OA therapeutic. Spherical PPBzymes were developed via nucleation and stabilization of Prussian blue inside Pluronic micelles. A uniformly distributed diameter of approximately 204 nm was obtained, which was maintained after storage in an aqueous solution and biological buffer. This indicates that PPBzymes are stable and could have biomedical applications. In vitro data revealed that PPBzymes promote cartilage generation and reduce cartilage degradation. Moreover, intra-articular injections with PPBzymes into mouse joints revealed their long-term stability and effective uptake into the cartilage matrix. Furthermore, intra-articular PPBzymes injections attenuated cartilage degradation without exhibiting cytotoxicity toward the synovial membrane, lungs, and liver. Notably, based on proteome microarray data, PPBzymes specifically block the JNK phosphorylation, which modulates inflammatory OA pathogenesis. These findings indicate that PPBzymes might represent a biocompatible and effective nanotherapeutic for obstructing JNK phosphorylation.
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Cartílago Articular , Osteoartritis , Ratones , Animales , Fosforilación , Poloxámero/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/uso terapéutico , Osteoartritis/patología , Cartílago Articular/metabolismo , Inyecciones IntraarticularesRESUMEN
Transcription factor CP2c (also known as TFCP2, α-CP2, LSF, and LBP-1c) is involved in diverse ubiquitous and tissue/stage-specific cellular processes and in human malignancies such as cancer. Despite its importance, many fundamental regulatory mechanisms of CP2c are still unclear. Here, we uncover an unprecedented mechanism of CP2c degradation via a previously unidentified SUMO1/PSME3/20S proteasome pathway and its biological meaning. CP2c is SUMOylated in a SUMO1-dependent way, and SUMOylated CP2c is degraded through the ubiquitin-independent PSME3 (also known as REGγ or PA28)/20S proteasome system. SUMOylated PSME3 could also interact with CP2c to degrade CP2c via the 20S proteasomal pathway. Moreover, precisely timed degradation of CP2c via the SUMO1/PSME3/20S proteasome axis is required for accurate progression of the cell cycle. Therefore, we reveal a unique SUMO1-mediated uncanonical 20S proteasome degradation mechanism via the SUMO1/PSME3 axis involving mutual SUMO-SIM interaction of CP2c and PSME3, providing previously unidentified mechanistic insights into the roles of dynamic degradation of CP2c in cell cycle progression.
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Neoplasias , Complejo de la Endopetidasa Proteasomal , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Sumoilación , Citoplasma/metabolismo , Neoplasias/metabolismo , Ciclo Celular , Proteínas de Unión al ADN/metabolismoRESUMEN
Osteoarthritis (OA) is a degenerative joint disorder associated with inflammation, functional disability, and high socioeconomic costs. The development of effective therapies against inflammatory OA has been limited owing to its complex and multifactorial nature. The efficacy of Prussian blue nanozymes coated with Pluronic (PPBzymes), US Food and Drug Administration-approved components, and their mechanisms of action have been described in this study, and PPBzymes have been characterized as a new OA therapeutic. Spherical PPBzymes were developed via nucleation and stabilization of Prussian blue inside Pluronic micelles. A uniformly distributed diameter of approximately 204 nm was obtained, which was maintained after storage in an aqueous solution and biological buffer. This indicates that PPBzymes are stable and could have biomedical applications. In vitro data revealed that PPBzymes promote cartilage generation and reduce cartilage degradation. Moreover, intra-articular injections with PPBzymes into mouse joints revealed their long-term stability and effective uptake into the cartilage matrix. Furthermore, intra-articular PPBzymes injections attenuated cartilage degradation without exhibiting cytotoxicity toward the synovial membrane, lungs, and liver. Notably, based on proteome microarray data, PPBzymes specifically block the JNK phosphorylation, which modulates inflammatory OA pathogenesis. These findings indicate that PPBzymes might represent a biocompatible and effective nanotherapeutic for obstructing JNK phosphorylation.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos , Osteoartritis , Estados Unidos , Animales , Ratones , Fosforilación , Poloxámero , Osteoartritis/tratamiento farmacológicoRESUMEN
X-ray angiography is commonly used in the diagnosis and treatment of coronary artery disease with the advantage of visualization of the inside of blood vessels in real-time. However, it has several disadvantages that occur in the acquisition process, which causes inconvenience and difficulty. Here, we propose a novel segmentation and nonrigid registration method to provide useful real-time assistive images and information. A convolutional neural network is used for the segmentation of coronary arteries in 2D X-ray angiography acquired from various angles in real-time. To compensate for errors that occur during the 2D X-ray angiography acquisition process, 3D CT angiography is used to analyze the topological structure. A novel energy function-based 3D deformation and optimization is utilized to implement real-time registration. We evaluated the proposed method for 50 series from 38 patients by comparing the ground truth. The proposed segmentation method showed that Precision, Recall, and F1 score were 0.7563, 0.6922, and 0.7176 for all vessels, 0.8542, 0.6003, and 0.7035 for markers, and 0.8897, 0.6389, and 0.7386 for bifurcation points, respectively. In the nonrigid registration method, the average distance of 0.8705, 1.06, and 1. 5706 mm for all vessels, markers, and bifurcation points was achieved. The overall process execution time was 0.179 s.
RESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Wiskott-Aldrich syndrome protein family member 2 (WASF2) is an integral member of the actin cytoskeleton pathway, which plays a crucial role in cell motility. In this study, we aimed to explore the role of WASF2 in HCC carcinogenesis and its regulatory mechanism. METHODS: WASF2 expression in HCC was analyzed using six public RNA-seq datasets and 66 paired tissues from patients with HCC. The role of WASF2 in normal hepatocyte cell phenotypes was evaluated using a WASF2 overexpression vector in vitro; it was evaluated in HCC cell phenotypes using small interfering RNA (siRNA) in vitro and in vivo. Epigenetic regulatory mechanism of WASF2 was assessed in the Cancer Genome Atlas liver hepatocellular carcinoma project (TCGA_LIHC) dataset and also validated in 38 paired HCC tissues. Site mutagenesis, bisulfite sequencing polymerase chain reaction (BSP), methylation-specific polymerase chain reaction (MSP), and quantitative MSP (qMSP) were used for evaluating WASF2 methylation status. RESULTS: WASF2 is overexpressed in HCC and is clinically correlated with its prognosis. WASF2 overexpression promoted normal hepatocyte proliferation. WASF2 inactivation decreased the viability, growth, proliferation, migration, and invasion of Huh-7 and SNU475 HCC cells by inducing G2/M phase arrest. This induced cell death and inhibited epithelial-mesenchymal transition, hindering actin polymerization. In addition, WASF2 knockdown using siWASF2 in a xenograft mouse model and a lung metastasis model exerted tumor suppressive effect. There was a negative correlation between WASF2 methylation status and mRNA expression. The methylation pattern of CpG site 2 (- 726 bp), located in the WASF2 promoter, plays an important role in the regulation of WASF2 expression. Furthermore, the cg242579 CpG island in the WASF2 5' promoter region was hypomethylated in HCC compared to that in the matched non-tumor samples. Patients with high WASF2 methylation and low WASF2 expression displayed the highest overall survival. CONCLUSIONS: WASF2 is overexpressed and hypomethylated in HCC and correlates with patient prognosis. WASF2 inactivation exerts anti-tumorigenic effects on HCC cells in vitro and in vivo, suggesting that WASF2 could be a potential therapeutic target for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/patología , Ratones , Regiones Promotoras Genéticas , Regulación hacia Arriba , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
PSMD14, a subunit of the 19S regulatory particles of the 26S proteasome, was recently identified as a potential prognostic marker and therapeutic target in diverse human cancers. Here, we show that the silencing and pharmacological blockade of PSMD14 in MDA-MB 435S breast cancer cells induce paraptosis, a non-apoptotic cell death mode characterized by extensive vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. The PSMD14 inhibitor, capzimin (CZM), inhibits proteasome activity but differs from the 20S proteasome subunit-inhibiting bortezomib (Bz) in that it does not induce aggresome formation or Nrf1 upregulation, which underlie Bz resistance in cancer cells. In addition to proteasome inhibition, the release of Ca2+ from the ER into the cytosol critically contributes to CZM-induced paraptosis. Induction of paraptosis by targeting PSMD14 may provide an attractive therapeutic strategy against cancer cells resistant to proteasome inhibitors or pro-apoptotic drugs.
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Neoplasias de la Mama , Calcio/metabolismo , Complejo de la Endopetidasa Proteasomal , Apoptosis , Bortezomib/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , TransactivadoresRESUMEN
DNA lesions impact on local transcription and the damage-induced transcriptional repression facilitates efficient DNA repair. However, how chromatin dynamics cooperates with these two events remained largely unknown. We here show that histone H2A acetylation at K118 is enriched in transcriptionally active regions. Under DNA damage, the RSF1 chromatin remodeling factor recruits HDAC1 to DSB sites. The RSF1-HDAC1 complex induces the deacetylation of H2A(X)-K118 and its deacetylation is indispensable for the ubiquitination of histone H2A at K119. Accordingly, the acetylation mimetic H2A-K118Q suppressed the H2A-K119ub level, perturbing the transcriptional repression at DNA lesions. Intriguingly, deacetylation of H2AX at K118 also licenses the propagation of γH2AX and recruitment of MDC1. Consequently, the H2AX-K118Q limits DNA repair. Together, the RSF1-HDAC1 complex controls the traffic of the DNA damage response and transcription simultaneously in transcriptionally active chromatins. The interplay between chromatin remodelers and histone modifiers highlights the importance of chromatin versatility in the maintenance of genome integrity.
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Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Epigénesis Genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Transactivadores/genética , Acetilación , Animales , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Células HEK293 , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Unión Proteica , Interferencia de ARN , Transactivadores/metabolismo , UbiquitinaciónRESUMEN
Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.
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Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/genética , Proteínas de Unión al ADN/genética , ADN/genética , Poli(ADP-Ribosa) Polimerasas/genética , Proteasas Ubiquitina-Específicas/genética , Secuencias de Aminoácidos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP)/metabolismo , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reparación del ADN por Recombinación , Transducción de Señal , Empalmosomas , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.
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Aurora Quinasa B/genética , Proteínas de Ciclo Celular/genética , Segregación Cromosómica , Mitosis , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Transactivadores/genética , Ácido Aspártico/metabolismo , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas Nucleares/deficiencia , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serina/metabolismo , Transactivadores/deficiencia , Quinasa Tipo Polo 1RESUMEN
Aberrant activation of the non-receptor kinase c-Abl is implicated in the development of pathogenic hallmarks of Parkinson's disease, such as α-synuclein aggregation and progressive neuronal loss. c-Abl-mediated phosphorylation and inhibition of parkin ligase function lead to accumulation of parkin interacting substrate (PARIS) that mediates α-synuclein pathology-initiated dopaminergic neurodegeneration. Here we show that, in addition to PARIS accumulation, c-Abl phosphorylation of PARIS is required for PARIS-induced cytotoxicity. c-Abl-mediated phosphorylation of PARIS at Y137 (within the Krüppel-associated box domain) drives its association with KAP1 and the repression of genes with diverse functions in pathways such as chromatin remodelling and p53-dependent cell death. One phosphorylation-dependent PARIS target, MDM4 (a p53 inhibitor that associates with MDM2; also known as MDMX), is transcriptionally repressed in a histone deacetylase-dependent manner via PARIS binding to insulin response sequence motifs within the MDM4 promoter. Virally induced PARIS transgenic mice develop c-Abl activity-dependent Parkinson's disease features such as motor deficits, dopaminergic neuron loss and neuroinflammation. PARIS expression in the midbrain resulted in c-Abl activation, PARIS phosphorylation, MDM4 repression and p53 activation, all of which are blocked by the c-Abl inhibitor nilotinib. Importantly, we also observed aberrant c-Abl activation and PARIS phosphorylation along with PARIS accumulation in the midbrain of adult parkin knockout mice, implicating c-Abl in recessive Parkinson's disease. Inhibition of c-Abl or PARIS phosphorylation by nilotinib or Y137F-PARIS expression in adult parkin knockout mice blocked MDM4 repression and p53 activation, preventing motor deficits and dopaminergic neurodegeneration. Finally, we found correlative increases in PARIS phosphorylation, MDM4 repression and p53 activation in post-mortem Parkinson's disease brains, pointing to clinical relevance of the c-Abl-PARIS-MDM4-p53 pathway. Taken together, our results describe a novel mechanism of epigenetic regulation of dopaminergic degeneration downstream of pathological c-Abl activation in Parkinson's disease. Since c-Abl activation has been shown in sporadic Parkinson's disease, PARIS phosphorylation might serve as both a useful biomarker and a potential therapeutic target to regulate neuronal loss in Parkinson's disease.
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Neuronas Dopaminérgicas/patología , Degeneración Nerviosa/patología , Trastornos Parkinsonianos/patología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Represoras/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Trastornos Parkinsonianos/metabolismo , FosforilaciónRESUMEN
Ubiquitin stress-induced NEDDylation leads to the formation of aggresome-like bodies (ALBs) in the perinuclear region of cells. Therefore, imaging analysis is essential for characterizing the biological phenotypes of ALBs. Here, we describe a protocol to monitor ALBs induced by ubiquitin stress using immunocytochemistry and to quantify cells containing ALBs. This optimized protocol details the use of readily available materials and reagents and can be applied to explore diverse molecules involved in stress-induced ALBs. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).
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Citosol , Microscopía Confocal/métodos , Biología Molecular/métodos , Técnica del Anticuerpo Fluorescente , Células HeLa , Histona Desacetilasa 6/inmunología , Histona Desacetilasa 6/metabolismo , Humanos , Inmunohistoquímica/métodos , Cuerpos de Inclusión/metabolismo , Proteína NEDD8/inmunología , Proteína NEDD8/metabolismo , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Necroptosis is emerging as a new target for cancer immunotherapy as it is now recognized as a form of cell death that increases tumor immunogenicity, which would be especially helpful in treating immune-desert tumors. De novo synthesis of inflammatory proteins during necroptosis appears especially important in facilitating increased anti-tumor immune responses. While late-stage transcription mediated by NF-κB during cell death is believed to play a role in this process, it is otherwise unclear what cell signaling events initiate this transactivation of inflammatory genes. METHODS: We employed tandem-affinity purification linked to mass spectrometry (TAP-MS), in combination with the analysis of RNA-sequencing (RNA-Seq) datasets to identify the Tripartite Motif Protein 28 (TRIM28) as a candidate co-repressor. Comprehensive biochemical and molecular biology techniques were used to characterize the role of TRIM28 in RIPK3 activation-induced transcriptional and immunomodulatory events. The cell composition estimation module was used to evaluate the correlation between RIPK3/TRIM28 levels and CD8+ T cells or dendritic cells (DC) in all TCGA tumors. RESULTS: We identified TRIM28 as a co-repressor that regulates transcriptional activity during necroptosis. Activated RIPK3 phosphorylates TRIM28 on serine 473, inhibiting its chromatin binding activity, thereby contributing to the transactivation of NF-κB and other transcription factors, such as SOX9. This leads to elevated cytokine expression, which then potentiates immunoregulatory processes, such as DC maturation. The expression of RIPK3 has a significant positive association with the tumor-infiltrating immune cells populations in various tumor type, thereby activating anti-cancer responses. CONCLUSION: Our data suggest that RIPK3 activation-dependent derepression of TRIM28 in cancer cells leads to increased immunostimulatory cytokine production in the tumor microenvironment, which then contributes to robust cytotoxic anti-tumor immunity.
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Regulación Neoplásica de la Expresión Génica , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , Microambiente Tumoral/genética , Animales , Sitios de Unión , Muerte Celular , Línea Celular , Citocinas/metabolismo , Humanos , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Necroptosis , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
Contact inhibition is a key cellular phenomenon that prevents cells from hyper-proliferating upon reaching confluence. Although not fully characterized, a critical driver of this process is the Hippo signaling pathway, whose downstream effector yes-associated protein plays pivotal roles in cell growth and differentiation. Here, we provide evidence that the E3 ligase WWP1 (WW-domain containing protein 1) mono-ubiquitinates AMOTL2 (angiomotin-like 2) at K347 and K408. Mono-ubiquitinated AMOTL2, in turn, interacts with the kinase LATS2, which facilitates recruitment of the upstream Hippo pathway component SAV1 and ultimately promotes yes-associated protein phosphorylation and subsequent cytoplasmic sequestration and/or degradation. Furthermore, contact inhibition induced by high cell density promoted the localization and stabilization of WWP1 at cell junctions, where it interacted with Crumbs polarity proteins. Notably, the Crumbs complex was functionally important for AMOTL2 mono-ubiquitination and LATS activation under high cell density conditions. These findings delineate a functionally important molecular mechanism in which AMOTL2 mono-ubiquitination by WWP1 at cell junctions and LATS activation are tightly coupled to upstream cell density cues.
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Angiomotinas/metabolismo , Inhibición de Contacto , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Angiomotinas/genética , Inhibición de Contacto/genética , Activación Enzimática , Humanos , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Accumulation of the parkin-interacting substrate (PARIS; ZNF746), due to inactivation of parkin, contributes to Parkinson's disease (PD) through repression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; PPARGC1A) activity. Here, we identify farnesol as an inhibitor of PARIS. Farnesol promoted the farnesylation of PARIS, preventing its repression of PGC-1α via decreasing PARIS occupancy on the PPARGC1A promoter. Farnesol prevented dopaminergic neuronal loss and behavioral deficits via farnesylation of PARIS in PARIS transgenic mice, ventral midbrain transduction of AAV-PARIS, adult conditional parkin KO mice, and the α-synuclein preformed fibril model of sporadic PD. PARIS farnesylation is decreased in the substantia nigra of patients with PD, suggesting that reduced farnesylation of PARIS may play a role in PD. Thus, farnesol may be beneficial in the treatment of PD by enhancing the farnesylation of PARIS and restoring PGC-1α activity.
Asunto(s)
Enfermedad de Parkinson , Animales , Dopamina , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Prenilación , Proteínas Represoras/metabolismo , Sustancia Negra/metabolismoRESUMEN
Stress-coupled NEDDylation potentially regulates the aggregation of nuclear proteins, which could protect the nuclear ubiquitin-proteasome system from proteotoxic stress. However, it remains unclear how NEDDylation controls protein-aggregation responses to diverse stress conditions. Here, we identified HDAC6 as a direct NEDD8-binding partner that regulates the formation of aggresome-like bodies (ALBs) containing NEDDylated cytosolic protein aggregates during ubiquitin stress. HDAC6 colocalizes with stress-induced ALBs, and HDAC6 inhibition suppresses ALBs formation, but not stress-induced NEDDylation, suggesting that HDAC6 carries NEDDylated-proteins to generate ALBs. Then, we monitored the ALBs-associated proteostasis network and found that p62 directly controls ALBs formation as an acceptor of NEDDylated cytosolic aggregates. Interestingly, we also observed that ALBs are highly condensed in chloroquine-treated cells with impaired autophagic flux, indicating that ALBs rely on autophagy. Collectively, our data suggest that NEDD8, HDAC6, and p62 are involved in the management of proteotoxic stress by forming cytosolic ALBs coupled to the aggresome-autophagy flux.