RESUMEN

A previous study on ovarian and hypothalami transcriptome analysis in white Muscovy duck revealed that MAP3K8 gene participated in MAPK signaling pathway that influence egg production. Additionally, MAP3K8 was predicted as a target gene of miRNA-509-3p that promotes the secretion of oestradiol which is an important hormone in egg ovulation. This suggested that MAP3K8 might have a functional role in the reproductive performance "egg production" of white Muscovy ducks. Herein, we focused on expression level of MAP3K8 in reproductive and non-reproductive tissues of highest (HP) and lowest (LP) egg producing white Muscovy ducks and identified the polymorphism in MAP3K8 and its association with three egg production traits; Age at first egg (AFE), number of eggs at 300 days (N300D) and 59 weeks (N59W). The results of expression level indicated that mRNA of MAP3K8 was significantly (p < 0.01) expressed in the oviduct than in the ovary and hypothalamus. Seven synonymous SNPs were detected, and association analysis showed that g.148303340 G>A and g.148290065 A>G were significantly (p < 0.05) associated with N300D and N59W. The results of this study might serve as molecular marker for marker-assisted selection of white Muscovy ducks for egg production.

Asunto(s)

Patos , Perfilación de la Expresión Génica , Quinasas Quinasa Quinasa PAM , Ovario , Polimorfismo de Nucleótido Simple , Animales , Patos/genética , Femenino , Ovario/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Hipotálamo/metabolismo , Oviductos/metabolismo

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

Triple-negative breast cancer (TNBC) is difficult to treat breast cancer subtype due to lack or insignificant expressions of targetable estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Therefore, finding a targetable protein or signaling pathway in TNBC would impact patient care. Here, we report that a member of the Mixed Lineage Kinase (MLK) family, MLK3, is an effector of G-protein-coupled protease-activated receptors 1 (PAR1) and targeting MLK3 by a small-molecule inhibitor prevented PAR1-mediated TNBC tumorigenesis. In silico and immunohistochemistry analysis of human breast tumors showed overexpression of PAR1 and MLK3 in TNBC tumors. Treating α-thrombin and PAR1 agonist increased MLK3 and JNK activities and induced cell migration in TNBC cells. The PAR1 positive/high (PAR1+/hi) population of TNBC cells showed aggressive tumor phenotype with increased MLK3 signaling. Moreover, combined inhibition of the PAR1 and MLK3 mitigated the TNBC tumor burden in preclinical TNBC models. Our data suggests that activation of the PAR1-MLK3 axis promotes TNBC tumorigenesis. Therefore, combinatorial therapy targeting MLK3 and PAR1 could effectively reduce TNBC tumor burden.

Asunto(s)

Quinasas Quinasa Quinasa PAM , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno , Receptor PAR-1 , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Humanos , Receptor PAR-1/metabolismo , Receptor PAR-1/genética , Femenino , Animales , Línea Celular Tumoral , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Movimiento Celular , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Carcinogénesis/metabolismo , Carcinogénesis/genética , Ratones , Proliferación Celular

RESUMEN

Pathological cardiac hypertrophy is the primary cause of heart failure, yet its underlying mechanisms remain incompletely understood. Transmembrane protein 100 (TMEM100) plays a role in various disorders, such as nervous system disease, pain and tumorigenesis, but its function in pathological cardiac hypertrophy is still unknown. In this study, we observed that TMEM100 is upregulated in cardiac hypertrophy. Functional investigations have shown that adeno-associated virus 9 (AAV9) mediated-TMEM100 overexpression mice attenuates transverse aortic constriction (TAC)-induced cardiac hypertrophy, including cardiomyocyte enlargement, cardiac fibrosis, and impaired heart structure and function. We subsequently demonstrated that adenoviral TMEM100 (AdTMEM100) mitigates phenylephrine (PE)-induced cardiomyocyte hypertrophy and downregulates the expression of cardiac hypertrophic markers in vitro, whereas TMEM100 knockdown exacerbates cardiomyocyte hypertrophy. The RNA sequences of the AdTMEM100 group and control group revealed that TMEM100 was involved in oxidative stress and the MAPK signaling pathway after PE stimulation. Mechanistically, we revealed that the transmembrane domain of TMEM100 (amino acids 53-75 and 85-107) directly interacts with the C-terminal region of TAK1 (amino acids 1-300) and inhibits the phosphorylation of TAK1 and its downstream molecules JNK and p38. TAK1-binding-defective TMEM100 failed to inhibit the activation of the TAK1-JNK/p38 pathway. Finally, the application of a TAK1 inhibitor (iTAK1) revealed that TAK1 is necessary for TMEM100-mediated cardiac hypertrophy. In summary, TMEM100 protects against pathological cardiac hypertrophy through the TAK1-JNK/p38 pathway and may serve as a promising target for the treatment of cardiac hypertrophy.

Asunto(s)

Cardiomegalia , Quinasas Quinasa Quinasa PAM , Proteínas de la Membrana , Miocitos Cardíacos , Animales , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Endogámicos C57BL , Masculino , Progresión de la Enfermedad , Humanos , Fenilefrina/farmacología , Sistema de Señalización de MAP Quinasas , Estrés Oxidativo

RESUMEN

TNF is a potent cytokine known for its involvement in physiology and pathology. In Rheumatoid Arthritis (RA), persistent TNF signals cause aberrant activation of synovial fibroblasts (SFs), the resident cells crucially involved in the inflammatory and destructive responses of the affected synovial membrane. However, the molecular switches that control the pathogenic activation of SFs remain poorly defined. Cyld is a major component of deubiquitination (DUB) machinery regulating the signaling responses towards survival/inflammation and programmed necrosis that induced by cytokines, growth factors and microbial products. Herein, we follow functional genetic approaches to understand how Cyld affects arthritogenic TNF signaling in SFs. We demonstrate that in spontaneous and induced RA models, SF-Cyld DUB deficiency deteriorates arthritic phenotypes due to increased levels of chemokines, adhesion receptors and bone-degrading enzymes generated by mutant SFs. Mechanistically, Cyld serves to restrict the TNF-induced hyperactivation of SFs by limiting Tak1-mediated signaling, and, therefore, leading to supervised NF-κB and JNK activity. However, Cyld is not critically involved in the regulation of TNF-induced death of SFs. Our results identify SF-Cyld as a regulator of TNF-mediated arthritis and inform the signaling landscape underpinning the SF responses.

Asunto(s)

Artritis Reumatoide , Enzima Desubiquitinante CYLD , Fibroblastos , Quinasa I-kappa B , Quinasas Quinasa Quinasa PAM , Transducción de Señal , Membrana Sinovial , Fibroblastos/metabolismo , Fibroblastos/patología , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Animales , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Ratones , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/genética , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Artritis Reumatoide/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Humanos , FN-kappa B/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

Asunto(s)

Caspasa 8 , Proteína 58 DEAD Box , Enzima Desubiquitinante CYLD , Inmunidad Innata , Virus de la Influenza A , Gripe Humana , Quinasas Quinasa Quinasa PAM , Ubiquitinación , Humanos , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , Caspasa 8/metabolismo , Caspasa 8/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Virus de la Influenza A/inmunología , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Células A549 , Animales , Transducción de Señal/inmunología , Receptores Inmunológicos

RESUMEN

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis of the skin and multiple vital organs, but the immunological pathogenesis of SSc remains unclear. We show here that miR-19b promotes Th9 cells that exacerbate SSc. Specifically, miR-19b and interleukin (IL)-9 increase in CD4+ T cells in experimental SSc in mice induced with bleomycin. Inhibiting miR-19b reduces Th9 cells and ameliorates the disease. Mechanistically, transforming growth factor beta (TGF-ß) plus IL-4 activates pSmad3-Ser213 and TRAF6-K63 ubiquitination by suppressing NLRC3. Activated TRAF6 sequentially promotes TGF-ß-activated kinase 1 (TAK1) and nuclear factor κB (NF-κB) p65 phosphorylation, leading to the upregulation of miR-19b. Notably, miR-19b activated Il9 gene expression by directly suppressing atypical E2F family member E2f8. In patients with SSc, higher levels of IL9 and MIR-19B correlate with worse disease progression. Our findings reveal miR-19b as a key factor in Th9 cell-mediated SSc pathogenesis and should have clinical implications for patients with SSc.

Asunto(s)

Interleucina-9 , MicroARNs , Esclerodermia Sistémica , MicroARNs/metabolismo , MicroARNs/genética , Animales , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/inmunología , Humanos , Ratones , Interleucina-9/metabolismo , Interleucina-9/genética , Ratones Endogámicos C57BL , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor de Crecimiento Transformador beta/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Proteína smad3/metabolismo , Femenino , Interleucina-4/metabolismo , Masculino , Bleomicina , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Transducción de Señal

RESUMEN

OBJECTIVE: Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1). METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB. RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells. CONCLUSION: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.

Asunto(s)

Apoptosis , Glucosa , Quinasas Quinasa Quinasa PAM , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Glucosa/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos no Esterificados/farmacología , Ácidos Grasos no Esterificados/efectos adversos , Proliferación Celular/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/genética , Endopeptidasas/metabolismo , Endopeptidasas/genética , Proteínas Quinasas

RESUMEN

Ovarian tumor domain-containing protease 1 (OTUD1) is a critical negative regulator that promotes innate immune homeostasis and is extensively involved in the pathogenesis of sepsis. In this study, we performed a powerful integration of multiomics analysis and an experimental mechanistic investigation to elucidate the immunoregulatory role of OTUD1 in sepsis at the clinical, animal and cellular levels. Our study revealed the upregulation of OTUD1 expression and the related distinctive alterations observed via multiomics profiling in clinical and experimental sepsis. Importantly, in vivo and in vitro, OTUD1 was shown to negatively regulate inflammatory responses and play a protective role in sepsis-induced pathological lung injury by mechanistically inhibiting the activation of the transforming growth factor-beta-activated kinase 1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in the present study. Subsequently, we probed the molecular mechanisms underlying OTUD1's regulation of NF-κB and MAPK pathways by pinpointing the target proteins that OTUD1 can deubiquitinate. Drawing upon prior research conducted in our laboratory, it has been demonstrated that tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) performs a protective function in septic lung injury and septic encephalopathy by suppressing the NF-κB and MAPK pathways. Hence, we hypothesized that TIPE2 might be a target protein of OTUD1. Additional experiments, including Co-IP, immunofluorescence co-localization, and Western blotting, revealed that OTUD1 indeed has the ability to deubiquitinate TIPE2. In summary, OTUD1 holds potential as an immunoregulatory and inflammatory checkpoint agent, and could serve as a promising therapeutic target for sepsis-induced lung injury.

Asunto(s)

Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Ratones Endogámicos C57BL , FN-kappa B , Sepsis , Proteasas Ubiquitina-Específicas , Animales , Sepsis/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , FN-kappa B/metabolismo , Ratones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Humanos , Proteasas Ubiquitina-Específicas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Transducción de Señal/fisiología , Ubiquitinación , Lesión Pulmonar/metabolismo , Lesión Pulmonar/etiología , Lesión Pulmonar/prevención & control , Sistema de Señalización de MAP Quinasas/fisiología

RESUMEN

Many DNA viruses develop various strategies to inhibit cell death to facilitate their replication. However, whether influenza A virus (IAV), a fast-replicating RNA virus, attenuates cell death remains unknown. Here, we report that IAV infection induces TAK1 phosphorylation in a murine alveolar epithelial cell line (LET1) and a murine fibroblastoma cell line (L929). The TAK1-specific inhibitor 5Z-7-Oxzeneonal (5Z) and TAK1 knockout significantly enhance IAV-induced apoptosis, as evidenced by increased PARP, caspase-8, and caspase-3 cleavage. TAK1 inhibition also increases necroptosis as evidenced by increased RIPK1S166, RIPK3T231/S232, and MLKLS345 phosphorylation. Mechanistically, TAK1 activates IKK, which phosphorylates RIPK1S25 and inhibits its activation. TAK1 also activates p38 and its downstream kinase MK2, which phosphorylates RIPK1S321 but does not affect RIPK1 activation. Further investigation revealed that the RIPK1 inhibitor Nec-1 and RIPK1 knockout abrogate IAV-induced apoptosis and necroptosis; re-expression of wild-type but not kinase-dead (KD)-RIPK1 restores IAV-induced cell death. ZBP1 knockout abrogates IAV-induced cell death, whereas RIPK3 knockout inhibits IAV-induced necroptosis but not apoptosis. 5Z treatment enhances IAV-induced cell death and slightly reduces the inflammatory response in the lungs of H1N1 virus-infected mice and prolongs the survival of IAV-infected mice. Our study provides evidence that IAV activates TAK1 to suppress RIPK1-dependent apoptosis and necroptosis, and that RIPK3 is required for IAV-induced necroptosis but not apoptosis in epithelial cells.

Asunto(s)

Apoptosis , Quinasas Quinasa Quinasa PAM , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Fosforilación , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/patología , Línea Celular , Virus de la Influenza A/fisiología , Ratones Endogámicos C57BL , Humanos

RESUMEN

Mixed-lineage kinase 3 (MLK3) is a serine/threonine kinase of the MAPK Kinase kinase (MAP3K) family that plays critical roles in various biological processes, including cancer. Upon activation, MLK3 differentially activates downstream MAPKs, such as JNK, p38, and ERK. In addition, it regulates various non-canonical signaling pathways, such as ß-catenin, AMPK, Pin1, and PAK1, to regulate cell proliferation, apoptosis, invasion, and metastasis. Recent studies have also uncovered other potentially diverse roles of MLK3 in malignancy, which include metabolic reprogramming, cancer-associated inflammation, and evasion of cancer-related immune surveillance. The role of MLK3 in cancer is complex and cancer-specific, and an understanding of its function at the molecular level aligned specifically with the cancer hallmarks will have profound therapeutic implications for diagnosing and treating MLK3-dependent cancers. This review summarizes the current knowledge about the effect of MLK3 on the hallmarks of cancer, providing insights into its potential as a promising anticancer drug target.

Asunto(s)

Quinasas Quinasa Quinasa PAM , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno , Neoplasias , Humanos , Neoplasias/patología , Neoplasias/genética , Neoplasias/enzimología , Neoplasias/tratamiento farmacológico , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Animales , Apoptosis , Transducción de Señal , Proliferación Celular

RESUMEN

RIPK1/TAK1 are important for programmed cell death, including liver death, necroptosis and apoptosis. However, there have been few published reports on the functions of RIPK1/TAK1 in invertebrates. In this study, full-length ChRIPK1 and ChTAK1 were cloned from C. hongkongensis through the rapid amplification of cDNA ends (RACE) technology. ChRIPK1 has almost no homology with human RIPK1 and lacks a kinase domain at the N-terminus but has a DD and RHIM domain. ChTAK1 is conserved throughout evolution. qRT‒PCR was used to analyze the mRNA expression patterns of ChRIPK1 in different tissues, developmental stages, and V. coralliilyticus-infected individuals, and both were highly expressed in the mantle and gills, while ChRIPK1 was upregulated in hemocytes and gills after V. coralliilyticus or S. aureus infection, which indicates that ChRIPK1 is involved in immune regulation. Fluorescence assays revealed that ChRIPK1 localized to the cytoplasm of HEK293T cells in a punctiform manner, but the colocalization of ChRIPK1 with ChTAK1 abolished the punctiform morphology. In the dual-luciferase reporter assay, both ChRIPK1 and ChRIPK1-RIHM activated the NF-κB signaling pathway in HEK293T cells, and ChTAK1 activated ChRIPK1 in the NF-κB signaling pathway. The apoptosis rate of the hemocytes was not affected by the necroptosis inhibitor Nec-1 but was significantly decreased, and ChRIPK1 expression was knocked down in the hemocytes of C. hongkongensis. These findings indicated that ChRIPK1 induces apoptosis but not necroptosis in oysters. This study provides a theoretical basis for further research on the molecular mechanism by which invertebrates regulate the programmed cell death of hemocytes in oysters.

Asunto(s)

Crassostrea , Necroptosis , Filogenia , Transducción de Señal , Animales , Crassostrea/genética , Crassostrea/inmunología , Necroptosis/inmunología , Transducción de Señal/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Regulación de la Expresión Génica/inmunología , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria , Secuencia de Aminoácidos , Inmunidad Innata/genética , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Staphylococcus aureus/fisiología , Dinoflagelados/fisiología , Dinoflagelados/genética

RESUMEN

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity.

Asunto(s)

Inmunidad Adaptativa , Cíclidos , Enfermedades de los Peces , Proteínas de Peces , Activación de Linfocitos , Quinasas Quinasa Quinasa PAM , Animales , Cíclidos/inmunología , Cíclidos/genética , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Enfermedades de los Peces/inmunología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/veterinaria , Edwardsiella/inmunología , Edwardsiella/fisiología , Regulación de la Expresión Génica/inmunología , Transducción de Señal/inmunología , Perfilación de la Expresión Génica/veterinaria , Filogenia , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos

RESUMEN

Colorectal cancer (CRC) remains a major global cause of cancer-related mortality, lacking effective biomarkers and therapeutic targets. Revealing the critical pathogenic factors of CRC and the underlying mechanisms would offer potential therapeutic strategies for clinical application. G protein signaling (RGS) protein family modulators play essential role within regulating downstream signaling of GPCR receptors, with function in cancers unclear. Our study focused on the expression patterns of RGS proteins in CRC, identifying Regulator of G protein signaling 16 (RGS16) as a prospective diagnostic and therapeutic target. Analyzing 899 CRC tissues revealed elevated RGS16 levels, correlating with clinicopathological features and CRC prognosis by immunohistochemistry (IHC) combined with microarray. We confirmed the elevated RGS16 protein level in CRC, and found that patients with RGS16-high tumors exhibited decreased disease-specific survival (DSS) and disease-free survival (DFS) compared to those with low RGS16 expression. Functional assays demonstrated that RGS16 promoted the CRC progression, knockdown of RGS16 led to significantly increased apoptosis rates of CRC in vitro and in vivo. Notably, we also confirmed these phenotypes of RGS16 in organoids originated from resected primary human CRC tissues. Mechanistically, RGS16 restrained JNK/P38-mediated apoptosis in CRC cells through disrupting the recruitment of TAB2/TAK1 to TRAF6. This study provides insights into addressing the challenges posed by CRC, offering avenues for clinical translation.

Asunto(s)

Apoptosis , Neoplasias Colorrectales , Quinasas Quinasa Quinasa PAM , Proteínas RGS , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas , Ratones Desnudos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas RGS/metabolismo , Proteínas RGS/genética , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/genética

RESUMEN

Recent studies have shown the crucial role of podocyte injury in the development of diabetic kidney disease (DKD). Deubiquitinating modification of proteins is widely involved in the occurrence and development of diseases. Here, we explore the role and regulating mechanism of a deubiquitinating enzyme, OTUD5, in podocyte injury and DKD. RNA-seq analysis indicates a significantly decreased expression of OTUD5 in HG/PA-stimulated podocytes. Podocyte-specific Otud5 knockout exacerbates podocyte injury and DKD in both type 1 and type 2 diabetic mice. Furthermore, AVV9-mediated OTUD5 overexpression in podocytes shows a therapeutic effect against DKD. Mass spectrometry and co-immunoprecipitation experiments reveal an inflammation-regulating protein, TAK1, as the substrate of OTUD5 in podocytes. Mechanistically, OTUD5 deubiquitinates K63-linked TAK1 at the K158 site through its active site C224, which subsequently prevents the phosphorylation of TAK1 and reduces downstream inflammatory responses in podocytes. Our findings show an OTUD5-TAK1 axis in podocyte inflammation and injury and highlight the potential of OTUD5 as a promising therapeutic target for DKD.

Asunto(s)

Nefropatías Diabéticas , Inflamación , Quinasas Quinasa Quinasa PAM , Ratones Noqueados , Podocitos , Ubiquitinación , Animales , Humanos , Masculino , Ratones , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/genética , Células HEK293 , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones Endogámicos C57BL , Fosforilación , Podocitos/metabolismo , Podocitos/patología , Proteasas Ubiquitina-Específicas/metabolismo , Proteasas Ubiquitina-Específicas/genética

RESUMEN

Acute lung injury (ALI), a critical complication observed in various clinical disorders, is characterized by widespread inflammation, neutrophil infiltration, and proinflammatory cytokine production. This study showed that the recently identified non-coding RNA ISIR and its human homolog gene AK131315 played a role in regulating lipopolysaccharide (LPS)-induced inflammatory responses. ISIR and AK131315 increased the production of inflammatory cytokines in LPS-stimulated macrophages, and exogenous ISIR aggravated LPS-induced lung inflammation in an animal model of ALI. Mechanistically, ISIR promoted LPS-triggered NF-κB and MAPK signaling and the transcription of proinflammatory cytokines by enhancing TAK1 activation. Furthermore, a significant correlation was observed between AK131315 expression and pulmonary infectious caused by Gram-negative bacteria, suggesting that AK131315 plays an important role in bacterial infections. Altogether, these findings indicate that ISIR regulates LPS-induced inflammation and AK131315 is involved in the pathogenesis of bacterial infections.

Asunto(s)

Lesión Pulmonar Aguda , Lipopolisacáridos , Quinasas Quinasa Quinasa PAM , FN-kappa B , Lipopolisacáridos/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , FN-kappa B/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Masculino , Citocinas/metabolismo , Citocinas/genética , Células RAW 264.7 , Inflamación/genética , Inflamación/inducido químicamente , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal , Modelos Animales de Enfermedad

RESUMEN

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. Analysis of TCGA databases suggested elevated MAP3K11 (MLK3 gene) expression, and TMA studies showed higher MLK3 activation in human HCCs. To understand MLK3's role in HCC, we utlized carcinogen-induced HCC model and compared between wild-type and MLK3 knockout (MLK3-/-) mice. Our studies showed that MLK3 kinase activity is upregulated in HCC, and MLK3 deficiency alleviates HCC progression. MLK3 deficiency reduced proliferation in vivo and MLK3 inhibition reduced proliferation and colony formation in vitro. To obtain further insight into the mechanism and identify newer targets mediating MLK3-induced HCCs, RNA-sequencing analysis was performed. These showed that MLK3 deficiency modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promoted EMT via autocrine TGFß signaling. Moreover, MLK3 deficiency attenuated activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes HSC activation via paracrine TGFß signaling. These findings reveal TGFß playing a key role at different steps of HCC, downstream of MLK3, implying MLK3-TGFß axis to be an ideal drug target for advanced HCC management.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Quinasas Quinasa Quinasa PAM , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno , Transducción de Señal , Factor de Crecimiento Transformador beta , Animales , Humanos , Masculino , Ratones , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones Noqueados , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Heat stress is becoming the major factor regarding dairy cow health and milk quality because of global warming. Circular RNAs (circRNAs) represent a special type of noncoding RNAs, which are related to regulating many biological processes. Nonetheless, little is known concerning their effects on heat-stressed bovine mammary epithelial cells (BMECs). Here, this study found a novel circRNA, circ_002033, using RNA sequencing (RNA-seq) and explored the role and underlying regulatory mechanism in proliferation, apoptosis, and oxidative damage in a heat-stressed bovine mammary epithelial cell line (MAC-T). According to the previous RNA-seq analysis, the abundance of circ_002033 in mammary gland tissue of heat-stressed cows increased relative to nonheat-stressed counterparts. This study found that the knockdown of circ_002033 promoted proliferation and alleviated apoptosis and oxidative damage in heat-stressed MAC-T. Mechanistically, circ_002033 localizes to miR-199a-5p in the cytoplasm of MAC-T to regulate mitogen-activated protein kinase kinase 11 (MAP3K11) expression. Meanwhile, miR-199a-5p and MAP3K11 are also involved in regulating the proliferation and apoptosis of heat-stressed MAC-T. Importantly, circ_002033 knockdown promoted the expression of miR-199a-5p while decreasing that of MAP3K11, thereby enhancing proliferation while alleviating apoptosis and oxidative damage in heat-stressed MAC-T. In summary, we found that circ_002033 regulates the proliferation, apoptosis, and oxidative damage of heat-stressed BMECs through the miR-199a-5p/MAP3K11 axis, providing the theoretical molecular foundation for mitigating heat stress of dairy cows.

Asunto(s)

Apoptosis , Proliferación Celular , Células Epiteliales , Respuesta al Choque Térmico , Quinasas Quinasa Quinasa PAM , Glándulas Mamarias Animales , MicroARNs , Estrés Oxidativo , ARN Circular , Animales , Bovinos , Células Epiteliales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/citología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno , Línea Celular

RESUMEN

Allostery is a fundamental mechanism driving biomolecular processes that holds significant therapeutic concern. Our study rigorously investigates how two distinct machine-learning algorithms uniquely classify two already close-to-active DFG-in states of TAK1, differing just by the presence or absence of its allosteric activator TAB1, from an ensemble mixture of conformations (obtained from 2.4 µs molecular dynamics (MD) simulations). The novelty, however, lies in understanding the deeper algorithmic potentials to systematically derive a diverse set of differential residue connectivity features that reconstruct the essential mechanistic architecture for TAK1-TAB1 allostery in such a close-to-active biochemical scenario. While the recursive, random forest-based workflow displays the potential of conducting discretized, hierarchical derivation of allosteric features, a multilayer perceptron-based approach gains considerable efficacy in revealing fluid connected patterns of features when hybridized with mutual information scoring. Interestingly, both pipelines benchmark similar directions of functional conformational changes for TAK1's activation. The findings significantly advance the depth of mechanistic understanding by highlighting crucial activation signatures along a directed C-lobe → activation loop → ATP pocket channel of information flow, including (1) the αF-αE biterminal alignments and (2) the "catalytic" drift of the activation loop toward kinase active site. Besides, some novel allosteric hotspots (K253, Y206, N189, etc.) are further recognized as TAB1 sensors, transducers, and responders, including a benchmark E70 mutation site, precisely mapping the important structural segments for sequential allosteric execution. Hence, our work demonstrates how to navigate through greater structural depths and dimensions of dynamic allosteric machineries just by leveraging standard ML methods in suitable streamlined workflows adaptive to the specific system and objectives.

Asunto(s)

Quinasas Quinasa Quinasa PAM , Aprendizaje Automático , Simulación de Dinámica Molecular , Regulación Alostérica , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Humanos , Conformación Proteica , Flujo de Trabajo , Algoritmos

RESUMEN

BACKGROUND: The innate immune system serves as the first line of host defense. Transforming growth factor-ß-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. RESULTS: In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. CONCLUSIONS: Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

Asunto(s)

Fosfoproteínas Fosfatasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Humanos , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Necroptosis , Inmunidad Innata