RESUMEN
Overactivation of the NLRP3 inflammasomes induces production of pro-inflammatory cytokines and drives pathological processes. Pharmacological inhibition of NLRP3 is an explicit strategy for the treatment of inflammatory diseases. Thus far no drug specifically targeting NLRP3 has been approved by the FDA for clinical use. This study was aimed to discover novel NLRP3 inhibitors that could suppress NLRP3-mediated pyroptosis. We screened 95 natural products from our in-house library for their inhibitory activity on IL-1ß secretion in LPS + ATP-challenged BMDMs, found that Britannin exerted the most potent inhibitory effect with an IC50 value of 3.630 µM. We showed that Britannin (1, 5, 10 µM) dose-dependently inhibited secretion of the cleaved Caspase-1 (p20) and the mature IL-1ß, and suppressed NLRP3-mediated pyroptosis in both murine and human macrophages. We demonstrated that Britannin specifically inhibited the activation step of NLRP3 inflammasome in BMDMs via interrupting the assembly step, especially the interaction between NLRP3 and NEK7. We revealed that Britannin directly bound to NLRP3 NACHT domain at Arg335 and Gly271. Moreover, Britannin suppressed NLRP3 activation in an ATPase-independent way, suggesting it as a lead compound for design and development of novel NLRP3 inhibitors. In mouse models of MSU-induced gouty arthritis and LPS-induced acute lung injury (ALI), administration of Britannin (20 mg/kg, i.p.) significantly alleviated NLRP3-mediated inflammation; the therapeutic effects of Britannin were dismissed by NLRP3 knockout. In conclusion, Britannin is an effective natural NLRP3 inhibitor and a potential lead compound for the development of drugs targeting NLRP3.
Asunto(s)
Inflamasomas , Lactonas , Proteína con Dominio Pirina 3 de la Familia NLR , Sesquiterpenos , Animales , Humanos , Ratones , Inflamasomas/agonistas , Interleucina-1beta/metabolismo , Lactonas/farmacología , Lactonas/uso terapéutico , Lipopolisacáridos/farmacología , Macrófagos , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Sesquiterpenos/farmacología , Sesquiterpenos/uso terapéuticoRESUMEN
Heart failure represents a major cause of death worldwide. Recent research has emphasized the potential role of protein ubiquitination/deubiquitination protein modification in cardiac pathology. Here, we investigate the role of the ovarian tumor deubiquitinase 1 (OTUD1) in isoprenaline (ISO)- and myocardial infarction (MI)-induced heart failure and its molecular mechanism. OTUD1 protein levels were raised markedly in murine cardiomyocytes after MI and ISO treatment. OTUD1 deficiency attenuated myocardial hypertrophy and cardiac dysfunction induced by ISO infusion or MI operation. In vitro, OTUD1 knockdown in neonatal rat ventricular myocytes (NRVMs) attenuated ISO-induced injuries, while OTUD1 overexpression aggravated the pathological changes. Mechanistically, LC-MS/MS and Co-IP studies showed that OTUD1 bound directly to the GAF1 and PDEase domains of PDE5A. OTUD1 was found to reverse K48 ubiquitin chain in PDE5A through cysteine at position 320 of OTUD1, preventing its proteasomal degradation. PDE5A could inactivates the cGMP-PKG-SERCA2a signaling axis which dysregulate the calcium handling in cardiomyocytes, and leading to the cardiomyocyte injuries. In conclusion, OTUD1 promotes heart failure by deubiquitinating and stabilizing PDE5A in cardiomyocytes. These findings have identified PDE5A as a new target of OTUD1 and emphasize the potential of OTUD1 as a target for treating heart failure.
Asunto(s)
Insuficiencia Cardíaca , Infarto del Miocardio , Ratones , Ratas , Animales , Isoproterenol/farmacología , Miocitos Cardíacos/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Insuficiencia Cardíaca/metabolismo , Infarto del Miocardio/metabolismoRESUMEN
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/genéticaRESUMEN
BACKGROUND: Cardiac hypertrophy is a crucial pathological characteristic of hypertensive heart disease and subsequent heart failure. Deubiquitinating enzymes (DUBs) have been found to be involved in the regulation of myocardial hypertrophy. OTU Domain-Containing Protein 6a (OTUD6a) is a recently identified DUB. To date, the potential role of OTUD6a in myocardial hypertrophy has not yet been revealed. METHODS AND RESULTS: We examined the up-regulated level of OTUD6a in mouse or human hypertrophic heart tissues. Then, transverse aortic constriction (TAC)- or angiotensin II (Ang II)- induced ventricular hypertrophy and dysfunction were significantly attenuated in OTUD6a gene knockout mice (OTUD6a-/-). In mechanism, we identified that the Stimulator of Interferon Genes (STING) is a direct substrate protein of OTUD6a via immunoprecipitation assay and mass spectrometry. OTUD6a maintains STING stability via clearing the K48-linked ubiquitin in cardiomyocytes. Subsequently, OTUD6a regulates the STING-downstream NF-κB signaling activation and inflammatory gene expression both in vivo and in vitro. Inhibition of STING blocked OTUD6a overexpression-induced inflammatory and hypertrophic responses in cardiomyocytes. CONCLUSION: This finding extends our understanding of the detrimental role of OTUD6a in myocardial hypertrophy and identifies STING as a deubiquinating substrate of OTUD6a, indicating that targeting OTUD6a could be a potential strategy for the treatment of cardiac hypertrophy.
Asunto(s)
Cardiomegalia , Insuficiencia Cardíaca , Animales , Humanos , Ratones , Cardiomegalia/metabolismo , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Insuficiencia Cardíaca/metabolismo , Inflamación/metabolismo , Ratones Noqueados , Miocitos Cardíacos/metabolismoRESUMEN
BACKGROUND: Chronic inflammation contributes to the progression of isoproterenol (ISO)-induced heart failure (HF). Caspase-associated recruitment domain (CARD) families are crucial proteins for initiation of inflammation in innate immunity. Nonetheless, the relevance of CARDs in ISO-driven cardiac remodelling is little explored. METHODS: This study utilized Card9-/- mice and reconstituted C57BL/6 mice with either Card9-/- or Otud1-/- marrow-derived cells. Mechanistic studies were conducted in primary macrophages, cardiomyocytes, fibroblasts and HEK-293T cells. RESULTS: Here, we demonstrated that CARD9 was substantially upregulated in murine hearts infused with ISO. Either whole-body CARD9 knockout or myeloid-specific CARD9 deletion inhibited ISO-driven murine cardiac inflammation, remodelling and dysfunction. CARD9 deficiency in macrophages prevented ISO-induced inflammation and alleviated remodelling changes in cardiomyocytes and fibroblasts. Mechanistically, we found that ISO enhances the activity of CARD9 by upregulating ovarian tumour deubiquitinase 1 (OTUD1) in macrophages. We further demonstrated that OTUD1 directly binds to the CARD9 and then removes the K33-linked ubiquitin from CARD9 to promote the assembly of the CARD9-BCL10-MALT1 (CBM) complex, without affecting CARD9 stability. The ISO-activated CBM complex results in NF-κB activation and macrophage-based inflammatory gene overproduction, which then enhances cardiomyocyte hypertrophy and fibroblast fibrosis, respectively. Myeloid-specific OTUD1 deletion also attenuated ISO-induced murine cardiac inflammation and remodelling. CONCLUSIONS: These results suggested that the OTUD1-CARD9 axis is a new pro-inflammatory signal in ISO-challenged macrophages and targeting this axis has a protective effect against ISO-induced HF. KEY POINTS: Macrophage CARD9 was elevated in heart tissues of mice under chronic ISO administration. Either whole-body CARD9 knockout or myeloid-specific CARD9 deficiency protected mice from ISO-induced inflammatory heart remodeling. ISO promoted the assembly of CBM complex and then activated NF-κB signaling in macrophages through OTUD1-mediated deubiquitinating modification. OTUD1 deletion in myeloid cells protected hearts from ISO-induced injuries in mice.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Isoproterenol , Macrófagos , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Ratones , Macrófagos/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , Ratones Endogámicos C57BL , Humanos , Inflamación/metabolismo , Inflamación/genética , Inflamación/inducido químicamente , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Remodelación Ventricular , Modelos Animales de EnfermedadRESUMEN
Dementia is a global health issue and the effects on caregivers are substantial. The study aimed to examine the associations of burden, coping, self-efficacy with quality of life among family caregivers of persons with dementia in Singapore. Structured interviews were conducted in a convenience sample of 84 family caregivers caring and seeking clinical care for the persons with dementia in an outpatient clinic of a public hospital in Singapore. The outcome measures included the Family Burden Interview Schedule, Family Crisis Oriented Personal Evaluation Scale, General Perceived Self-Efficacy Scale, and World Health Organization Quality of Life Scale - Brief Version. In general, significant correlations were observed between the quality of life scores with coping strategy and family burden scores, but not between the coping strategy and family burden scores. Compared to demographic factors such as caregiver age and household income, psychosocial factors including family burden, coping strategies, and self-efficacy demonstrated greater association with quality of life in the participants. However, the dynamics of these associations will change with an increasing population of persons with dementia, decreasing nuclear family size, and predicted changes in family living arrangements for the persons with dementia in future. As such, it necessitates continuous study examining the needs and concerns of family caregivers and the relevance of ongoing interventions specific to caregivers of persons with dementia.