RESUMO
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.
Assuntos
Imunidade Inata , Interferon Tipo I , Transdução de Sinais , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Interferon Tipo I/metabolismo , Viroses/imunologia , Viroses/genética , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genética , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genéticaRESUMO
TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity.
Assuntos
Ligação Proteica , RNA Viral , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , RNA Viral/metabolismo , RNA Viral/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células HEK293 , Imunidade Inata , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Antivirais/metabolismo , Antivirais/farmacologia , Vírus de RNA/genética , Sítios de LigaçãoRESUMO
Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance.
Assuntos
Apresentação de Antígeno , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Antígenos de Histocompatibilidade Classe I , Proteínas de Membrana Transportadoras , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
The fusion of autophagosomes and lysosomes is essential for the prevention of nonalcoholic fatty liver disease (NAFLD). Here, we generate a hepatocyte-specific CHIP knockout (H-KO) mouse model that develops NAFLD more rapidly in response to a high-fat diet (HFD) or high-fat, high-fructose diet (HFHFD). The accumulation of P62 and LC3 in the livers of H-KO mice and CHIP-depleted cells indicates the inhibition of autophagosome-lysosome fusion. AAV8-mediated overexpression of CHIP in the murine liver slows the progression of NAFLD induced by HFD or HFHFD feeding. Mechanistically, CHIP induced K63- and K27-linked polyubiquitination at the lysine 198 residue of STX17, resulting in increased STX17-SNAP29-VAMP8 complex formation. The STX17 K198R mutant was not ubiquitinated by CHIP; it interfered with its interaction with VAMP8, rendering STX17 incapable of inhibiting steatosis development in mice. These results indicate that a signaling regulatory mechanism involving CHIP-mediated non-degradative ubiquitination of STX17 is necessary for autophagosome-lysosome fusion.
Assuntos
Autofagossomos , Lisossomos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Lisossomos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Autofagossomos/metabolismo , Camundongos , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Dieta Hiperlipídica/efeitos adversos , Masculino , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Hepatócitos/metabolismo , Modelos Animais de Doenças , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Fusão de Membrana , Autofagia , Fator de Transcrição TFIIHRESUMO
Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.
Assuntos
Ligação Proteica , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/química , Humanos , Fosforilação , Cristalografia por Raios X , Modelos Moleculares , Ubiquitina/metabolismo , CinéticaRESUMO
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.
Assuntos
Cisplatino , MAP Quinase Quinase Quinases , Camundongos Knockout , Transdução de Sinais , Cisplatino/efeitos adversos , Cisplatino/toxicidade , Animais , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Camundongos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Antineoplásicos/farmacologia , Antineoplásicos/efeitos adversos , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de SinalRESUMO
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.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Histona Desmetilases , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Ubiquitina-Proteína Ligases , Animais , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Camundongos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias Murinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/genética , Histonas/metabolismo , Proliferação de Células , UbiquitinaçãoRESUMO
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.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais , Neoplasias Hepáticas , Nomogramas , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Masculino , Feminino , Prognóstico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Idoso , Período Pós-Operatório , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Segunda Neoplasia Primária/patologia , Segunda Neoplasia Primária/metabolismo , Segunda Neoplasia Primária/genética , Proteínas com Motivo Tripartido , Proteínas de Ligação a DNA , Proteínas NuclearesRESUMO
BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified. METHODS: We confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclearâcytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model. RESULTS: This study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration. CONCLUSIONS: These findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.
Assuntos
Neoplasias dos Ductos Biliares , Proteínas Estimuladoras de Ligação a CCAAT , Colangiocarcinoma , Metilação de DNA , Progressão da Doença , Ubiquitina-Proteína Ligases , Ubiquitinação , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Masculino , Proliferação de Células , Feminino , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genéticaRESUMO
Van Gogh-like 2 (Vangl2), a core planar cell polarity component, plays an important role in polarized cellular and tissue morphology induction, growth development, and cancer. However, its role in regulating inflammatory responses remains elusive. Here, we report that Vangl2 is upregulated in patients with sepsis and identify Vangl2 as a negative regulator of The nuclear factor-kappaB (NF-κB) signaling by regulating the protein stability and activation of the core transcription component p65. Mice with myeloid-specific deletion of Vangl2 (Vangl2ΔM) are hypersusceptible to lipopolysaccharide (LPS)-induced septic shock. Vangl2-deficient myeloid cells exhibit enhanced phosphorylation and expression of p65, therefore, promoting the secretion of proinflammatory cytokines after LPS stimulation. Mechanistically, NF-κB signaling-induced-Vangl2 recruits E3 ubiquitin ligase PDLIM2 to catalyze K63-linked ubiquitination on p65, which serves as a recognition signal for cargo receptor NDP52-mediated selective autophagic degradation. Taken together, these findings demonstrate Vangl2 as a suppressor of NF-κB-mediated inflammation and provide insights into the crosstalk between autophagy and inflammatory diseases.
Assuntos
Autofagia , Sepse , Transdução de Sinais , Fator de Transcrição RelA , Animais , Camundongos , Humanos , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Sepse/metabolismo , NF-kappa B/metabolismo , Lipopolissacarídeos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos Endogâmicos C57BL , Ubiquitinação , Proteínas do Tecido Nervoso , Proteínas Adaptadoras de Transdução de Sinal , Proteínas com Domínio LIMRESUMO
Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease.
Assuntos
Doença de Parkinson , Ubiquitina-Proteína Ligases , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/química , Humanos , Doença de Parkinson/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/química , Cristalografia por Raios X , Mutação , Fosforilação , Regulação Alostérica , Mitofagia/efeitos dos fármacos , Ubiquitina/metabolismo , Modelos Moleculares , Ligação Proteica , Células HEK293RESUMO
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovitis, degradation of articular cartilage, and bone destruction. Fibroblast-like synoviocytes (FLS) play a central role in RA, producing a significant amount of inflammatory mediators such as tumor necrosis factor(TNF)-α and IL-6, which promote inflammatory responses within the joints. Moreover, FLS exhibit tumor-like behavior, including aggressive proliferation and enhanced anti-apoptotic capabilities, which collectively drive chronic inflammation and joint damage in RA. TNF is a major pro-inflammatory cytokine that mediates a series of signaling pathways through its receptor TNFR1, including NF-κB and MAPK pathways, which are crucial for inflammation and cell survival in RA. The abnormal proliferation and anti-apoptotic characteristics of FLS in RA may result from dysregulation in TNF-mediated cell death pathways such as apoptosis and necroptosis. Ubiquitination is a critical post-translational modification regulating these signaling pathways. E3 ubiquitin ligases, such as cIAP1/2, promote the ubiquitination and degradation of target proteins within the TNF receptor complex, modulating the signaling proteins. The high expression of the BIRC3 gene and its encoded protein, cIAP2, in RA regulates various cellular processes, including apoptosis, inflammatory signaling, immune response, MAPK signaling, and cell proliferation, thereby promoting FLS survival and inflammatory responses. Inhibiting BIRC3 expression can reduce the secretion of inflammatory cytokines by RA-FLS under both basal and inflammatory conditions and inhibit their proliferation. Although BIRC3 inhibitors show potential in RA treatment, their possible side effects must be carefully considered. Further research into the specific mechanisms of BIRC3, including its roles in cell signaling, apoptosis regulation, and immune evasion, is crucial for identifying new therapeutic targets and strategies.
Assuntos
Artrite Reumatoide , Proteína 3 com Repetições IAP de Baculovírus , Proliferação de Células , Fibroblastos , Transdução de Sinais , Sinoviócitos , Fator de Necrose Tumoral alfa , Humanos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Sinoviócitos/imunologia , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Proteína 3 com Repetições IAP de Baculovírus/genética , Fator de Necrose Tumoral alfa/metabolismo , Fibroblastos/metabolismo , Apoptose , Ubiquitinação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Astrócitos , Encefalomielite Autoimune Experimental , Ribonucleoproteínas , Regulação para Cima , Animais , Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/genética , Camundongos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Ligação a Hormônio da Tireoide , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Transporte Ativo do Núcleo Celular , Feminino , Glicólise , Ubiquitinação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Núcleo Celular/metabolismoRESUMO
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.
Assuntos
Doença de Alzheimer , Ligação Proteica , Ubiquitina-Proteína Ligases , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/química , Fosforilação , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Animais , Células HEK293 , Cristalografia por Raios X , Processamento de Proteína Pós-TraducionalRESUMO
Exposure to fine particulate matter (PM) disrupts the function of airway epithelial barriers causing cellular stress and damage. However, the precise mechanisms underlying PM-induced cellular injury and the associated molecular pathways remain incompletely understood. In this study, we used intratracheal instillation of PM in C57BL6 mice and PM treatment of the BEAS-2B cell line as in vivo and in vitro models, respectively, to simulate PM-induced cellular damage and inflammation. We collected lung tissues and bronchoalveolar lavage fluids to assess histopathological changes, necroptosis, and airway inflammation. Our findings reveal that PM exposure induces necroptosis in mouse airway epithelial cells. Importantly, concurrent administration of a receptor interacting protein kinases 3 (RIPK3) inhibitor or the deletion of the necroptosis effector mixed-lineage kinase domain-like protein (MLKL) effectively attenuated PM-induced airway inflammation. PM exposure dose-dependently induces the expression of Parkin, an E3 ligase we recently reported to play a pivotal role in necroptosis through regulating necrosome formation. Significantly, deletion of endogenous Parkin exacerbates inflammation by enhancing epithelial necroptosis. These results indicate that PM-induced Parkin expression plays a crucial role in suppressing epithelial necroptosis, thereby reducing airway inflammation. Overall, these findings offer valuable mechanistic insights into PM-induced airway injury and identify a potential target for clinical intervention.
Assuntos
Camundongos Endogâmicos C57BL , Necroptose , Material Particulado , Ubiquitina-Proteína Ligases , Necroptose/efeitos dos fármacos , Animais , Material Particulado/toxicidade , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Poluentes Atmosféricos/toxicidade , Células Epiteliais/efeitos dos fármacos , Inflamação , Linhagem CelularRESUMO
BACKGROUND: Sarcopenic obesity, which is associated with a poorer prognosis than that of sarcopenia alone, may be positively affected by soy isoflavones, known inhibitors of muscle atrophy. Herein, we hypothesize that these compounds may prevent sarcopenic obesity by upregulating the gut metabolites with anti-inflammatory effects. METHODS: To explore the effects of soy isoflavones on sarcopenic obesity and its mechanisms, we employed both in vivo and in vitro experiments. Mice were fed a high-fat, high-sucrose diet with or without soy isoflavone supplementation. Additionally, the mouse C2C12 myotube cells were treated with palmitic acid and daidzein in vitro. RESULTS: The isoflavone considerably reduced muscle atrophy and the expression of the muscle atrophy genes in the treated group compared to the control group (Fbxo32, p = 0.0012; Trim63, p < 0.0001; Foxo1, p < 0.0001; Tnfa, p = 0.1343). Elevated levels of daidzein were found in the muscles and feces of the experimental group compared to the control group (feces, p = 0.0122; muscle, p = 0.0020). The real-time PCR results demonstrated that the daidzein decreased the expression of the palmitate-induced inflammation and muscle atrophy genes in the C2C12 myotube cells (Tnfa, p = 0.0201; Il6, p = 0.0008; Fbxo32, p < 0.0001; Hdac4, p = 0.0002; Trim63, p = 0.0114; Foxo1, p < 0.0001). Additionally, it reduced the palmitate-induced protein expression related to the muscle atrophy in the C2C12 myotube cells (Foxo1, p = 0.0078; MuRF1, p = 0.0119). CONCLUSIONS: The daidzein suppressed inflammatory cytokine- and muscle atrophy-related gene expression in the C2C12 myotubes, thereby inhibiting muscle atrophy.
Assuntos
Citocinas , Isoflavonas , Atrofia Muscular , Isoflavonas/farmacologia , Animais , Camundongos , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Masculino , Citocinas/metabolismo , Citocinas/genética , Linhagem Celular , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sarcopenia/prevenção & controle , Sarcopenia/metabolismo , Sarcopenia/tratamento farmacológico , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Dieta Hiperlipídica/efeitos adversos , Obesidade/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Glycine max/química , Modelos Animais de Doenças , Ácido Palmítico/farmacologiaRESUMO
Endoplasmic reticulum (ER) homeostasis in the hypothalamus has been implicated in the pathogenesis of diet-induced obesity (DIO) and type 2 diabetes; however, the underlying molecular mechanism remain vague and debatable. Here we report that SEL1L-HRD1 protein complex of the highly conserved ER-associated protein degradation (ERAD) machinery in POMC-expressing neurons ameliorates diet-induced obesity and its associated complications, partly by regulating the turnover of the long isoform of Leptin receptors (LepRb). Loss of SEL1L in POMC-expressing neurons attenuates leptin signaling and predisposes mice to HFD-associated pathologies including fatty liver, glucose intolerance, insulin and leptin resistance. Mechanistically, nascent LepRb, both wildtype and disease-associated Cys604Ser variant, are misfolding prone and bona fide substrates of SEL1L-HRD1 ERAD. In the absence of SEL1L-HRD1 ERAD, LepRb are largely retained in the ER, in an ER stress-independent manner. This study uncovers an important role of SEL1L-HRD1 ERAD in the pathogenesis of central leptin resistance and leptin signaling.
Assuntos
Dieta Hiperlipídica , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Leptina , Neurônios , Obesidade , Pró-Opiomelanocortina , Receptores para Leptina , Transdução de Sinais , Ubiquitina-Proteína Ligases , Animais , Obesidade/metabolismo , Obesidade/genética , Obesidade/etiologia , Obesidade/patologia , Neurônios/metabolismo , Leptina/metabolismo , Receptores para Leptina/metabolismo , Receptores para Leptina/genética , Camundongos , Pró-Opiomelanocortina/metabolismo , Pró-Opiomelanocortina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Dieta Hiperlipídica/efeitos adversos , Masculino , Retículo Endoplasmático/metabolismo , Hipotálamo/metabolismo , Estresse do Retículo Endoplasmático , Camundongos Endogâmicos C57BL , Proteínas/metabolismo , Proteínas/genética , Camundongos Knockout , Humanos , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Mitochondrial dysfunction is a significant contributor to podocyte injury in diabetic kidney disease (DKD). While previous studies have shown that PVT1 might play a vital role in DKD, the precise molecular mechanisms are largely unknown. By analyzing the plasma and kidney tissues of DKD patients, we observed a significant upregulation of PVT1 expression, which exhibited a positive correlation with albumin/creatinine ratios and serum creatinine levels. Then, we generated mice with podocyte-specific deletion of PVT1 (Nphs2-Cre/Pvt1flox/flox) and confirmed that the deletion of PVT1 suppressed podocyte mitochondrial dysfunction and inflammation in addition to ameliorating diabetes-induced podocyte injury, glomerulopathy, and proteinuria. Subsequently, we cultured podocytes in vitro and observed that PVT1 expression was upregulated under hyperglycemic conditions. Mechanistically, we demonstrated that PVT1 was involved in mitochondrial dysfunction by interacting with TRIM56 post-transcriptionally to modulate the ubiquitination of AMPKα, leading to aberrant mitochondrial biogenesis and fission. Additionally, the release of mtDNA and mtROS from damaged mitochondria triggered inflammation in podocytes. Subsequently, we verified the important role of TRIM56 in vivo by constructing Nphs2-Cre/Trim56flox/flox mice, consistently with the results of Nphs2-Cre/Pvt1flox/flox mice. Together, our results revealed that upregulation of PVT1 could promote mitochondrial dysfunction and inflammation of podocyte by modulating TRIM56, highlighting a potential novel therapeutic target for DKD.
Assuntos
Nefropatias Diabéticas , Mitocôndrias , Podócitos , RNA Longo não Codificante , Podócitos/metabolismo , Podócitos/patologia , Animais , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Mitocôndrias/metabolismo , Camundongos , Humanos , Masculino , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , FemininoRESUMO
Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.
Assuntos
Dano ao DNA , Proteína Quinase Ativada por DNA , Estresse do Retículo Endoplasmático , Ubiquitina-Proteína Ligases , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteína Quinase Ativada por DNA/genética , Retículo Endoplasmático/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/genética , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
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.