RESUMEN
p62 bodies are ubiquitin-positive cytoplasmic condensates formed by liquid-liquid phase separation. They are targeted by selective autophagy and play important roles in intracellular quality control and stress responses. However, little is known about their constituents. In this chapter, we describe a method for purifying p62 bodies using fluorescence-activated particle sorting. This method contributes to the identification of novel components of p62 bodies under various physiological and stress conditions.
Asunto(s)
Autofagia , Citometría de Flujo , Humanos , Citometría de Flujo/métodos , Ubiquitina/metabolismo , Proteína Sequestosoma-1/metabolismoRESUMEN
OBJECTIVE: Cartilage degeneration is a key feature of osteoarthritis (OA) and rheumatoid arthritis and is thought to negatively impact patients' quality of life. Diclofenac etalhyaluronate (DEH, SI-613/ONO-5704) is a hyaluronic acid (HA) derivative chemically bound to diclofenac (DF) that has been reported to improve OA symptoms; however, its effect on cartilage degeneration remains unknown. In the present study, we investigated the chondroprotective effect of DEH in rats with collagen-induced arthritis and interleukin-1ß-stimulated human chondrocytes. DESIGN: Rats with collagen-induced arthritis were administered DEH and HA intra-articularly, and DF orally. Knee joint swelling, histological scores of articular cartilage, and inflammatory (Il1b) and catabolic (Mmp3 and Mmp13) gene expression in the synovial tissue and cartilage were evaluated. In vitro direct effects of DEH on matrix metalloproteinase (MMP)-3 and MMP-13 expression were examined in interleukin-1ß-stimulated human chondrocytes. RESULTS: In a rat model of collagen-induced arthritis, a single intra-articular dose of DEH inhibited knee joint inflammation and cartilage degeneration. Daily oral administration of DF had similar effects. Conversely, HA administered as a single intra-articular dose had no effect. Only DEH inhibited Mmp3 gene expression in the cartilage, whereas DEH and DF inhibited Mmp3 and Mmp13 mRNA expression in the synovial tissue. In interleukin-1ß-stimulated human chondrocytes, DEH and HA inhibited MMP-3 and MMP-13 production, whereas DF had no effect. CONCLUSIONS: In this study, we demonstrated the chondroprotective effect of DEH in rats with collagen-induced arthritis and in interleukin-1ß-stimulated human chondrocytes. Thus, DEH may suppress cartilage degeneration in patients with musculoskeletal diseases, such as OA.
RESUMEN
OBJECTIVE: To investigate mechanisms of anaphylaxis in patients with osteoarthritis (OA) of knee and hip after diclofenac etalhyaluronate (product name: JOYCLU® [JCL]) intra-articular injection, and to determine the utility of tests to investigate the mechanism involved. METHODS: In this observational study in Japan, patients aged ≥20 years with knee or hip OA who received JCL intra-articular injection, experienced anaphylactic symptoms considered related to JCL ("experienced patients") or did not experience allergic symptoms considered related to JCL ("non-experienced patients"). Basophil activation tests (BAT), specific immunoglobulin E (IgE) antibody testing by enzyme-linked immunosorbent assays (ELISA) or immunochromatographic kit, and genome-wide association studies (GWAS) were conducted using patient blood and saliva. RESULTS: Thirteen experienced patients and 14 non-experienced patients were tested. Seven experienced patients tested positive by BAT using diclofenac etalhyaluronate (DEH)-containing test substances. Diclofenac (DF)-specific IgE antibodies were detected in 4 of 7 BAT-positive patients, but not in the non-experienced patients. Specific IgE antibody testing by immunochromatographic kit and GWAS showed no clear results. CONCLUSIONS: These findings suggest that anaphylaxis occurs after JCL administration via an IgE-mediated mechanism and that DEH may be involved in this mechanism. BAT and DF-specific IgE ELISA may be useful tests for investigating the mechanisms of anaphylactic reactions after JCL administration.
RESUMEN
NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Humanos , Animales , Ratones , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Fosforilación , Proteína Sequestosoma-1/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.
Asunto(s)
Neoplasias Hepáticas , Proteómica , Animales , Humanos , Ratones , Proteína Sequestosoma-1/metabolismo , Autofagia , Orgánulos/metabolismoRESUMEN
Acetaminophen-induced liver injury in mice is a model system of human acetaminophen overdose and oxidative stress in vivo. The system is technically established, and we usually obtain severe liver damage in the treated mice; however, it is possible that the degree of liver damage is affected by the type of chow fed to mice. Thus, in this experiment, we investigated the effect of different chows on mice by comparing acetaminophen-induced liver damage, liver antioxidant level, and serum amino-acid concentrations. The results showed that differences in chows, even standard ones, affected mouse physiological conditions, with the response to oxidative stress greatly affected.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatopatías , Enfermedades de los Roedores , Acetaminofén/toxicidad , Animales , Antioxidantes/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/veterinaria , Glutatión , Hígado/metabolismo , Hepatopatías/tratamiento farmacológico , Hepatopatías/veterinaria , Ratones , Ratones Endogámicos C57BL , Estrés OxidativoRESUMEN
Karyolysis is the complete dissolution of nuclear components of a dying cell. However, the generation mechanism has not been clarified. We studied a necrotic DNA fragmentation factor DNase γ (also known as DNase1L3) and previously found that karyolysis was inhibited in DNase γ deficient (DNase γ-/-) mice. To confirm this, we transiently expressed DNase γ in the liver of DNase γ-/- mice and caused hepatocyte necrosis by acetaminophen overdose. As expected, karyolysis was induced in the necrotic hepatocytes. We also found that the depletion of Kupffer cells from wild type mice reduced the expression and activity of DNase γ in the liver. Thus, we concluded that DNase γ produced from Kupffer cells caused karyolysis of necrotic hepatocytes.
Asunto(s)
Muerte Celular , Fragmentación del ADN , Hepatocitos/patología , Acetaminofén/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Endodesoxirribonucleasas , Macrófagos del Hígado/metabolismo , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Cell-free DNA (cfDNA) (e.g. fetal- or tumor-derived DNA) is DNA found in the blood circulation. It is now widely investigated as a biomarker for prenatal screening, tumor diagnosis, and tumor monitoring as "liquid biopsies". However, the biological and biochemical aspects of cfDNA remain unclear. Although cfDNA is considered to be mainly derived from dead cells, information is scarce as to whether it is apoptotic or necrotic and what kinds of endonucleases or DNases are involved. We induced in vivo hepatocyte necrosis and apoptosis in mice deficient in DNase1L3 (also named DNase γ) and/or caspase-activated DNase (CAD) genes with acetaminophen overdose and anti-Fas antibody treatments. We found that (i) DNase1L3 was the endonuclease responsible for generating cfDNA in acetaminophen-induced hepatocyte necrosis and (ii) CAD and DNase1L3 cooperated in producing cfDNA for anti-Fas mediated hepatocyte apoptosis.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Desoxirribonucleasas/genética , Endodesoxirribonucleasas/genética , Necrosis/genética , Receptor fas/genética , Acetaminofén/administración & dosificación , Animales , Anticuerpos Neutralizantes/farmacología , Ácidos Nucleicos Libres de Células/sangre , Desoxirribonucleasas/sangre , Endodesoxirribonucleasas/sangre , Trampas Extracelulares/metabolismo , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis/sangre , Necrosis/inducido químicamente , Necrosis/patología , Transducción de Señal , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
Sialic acids (Sias) are often conjugated to the termini of cellular glycans and are key mediators of cellular recognition. Sias are nine-carbon acidic sugars, and, in vertebrates, the major species are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), differing in structure at the C5 position. Previously, we described a positive feedback loop involving regulation of Neu5Gc expression in mouse B cells. In this context, Neu5Gc negatively regulated B-cell proliferation, and Neu5Gc expression was suppressed upon activation. Similarly, resting mouse T cells expressed principally Neu5Gc, and Neu5Ac was induced upon activation. In the present work, we used various probes to examine sialoglycan expression by activated T cells in terms of the Sia species expressed and the linkages of Sias to glycans. Upon T-cell activation, sialoglycan expression shifted from Neu5Gc to Neu5Ac, and the linkage shifted from α2,6 to α2,3. These changes altered the expression levels of sialic acid-binding immunoglobulin-like lectin (siglec) ligands. Expression of sialoadhesin and Siglec-F ligands increased, and that of CD22 ligands decreased. Neu5Gc exerted a negative effect on T-cell activation, both in terms of the proliferative response and in the context of activation marker expression. Suppression of Neu5Gc expression in mouse T and B cells prevented the development of nonspecific CD22-mediated T cell-B cell interactions. Our results suggest that an activation-dependent shift from Neu5Gc to Neu5Ac and replacement of α2,6 by α2,3 linkages may regulate immune cell interactions at several levels.