RESUMO
The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.
Assuntos
Fatores de Transcrição Kruppel-Like , Proteína C , Animais , Camundongos , Proteína C/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Diferenciação Celular/genética , Transporte Proteico , Células Eritroides/metabolismoRESUMO
Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.
Assuntos
Eritropoese/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Sequência Conservada , Células Precursoras Eritroides/citologia , Técnicas de Silenciamento de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/química , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Transplante Heterólogo , Ubiquitinação , Regulação para CimaRESUMO
The nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome is involved in various acute and chronic liver diseases, however, it is not clear whether NLRP3 contributes to d-Galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced acute liver failure (ALF). This study aims to investigate the role of NLRP3 inflammasome in D-GalN/LPS-induced fatal hepatitis. We found that Nlrp3-/- and WT mice showed similar mortality against a lethal dose of D-GalN/LPS treatment. Serum ALT and AST levels, as well as liver necrosis area and hepatocyte apoptosis, were not significantly different between Nlrp3-/- and WT mice at 6 h after D-GalN/LPS injection. Moreover, the numbers of intrahepatic F4/80+ cells and Ly6G+ cells were comparable in two genotype mice following D-GalN/LPS treatment. Besides, Nlrp3-/- mice had reduced IL-1ß levels but similar TNF-α, IL-6, and MCP-1 levels compared with WT mice upon D-GalN/LPS administration. Our findings revealed that NLRP3 ablation does not protect mice from D-GalN/LPS-induced fatal hepatitis and has a marginal effect on intrahepatic inflammatory response upon D-GalN/LPS treatment. This suggests that NLRP3 inflammasome does not appear to be a major contributor to D-GalN/LPS-induced ALF.
Assuntos
Falência Hepática Aguda/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Galactosamina , Inflamassomos/metabolismo , Inflamassomos/fisiologia , Interleucina-1beta/sangue , Lipopolissacarídeos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/imunologia , Falência Hepática Aguda/metabolismo , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Necrose Tumoral alfa/sangueRESUMO
Salmonids can be co-infected by infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) under natural or experimental conditions. To reveal the influence of IPNV on IHNV in co-infections, CHSE-214 cells were inoculated with IPNV at different time intervals prior to or after IHNV infection. Propagation of IHNV was determined by an immunofluorescence antibody test, real-time quantitative polymerase chain reaction, flow cytometry, and virus titration. The results showed that when cells were inoculated with IPNV prior to IHNV, IHNV multiplication was inhibited. This inhibitory effect became stronger with increasing time intervals (P < 0.05). When cells were inoculated with IPNV after IHNV, the inhibitory effect became weaker with increasing time intervals (P < 0.05), and no significant inhibition was observed at 12 h (P > 0.05) compared with the single IHNV infection group. The findings suggest that IHNV is inhibited at the early stage of infection by IPNV and in a time dependent manner during co-infection. Furthermore, the effect of IPNV on IHNV entry and expression of IHNV entry-related genes clathrin, dynamin-2, adaptor protein 2, and vacuolar protein sorting 35 were also determined. The results showed that IPNV did not affect the amount of IHNV entering the cells. However, the expression levels of clathrin and dynamin-2 were significantly lower in co-infection than those in single IHNV infection, which suggests that IPNV likely inhibits IHNV by affecting IHNV invasion via downregulating IHNV entry-related genes clathrin and dynamin-2.
Assuntos
Infecções por Birnaviridae/veterinária , Coinfecção/veterinária , Doenças dos Peixes/imunologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Infecções por Rhabdoviridae/veterinária , Salmão , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Coinfecção/imunologia , Coinfecção/virologia , Regulação para Baixo , Embrião não Mamífero , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologiaRESUMO
Based on extensive global-minimum searches and first-principles electronic structure calculations, we present the viability of an endohedral metalloborospherene Cs Ca@B38 () which contains a Cs B38(2-) () dianion composed of interwoven boron double chains with a σ + π double delocalization bonding pattern, extending the Bn(q) (q = n - 40) borospherene family from n = 39-42 to n = 38. Transition metal endohedral complexes Cs M@B38 (M = Sc, Y, Ti) (, , ) based on Cs B38(2-) () are also predicted.
RESUMO
The diagonal relationship between beryllium and aluminum and the isoelectronic relationship between BeH unit and Al atom were utilized to design a new series ppC- or quasi-ppC-containing species C5v CBe5H5(+), Cs CBe5H4, C2v CBe5H3(-), and C2v CBe5H2(2-) by replacing the Al atoms in previously reported global minima planar pentacoordinate carbon (ppC) species D5h CAl5(+), C2v CAl4Be, C2v CAl3Be2(-), and C2v CAl2Be3(2-) with BeH units. The three-center two-electron (3c-2e) bonds formed between Be and bridging H atoms were crucial for the stabilization of these ppC species. The natural bond orbital (NBO) and adaptive natural density partitioning (AdNDP) analyses revealed that the central ppCs or quasi-ppCs possess the stable eight electron-shell structures. The AdNDP analyses also disclosed that these species are all 6σ+2π double-aromatic in nature. The aromaticity was proved by the calculated negative nucleus-independent chemical shifts (NICS) values. DFT and high-level CCSD(T) calculations revealed that these ppC- or quasi-ppC species are the global minimum or competitive low-lying local minimum (Cs CBe5H4) on their potential energy surfaces. The Born-Oppenheimer molecular dynamic (BOMD) simulations revealed that the H atoms in C2v CBe5H3(-) and C2v CBe5H2(2-) can easily rotate around the CBe5 cores and the structure of quasi-planar C5v CBe5H5(+) will become the planar structure at room temperature; however, these interesting dynamic behaviors did not indicate the kinetic instability as the basic ppC structures were maintained during the simulations. Therefore, it would be potentially possible to realize these interesting ppC- or quasi-ppc-species in future experiments.
RESUMO
Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro, which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na+ concentration and inversely proportional to extracellular K+ concentration, and bufalin may inhibit IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.
Assuntos
Bufanolídeos , Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Oncorhynchus mykiss , Vacinas de DNA , Animais , Vírus da Necrose Hematopoética Infecciosa/genética , Medicina Tradicional Chinesa , Antivirais/farmacologia , Antivirais/uso terapêutico , Adenosina Trifosfatases , Necrose , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/prevenção & controleRESUMO
G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.
Assuntos
Diferenciação Celular , Eritropoese , Hemina , Leucemia Eritroblástica Aguda , Correpressor 1 de Receptor Nuclear , Humanos , Células K562 , Eritropoese/genética , Leucemia Eritroblástica Aguda/patologia , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 1 de Receptor Nuclear/genética , Hemina/farmacologia , Células Eritroides/metabolismo , Células Eritroides/citologia , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Hemoglobinas/metabolismo , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1ß, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Animais , Camundongos , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Células de Kupffer/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fígado/metabolismo , Inflamação/metabolismo , Galactosamina , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismoRESUMO
DDX3, a member of the DEAD-box RNA helicase family and has highly conserved ATP-dependent RNA helicase activity, has important roles in RNA metabolism and innate anti-viral immune responses. In this study, five transcript variants of the DDX3 gene were cloned and characterized from rainbow trout (Oncorhynchus mykiss). These five transcript variants of DDX3 encoded proteins were 74.2 kDa (686 aa), 76.4 kDa (709 aa), 77.8 kDa (711 aa), 78.0 kDa (718 aa), and 78.8 kDa (729 aa) and the predicted isoelectric points were 6.91, 7.63, 7.63, 7.18, and 7.23, respectively. All rainbow trout DDX3 proteins contained two conserved RecA-like domains that were similar to the DDX3 protein reported in mammals. Phylogenetic analysis showed that the five cloned rainbow trout DDX3 were separate from mammals but clustered with fish, especially Northern pike (Esox lucius) and Nile tilapia (Oreochromis niloticus). RT-qPCR analysis showed that the DDX3 gene was broadly expressed in all tissues studied. The expression of DDX3 after infectious hematopoietic necrosis virus (IHNV) infection increased gradually after the early stage of IHNV infection, decreased gradually with the proliferation of IHNV in vivo (liver, spleen, and kidney), and was significantly decreased after the in vitro infection of epithelioma papulosum cyprini (EPC) and rainbow trout gonad cell line-2 (RTG-2) cell lines. We also found that rainbow trout DDX3 was significantly increased by a time-dependent mechanism after the poly I:C treatment of EPC and RTG cells; however no significant changes were observed with lipopolysaccharide (LPS) treatment. Knockdown of DDX3 by siRNA showed significantly increased IHNV replication in infected RTG cells. This study suggests that DDX3 has an important role in host defense against IHNV infection and these results may provide new insights into IHNV pathogenesis and antiviral drug research.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Antivirais , RNA Helicases DEAD-box/genética , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Mamíferos , Filogenia , Proteínas/genéticaRESUMO
Infectious hematopoietic necrosis virus (IHNV) is the causative pathogen of infectious hematopoietic necrosis, outbreaks of which are responsible for significant losses in rainbow trout aquaculture. Strains of IHNV isolated worldwide have been classified into five major genogroups, J, E, L, M, and U. To date, comparative transcriptomic analysis has only been conducted individually for the J and M genogroups. In this study, we compared the transcriptome profiles in U genogroup and J genogroup IHNV-infected RTG-2 cells with mock-infected RTG-2 cells. The RNA-seq results revealed 17,064 new genes, of which 7,390 genes were functionally annotated. Differentially expressed gene (DEG) analysis between U and J IHNV-infected cells revealed 2,238 DEGs, including 1,011 downregulated genes and 1,227 upregulated genes. Among the 2,238 DEGs, 345 new genes were discovered. The DEGs related to immune responses, cellular signal transduction, and viral diseases were further analyzed. RT-qPCR validation confirmed that the changes in expression of the immune response-related genes trpm2, sting, itgb7, ripk2, and irf1, cellular signal transduction-related genes irl, cacnb2, bmp2l, gadd45α, and plk2, and viral disease-related genes mlf1, mtor, armc5, pik3r1, and c-myc were consistent with the results of transcriptome analysis. Taken together, our findings provide a comprehensive transcriptional analysis of the differential virulence of the U and J genogroups of IHNV, and shed new light on the pathogenic mechanisms of IHNV strains.
RESUMO
BACKGROUND: Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aimed to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration. METHODS: We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice, as a model of liver regeneration. Bacterial flagellin content was measured with ELISA, and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, we analyzed bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression with immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels, and with Western blotting analysis of hepatic NF-κB and STAT3 activation. Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. RESULTS: The bacterial flagellin content in the serum and liver increased, and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx. TLR5-deficient mice exhibited diminished numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokine and growth factor production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5-/- mice, as compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation, which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Furthermore, Tlr5-/- mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx. CONCLUSION: We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.
Assuntos
Regeneração Hepática/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor 5 Toll-Like/uso terapêutico , Animais , Modelos Animais de Doenças , Regeneração Hepática/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Estatísticas não Paramétricas , Receptor 5 Toll-Like/metabolismoRESUMO
BRCA1/BRCA2-containing complex subunit 3 (BRCC3) is a lysine 63-specific deubiquitinase involved in multiple biological processes, such as DNA repair and immune responses. However, the regulation mechanism for BRCC3 protein stability is still unknown. Here, we demonstrate that BRCC3 is mainly degraded through the ubiquitin-proteasome pathway. The HECT-type E3 ubiquitin ligase WWP2 modulates BRCC3 ubiquitination and degradation. ABRO1, a subunit of the BRCC36 isopeptidase complex (BRISC), competes with WWP2 to bind to BRCC3, thereby preventing WWP2-mediated BRCC3 ubiquitination and enhancing BRCC3 stability. Functionally, we show that lentivirus-mediated overexpression of WWP2 in murine macrophages inhibits NLRP3 inflammasome activation by decreasing BRCC3 protein level. This study provides the first insights into the regulation of BRCC3 stability and expands our knowledge about the physiological function of WWP2.
Assuntos
Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Enzimas Desubiquitinantes/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteínas Associadas à Matriz Nuclear/genética , Estabilidade Proteica , Proteólise , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
Pharmacologically inhibiting nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome activation results in potent therapeutic effects in a wide variety of preclinical inflammatory disease models. NLRP3 deubiquitination is essential for efficient NLRP3 inflammasome activity, but it remains unclear whether this process can be harnessed for therapeutic benefit. Here, we show that thiolutin (THL), an inhibitor of the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease, blocks NLRP3 inflammasome activation by canonical, noncanonical, alternative, and transcription-independent pathways at nanomolar concentrations. In addition, THL potently inhibited the activation of multiple NLRP3 mutants linked with cryopyrin-associated periodic syndromes (CAPS). Treatment with THL alleviated NLRP3-related diseases in mouse models of lipopolysaccharide-induced sepsis, monosodium urate-induced peritonitis, experimental autoimmune encephalomyelitis, CAPS, and methionine-choline-deficient diet-induced nonalcoholic fatty liver disease. Mechanistic studies revealed that THL inhibits the BRCC3-containing isopeptidase complex (BRISC)-mediated NLRP3 deubiquitination and activation. In addition, we show that holomycin, a natural methyl derivative of THL, displays an even higher inhibitory activity against NLRP3 inflammasome than THL. Our study validates that posttranslational modification of NLRP3 can be pharmacologically targeted to prevent or treat NLRP3-associated inflammatory diseases. Future clinical development of derivatives of THL may provide new therapies for NLRP3-related diseases.
Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Sangue Fetal , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lactamas/farmacologia , Lactamas/uso terapêutico , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/imunologia , Gravidez , Cultura Primária de Células , Pirrolidinonas/farmacologia , Pirrolidinonas/uso terapêutico , Células THP-1 , Ubiquitinação/efeitos dos fármacosRESUMO
Transition metal-boron complexes BnM have been predicted at density functional theory level to be molecular bowls (n = 8-14) hosting a transition metal atom (M) inside or molecular tires (n = 14) centered with a transition metal atom. Small Bn clusters prove to be effective inorganic ligands to all the VB-VIIIB transition metal elements in the periodic table. Density functional evidences obtained in this work strongly suggest that bowl-shaped fullerene analogues of Bn units exist in small BnM complexes and the bowl-to-tire structural transition occur to the first-row transition metal complexes BnM (M = Mn, Fe, Co) at n = 14, a size obviously smaller than n = 20 where the 2D-3D structural transition occurs to bare Bn. The half-sandwich-type B12Cr (C3v), full sandwich-type (B12)2Cr (D3d), bowl-shaped B14Fe (C2), and tire-shaped B14Fe (D7d) and B14Fe- (C7v) are the most interesting prototypes to be targeted in future experiments. These BnM complexes may serve as building blocks to form extended boron-rich BnMm tubes or cages (m > or = 2) or as structural units to be placed inside carbon nanotubes with suitable diameters.
RESUMO
Geometrical optimizations and electronic structural analyses of the -O(2)B(2)- bridged hydrometal complexes (M(4)H(3)C)(2)B(2)O(2) and (M(4)H(3)N)(2)B(2)O(2)(2+) (M = Ni, Mg) containing double tetracoordinate planar nonmetals (TPN) have been performed using the density functional theory at the B3LYP/6-311+G(d,p) level. Theoretical evidence of the possibility of double TPN centers coexisting in one planar molecule is presented.
RESUMO
A density functional theory investigation on a series of S-shaped or cyclic (BnEmSi)2H2 molecules (E = B, C, Si; n = 3-6; m = 1, 2) containing double planar tetra-, penta-, and hexacoordinate silicons has been presented in this work. Further theoretical evidence is provided to support the previously proposed structural pattern to host planar hypercoordinate silicons in small aromatic molecules.
RESUMO
Density functional theory investigations indicate that carbon boronyls (CBO)n (n = 3-7) are considerably more stable in thermodynamics than their boron carbonyl isomers (BCO)n and exhibit aromaticity throughout the whole series. The extra stabilities of (CBO)n originate from their frontier pi molecular orbitals delocalized over the Dnh Cn central rings which are absent in (BCO)n. It is expected that experimental characterization of these (CBO)n species may open a new branch of chemistry on carbon boronyls.