RESUMEN

Antiviral innate immunity is a complicated system initiated by the induction of type I interferon (IFN-I) and downstream interferon-stimulated genes (ISGs) and is finely regulated by numerous positive and negative factors at different signaling adaptors. During this process, posttranslational modifications, especially ubiquitination, are the most common regulatory strategy used by the host to switch the antiviral innate signaling pathway and are mainly controlled by E3 ubiquitin ligases from different protein families. A comprehensive understanding of the regulatory mechanisms and a novel discovery of regulatory factors involved in the IFN-I signaling pathway are important for researchers to identify novel therapeutic targets against viral infectious diseases based on innate immunotherapy. In this section, we use the E3 ubiquitin ligase as an example to guide the identification of a protein belonging to the RING Finger (RNF) family that regulates the RIG-I-mediated IFN-I pathway through ubiquitination.

Asunto(s)

Inmunidad Innata , Interferón Tipo I , Transducción de Señal , Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Interferón Tipo I/metabolismo , Virosis/inmunología , Virosis/genética , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética

RESUMEN

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

Asunto(s)

Proteínas Inmediatas-Precoces , Inmunoprecipitación , Ubiquitina-Proteína Ligasas , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Inmunoprecipitación/métodos , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Unión Proteica , Proteínas Ubiquitinadas/metabolismo , Herpesvirus Humano 1/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas Virales/metabolismo

RESUMEN

Centromere pairing is crucial for synapsis in meiosis. This study delves into the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, specifically focusing on F-box protein 47 (FBXO47), in mouse meiosis. Here, we revealed that FBXO47 is localized at the centromere and it regulates centromere pairing cooperatively with SKP1 to ensure proper synapsis in pachynema. The absence of FBXO47 causes defective centromeres, resulting in incomplete centromere pairing, which leads to corruption of SC at centromeric ends and along chromosome axes, triggering premature dissociation of chromosomes and pachytene arrest. FBXO47 deficient pachytene spermatocytes exhibited drastically reduced SKP1 expression at centromeres and chromosomes. Additionally, FBXO47 stabilizes SKP1 by down-regulating its ubiquitination in HEK293T cells. In essence, we propose that FBXO47 collaborates with SKP1 to facilitate centromeric SCF formation in spermatocytes. In summary, we posit that the centromeric SCF E3 ligase complex regulates centromere pairing for pachynema progression in mice.

Asunto(s)

Centrómero , Emparejamiento Cromosómico , Proteínas F-Box , Espermatocitos , Animales , Masculino , Centrómero/metabolismo , Centrómero/genética , Ratones , Espermatocitos/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Humanos , Células HEK293 , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Meiosis , Ratones Noqueados , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Ratones Endogámicos C57BL

RESUMEN

Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-ß-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage.

Asunto(s)

Cisplatino , Quinasas Quinasa Quinasa PAM , Ratones Noqueados , Transducción de Señal , Cisplatino/efectos adversos , Cisplatino/toxicidad , Animales , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Transducción de Señal/efectos de los fármacos , Ratones , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Antineoplásicos/farmacología , Antineoplásicos/efectos adversos , Túbulos Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales

RESUMEN

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to NaV1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Asunto(s)

Inteligencia Artificial , Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Descubrimiento de Drogas/métodos , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/química , Unión Proteica , Cristalografía por Rayos X , Ligandos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Evaluación Preclínica de Medicamentos/métodos

RESUMEN

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT , Diferenciación Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Histona Demetilasas , Ratones Noqueados , Células Madre Embrionarias de Ratones , Ubiquitina-Proteína Ligasas , Animales , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Ratones , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Células Madre Embrionarias de Ratones/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 1/genética , Histonas/metabolismo , Proliferación Celular , Ubiquitinación

RESUMEN

OBJECTIVE: The E3 ubiquitin ligase is well recognized as a significant contributor to glioblastoma (GBM) progression and has promise as a prospective therapeutic target. This study explores the contribution of E3 ubiquitin ligase RNF122 in the GBM progression and the related molecular mechanisms. METHODS: RNF122 expression levels were evaluated using qRT-PCR, WB, and IHC, while functional assays besides animal experiments were used to assess RNF122's effect on GBM progression. We also tested the RNF122 impact on JAK2/STAT3/c-Myc signaling using WB. RESULTS: RNF122 was upregulated in GBM and correlated to the advanced stage and poor clinical outcomes, representing an independent prognostic factor. Based on functional assays, RNF122 promotes GBM growth and cell cycle, which was validated further in subsequent analyses by JAK2/STAT3/c-Myc pathway activation. Moreover, JAK2/STAT3 signaling pathway inhibitor WP1066 can weaken the effect of overexpression RNF122 on promoting GBM progression. CONCLUSION: Our results revealed that RNF122 caused an aggressive phenotype to GBM and was a poor prognosticator; thus, targeting RNF122 may be effectual in GBM treatment.

Asunto(s)

Neoplasias Encefálicas , Glioblastoma , Janus Quinasa 2 , Proteínas Proto-Oncogénicas c-myc , Factor de Transcripción STAT3 , Transducción de Señal , Ubiquitina-Proteína Ligasas , Humanos , Glioblastoma/metabolismo , Glioblastoma/patología , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal/fisiología , Transducción de Señal/efectos de los fármacos , Masculino , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Femenino , Animales , Línea Celular Tumoral , Ratones Desnudos , Persona de Mediana Edad , Ratones , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Ratones Endogámicos BALB C , Péptidos y Proteínas de Señalización Intracelular

RESUMEN

BACKGROUND: Targeted protein degradation of neosubstrates plays a crucial role in hematological cancer treatment involving immunomodulatory imide drugs (IMiDs) therapy. Nevertheless, the persistence of inevitable drug resistance and hematological toxicities represents a significant obstacle to their clinical effectiveness. METHODS: Phenotypic profiling of a small molecule compounds library in multiple hematological cancer cell lines was conducted to screen for hit degraders. Molecular dynamic-based rational design and cell-based functional assays were conducted to develop more potent degraders. Multiple myeloma (MM) tumor xenograft models were employed to investigate the antitumor efficacy of the degraders as single or combined agents with standard of care agents. Unbiased proteomics was employed to identify multiple therapeutically relevant neosubstrates targeted by the degraders. MM patient-derived cell lines (PDCs) and a panel of solid cancer cell lines were utilized to investigate the effects of candidate degrader on different stage of MM cells and solid malignancies. Unbiased proteomics of IMiDs-resistant MM cells, cell-based functional assays and RT-PCR analysis of clinical MM specimens were utilized to explore the role of BRD9 associated with IMiDs resistance and MM progression. RESULTS: We identified a novel cereblon (CRBN)-dependent lead degrader with phthalazinone scaffold, MGD-4, which induced the degradation of Ikaros proteins. We further developed a novel potent candidate, MGD-28, significantly inhibited the growth of hematological cancer cells and induced the degradation of IKZF1/2/3 and CK1α with nanomolar potency via a Cullin-CRBN dependent pathway. Oral administration of MGD-4 and MGD-28 effectively inhibited MM tumor growth and exhibited significant synergistic effects with standard of care agents. MGD-28 exhibited preferentially profound cytotoxicity towards MM PDCs at different disease stages and broad antiproliferative activity in multiple solid malignancies. BRD9 modulated IMiDs resistance, and the expression of BRD9 was significant positively correlated with IKZF1/2/3 and CK1α in MM specimens at different stages. We also observed pronounced synergetic efficacy between the BRD9 inhibitor and MGD-28 for MM treatment. CONCLUSIONS: Our findings present a strategy for the multi-targeted degradation of Ikaros proteins and CK1α against hematological cancers, which may be expanded to additional targets and indications. This strategy may enhance efficacy treatment against multiple hematological cancers and solid tumors.

Asunto(s)

Neoplasias Hematológicas , Humanos , Animales , Línea Celular Tumoral , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/metabolismo , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Factor de Transcripción Ikaros/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales

RESUMEN

The ubiquitin-proteasome system is frequently employed to degrade viral proteins, thereby inhibiting viral replication and pathogenicity. Through an analysis of the degradation kinetics of all the SARS-CoV-2 proteins, our study revealed rapid degradation of several proteins, particularly NSP5. Additionally, we identified FBXO22, an E3 ubiquitin ligase, as the primary regulator of NSP5 ubiquitination. Moreover, we validated the interaction between FBXO22 and NSP5, demonstrating that FBXO22-mediated ubiquitination of NSP5 facilitated its recognition by the proteasome, leading to subsequent degradation. Specifically, FBXO22 catalyzed the formation of K48-linked polyubiquitin chains on NSP5 at lysine residues 5 and 90. Knockdown of FBXO22 resulted in decreased NSP5 ubiquitination levels, increased stability, and enhanced ability to evade the host innate immune response. Notably, the protein level of FBXO22 were negatively correlated with SARS-CoV-2 load, highlighting its importance in inhibiting viral replication. This study elucidates the molecular mechanism by which FBXO22 mediates the degradation of NSP5 and underscores its critical role in limiting viral replication. The identification of FBXO22 as a regulator of NSP5 stability provides new insights and potential avenues for targeting NSP5 in antiviral strategies.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , SARS-CoV-2 , Ubiquitinación , Replicación Viral , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , COVID-19/virología , COVID-19/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Células HEK293 , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteolisis , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Receptores Citoplasmáticos y Nucleares

RESUMEN

Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.

Asunto(s)

Astrocitos , Encefalomielitis Autoinmune Experimental , Ribonucleoproteínas , Regulación hacia Arriba , Animales , Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Ratones , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hormonas Tiroideas/metabolismo , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona Tiroide , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones Endogámicos C57BL , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Transporte Activo de Núcleo Celular , Femenino , Glucólisis , Ubiquitinación , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Núcleo Celular/metabolismo

RESUMEN

Rationale: Parkin (an E3 ubiquitin protein ligase) is an important regulator of mitophagy. However, the role of Parkin in viral myocarditis (VMC) remains unclear. Methods: Coxsackievirus B3 (CVB3) infection was induced in mice to create VMC. Cardiac function and inflammatory response were evaluated by echocardiography, histological assessment, and molecular analyses. AAV9 (adeno-associated virus 9), transmission electron microscopy (TEM) and western blotting were used to investigate the mechanisms by which Parkin regulates mitophagy and cardiac inflammation. Results: Our data indicated that Parkin- and BNIP3 (BCL2 interacting protein 3 like)-mediated mitophagy was activated in VMC mice and neonatal rat cardiac myocytes (NRCMs) infected with CVB3, which blocked autophagic flux by inhibiting autophagosome-lysosome fusion. Parkin silencing aggravated mortality and accelerated the development of cardiac dysfunction in CVB3-treated mice. While silencing of Parkin did not significantly increase inflammatory response through activating NF-κB pathway and production of inflammatory cytokines post-VMC, the mitophagy activity were reduced, which stimulated the accumulation of damaged mitochondria. Moreover, Parkin silencing exacerbated VMC-induced apoptosis. We consistently found that Parkin knockdown disrupted mitophagy activity and inflammatory response in NRCMs. Conclusion: This study elucidated the important role of Parkin in maintaining cardiac function and inflammatory response by regulating mitophagy activity and the NF-κB pathway during acute VMC. Although the functional impact of mitophagy remains unclear, our findings suggest that Parkin silencing may accelerate VMC development.

Asunto(s)

Infecciones por Coxsackievirus , Mitofagia , Miocarditis , Miocitos Cardíacos , Ubiquitina-Proteína Ligasas , Animales , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Miocarditis/virología , Miocarditis/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Masculino , Ratas , Enterovirus Humano B/fisiología , Apoptosis , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , FN-kappa B/metabolismo , Ratones Endogámicos BALB C

Asunto(s)

Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética

RESUMEN

The Ideal Plant Architecture 1 (IPA1) transcription factor promotes rice yield and immunity through phosphorylation at its amino acid residue Ser163 as a switch. Although phosphorylated IPA1 mimic, IPA1(S163D), directly targets the promoter of immune response gene WRKY45, it cannot activate its expression. Here, we identified a co-activator of IPA1(S163D), a RING-finger E3 ligase IPA1 interactor 7 (IPI7), which fine-tunes the transcriptional activity of IPA1 to timely promote plant immunity and simultaneously maintain growth for yield. IPI7 interacts with IPA1 and promotes K29-polyubiquitination of IPA1 in vitro and in vivo. However, the stability of IPA1 protein is not affected by IPI7-mediated ubiquitination. The IPI7-promoted K29-polyubiquitination of IPA1 is induced by Magnaporthe oryzae infection and required for phosphorylated IPA1 to transactivate WRKY45 expression for immune response but not for plain IPA1 to transactivate DENSE AND ERECT PANICLES 1 (DEP1) expression for panicle development. IPI7 knockout impairs IPA1-mediated immunity but not yield. Our study reveals that plants utilize non-proteolytic K29-ubiquitination as a response to pathogen infection to fine-tune IPA1 transactivation activity for promoting immunity.

Asunto(s)

Oryza , Enfermedades de las Plantas , Proteínas de Plantas , Activación Transcripcional , Ubiquitina-Proteína Ligasas , Ubiquitinación , Enfermedades de las Plantas/microbiología , Oryza/microbiología , Oryza/metabolismo , Oryza/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fosforilación , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Inmunidad de la Planta/genética , Ascomicetos

RESUMEN

The HECT E3 ubiquitin ligases 1 (WWP1) and 2 (WWP2) are responsible for the ubiquitin-mediated degradation of key tumour suppressor proteins and are dysregulated in various cancers and diseases. Here we expand their limited inhibitor space by identification of NSC-217913 displaying a WWP1 IC50 of 158.3 µM (95% CI = 128.7, 195.1 µM). A structure-activity relationship by synthesis approach aided by molecular docking led to compound 11 which displayed increased potency with an IC50 of 32.7 µM (95% CI = 24.6, 44.3 µM) for WWP1 and 269.2 µM (95% CI = 209.4, 347.9 µM) for WWP2. Molecular docking yielded active site-bound poses suggesting that the heterocyclic imidazo[4,5-b]pyrazine scaffold undertakes a π-stacking interaction with the phenolic group of tyrosine, and the ethyl ester enables strong ion-dipole interactions. Given the therapeutic potential of WWP1 and WWP2, we propose that compound 11 may provide a basis for future lead compound development.

Asunto(s)

Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Relación Estructura-Actividad , Estructura Molecular , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química

RESUMEN

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.

Asunto(s)

Cápside , Proteínas de Ciclo Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Virus de la Hepatitis B , Ubiquitina-Proteína Ligasas Nedd4 , Ubiquitina-Proteína Ligasas , Ubiquitinación , Liberación del Virus , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Cápside/metabolismo , Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Liberación del Virus/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Hepatitis B/metabolismo , Hepatitis B/virología , Proteínas de Unión al Calcio

RESUMEN

AIMS: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease that affects the hepatic bile ducts, leading to hepatic inflammation and fibrosis. PSC can also impact skeletal muscle through the muscle-liver axis, resulting in sarcopenia, a complication characterized by a generalized loss of muscle mass and strength. The underlying mechanisms and therapy of PSC-induced sarcopenia are not well understood, but one potential regulator is the transcription factor forkhead box protein O1 (FOXO1), which is involved in the ubiquitin proteasome system. Thus, the aim of this study is to assess the pharmacological potential of FOXO1 inhibition for treating PSC-induced sarcopenia. MATERIALS AND METHODS: To establish diet-induced PSC model, we provided mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 4 weeks. Mice were intramuscularly injected with AS1842856 (AS), a FOXO1 inhibitor, at a dose of 3.5 mg/kg twice a week for last two weeks. C2C12 myotubes with cholic acid (CA) or deoxycholic acid (DCA) were treated with AS. KEY FINDINGS: We observed a decrease in muscle size and performance in DDC-fed mice with upregulated expression of FOXO1 and E3 ligases such as ATROGIN1 and MuRF1. We found that myotube diameter and MyHC protein level were decreased by CA or DCA in C2C12 myotubes, but treatment of AS reversed these reductions. We observed that intramuscular injection of AS effectively mitigates DDC diet-induced sarcopenia in a rodent PSC model. SIGNIFICANCE: Our study suggests that a FOXO1 inhibitor could be a potential leading therapeutic drug for relieving PSC-induced sarcopenia.

Asunto(s)

Colangitis Esclerosante , Modelos Animales de Enfermedad , Proteína Forkhead Box O1 , Sarcopenia , Transducción de Señal , Animales , Sarcopenia/metabolismo , Sarcopenia/etiología , Sarcopenia/tratamiento farmacológico , Sarcopenia/prevención & control , Sarcopenia/patología , Ratones , Proteína Forkhead Box O1/metabolismo , Colangitis Esclerosante/complicaciones , Colangitis Esclerosante/tratamiento farmacológico , Colangitis Esclerosante/metabolismo , Colangitis Esclerosante/patología , Transducción de Señal/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Proteínas Musculares/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Piridinas/farmacología , Quinolonas

RESUMEN

With emerging genetic association studies, new genes and pathways are revealed as causative factors in the development of Parkinson's disease (PD). However, many of these PD genes are poorly characterized in terms of their function, subcellular localization, and interaction with other components in cellular pathways. This represents a major obstacle towards a better understanding of the molecular causes of PD, with deeper molecular studies often hindered by a lack of high-quality, validated antibodies for detecting the corresponding proteins of interest. In this study, we leveraged the nanoluciferase-derived LgBiT-HiBiT system by generating a cohort of tagged PD genes in both induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells. To promote luminescence signals within cells, a master iPSC line was generated, in which LgBiT expression is under the control of a doxycycline-inducible promoter. LgBiT could bind to HiBiT when present either alone or when tagged onto different PD-associated proteins encoded by the genes GBA1, GPNMB, LRRK2, PINK1, PRKN, SNCA, VPS13C, and VPS35. Several HiBiT-tagged proteins could already generate luminescence in iPSCs in response to the doxycycline induction of LgBiT, with the enzyme glucosylceramidase beta 1 (GCase), encoded by GBA1, being one such example. Moreover, the GCase chaperone ambroxol elicited an increase in the luminescence signal in HiBiT-tagged GBA1 cells, correlating with an increase in the levels of GCase in dopaminergic cells. Taken together, we have developed and validated a Doxycycline-inducible luminescence system to serve as a sensitive assay for the quantification, localization, and activity of HiBiT-tagged PD-associated proteins with reliable sensitivity and efficiency.

Asunto(s)

Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas Quinasas

RESUMEN

Neuroinflammation is considered to be one of the driving factors in Parkinson's disease (PD). This study was conducted using neuronal and glial cell cultures differentiated from induced pluripotent stem cells (iPSC) of healthy donors (HD) and PD patients with different PARK2 mutations (PD). Based on the results of RNA sequencing, qPCR and ELISA, we revealed transcriptional and post-transcriptional changes in HD and PD neurons cultivated in HD and PD glial-conditioned medium. We demonstrated that if one or both of the components of the system, neurons or glia, is Parkin-deficient, the interaction resulted in the down-regulation of a number of key genes related to inflammatory intracellular pathways and negative regulation of apoptosis in neurons, which might be neuroprotective. In PD neurons, the stress-induced up-regulation of APLNR was significantly stronger compared to HD neurons and was diminished by glial soluble factors, both HD and PD. PD neurons in PD glial conditioned medium increased APLN expression and also up-regulated apelin synthesis and release into intracellular fluid, which represented another compensatory action. Overall, the reported results indicate that neuronal self-defense mechanisms contribute to cell survival, which might be characteristic of PD patients with Parkin-deficiency.

Asunto(s)

Células Madre Pluripotentes Inducidas , Neuroglía , Neuronas , Enfermedad de Parkinson , Ubiquitina-Proteína Ligasas , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Neuroglía/metabolismo , Neuronas/metabolismo , Transducción de Señal , Medios de Cultivo Condicionados/farmacología , Células Cultivadas , Inflamación/metabolismo , Inflamación/genética , Diferenciación Celular

RESUMEN

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer's disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau's accumulation or what mechanisms might be involved. To explore these questions, we focus on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to weaken its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 reveals the mechanism of this clash, allowing design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We confirm that, in our models, pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a molecular mechanism for how phosphorylation at a discrete site contributes to accumulation of a tau proteoform.

Asunto(s)

Enfermedad de Alzheimer , Unión Proteica , Ubiquitina-Proteína Ligasas , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/química , Fosforilación , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Animales , Células HEK293 , Cristalografía por Rayos X , Procesamiento Proteico-Postraduccional

RESUMEN

BACKGROUND: Colorectal cancer ranks among the most prevalent malignancies globally. Accurate prediction of metachronous liver metastasis is crucial for optimizing postoperative management. Tripartite motif-containing protein 27 (TRIM27), an E3 ubiquitin ligase, is implicated in diverse cellular functions and tumorigenesis. METHODS: This study aimed to develop and validate a TRIM27-based nomogram for prognostication in colorectal cancer patients. Transcriptome sequencing of five paired tumor and normal tissue samples identified TRIM27 as a potential prognostic biomarker. Immunohistochemistry was employed to assess TRIM27 expression in colorectal cancer cohorts from two institutions. RESULTS: TRIM27 expression correlated significantly with both the prognosis of colorectal cancer patients and the occurrence of metachronous liver metastasis. A nomogram incorporating TRIM27 and clinical factors was constructed and demonstrated robust predictive accuracy in an independent validation cohort. CONCLUSION: The TRIM27-based nomogram is a valuable prognostic tool for predicting prognosis and metachronous liver metastasis in colorectal cancer patients, aiding in personalized treatment decisions.

Asunto(s)

Biomarcadores de Tumor , Neoplasias Colorrectales , Neoplasias Hepáticas , Nomogramas , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Masculino , Femenino , Pronóstico , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Anciano , Periodo Posoperatorio , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Neoplasias Primarias Secundarias/patología , Neoplasias Primarias Secundarias/metabolismo , Neoplasias Primarias Secundarias/genética , Proteínas de Motivos Tripartitos , Proteínas de Unión al ADN , Proteínas Nucleares