RESUMO
Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.
Assuntos
Inflamassomos , Proteínas NLR , Tiorredoxinas , Humanos , Microscopia Crioeletrônica , Inflamassomos/metabolismo , Proteínas NLR/antagonistas & inibidores , Proteínas NLR/química , Proteínas NLR/metabolismo , Proteínas NLR/ultraestrutura , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Spodoptera , Proteínas de Insetos , Oxirredução , Cisteína/metabolismo , Imunidade InataRESUMO
Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.
Assuntos
Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Bovinos , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Ratos , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Sódio/metabolismo , Simportadores/química , Simportadores/genética , Simportadores/metabolismo , Simportadores/ultraestruturaRESUMO
Toll-like receptor 7 (TLR7) is a single-stranded RNA (ssRNA) sensor in innate immunity and also responds to guanosine and chemical ligands, such as imidazoquinoline compounds. However, TLR7 activation mechanism by these ligands remain largely unknown. Here, we generated crystal structures of three TLR7 complexes, and found that all formed an activated m-shaped dimer with two ligand-binding sites. The first site conserved in TLR7 and TLR8 was used for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 was used for a ssRNA-binding that enhanced the affinity of the first-site ligands. The first site preferentially recognized guanosine and the second site specifically bound to uridine moieties in ssRNA. Our structural, biochemical, and mutagenesis studies indicated that TLR7 is a dual receptor for guanosine and uridine-containing ssRNA. Our findings have important implications for understanding of TLR7 function, as well as for therapeutic manipulation of TLR7 activation.
Assuntos
Guanosina/metabolismo , RNA/metabolismo , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Drosophila , Guanosina/imunologia , Células HEK293 , Humanos , Imunidade Inata/imunologia , Ligantes , Macaca mulatta , RNA/imunologia , Receptor 7 Toll-Like/imunologiaRESUMO
SignificanceThe nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that forms an inflammasome. The cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950 has established the structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation and the mechanism of action of the NLRP3 specific inhibitor. The inactive NLRP3 oligomer represents the NLRP3 resting state, capable of binding to membranes and is likely disrupted for its activation. Visualization of the inhibitor binding mode will enable optimization of the activity of NLRP3 inflammasome inhibitor drugs.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Multimerização Proteica , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Camundongos , Modelos Moleculares , Mutação , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Sedatives/anesthetics are important medical tools to facilitate medical care and increase patients' comfort. Increasingly, there is recognition that sedatives/anesthetics can modulate immune functions. Toll-like receptors (TLRs) are major pattern recognition receptors involved in the recognition of microbial components. TLR7 recognizes single-strand RNA virus such as influenza and SARS-CoV2 viruses and initiates interferon (IFN) responses. IFN production triggered by TLR7 stimulation is a critical anti-viral response. For example, patients with TLR7 variants including loss-of- function variants were associated with severe COVID-19. Taken together, it is important to determine if sedatives/anesthetics mitigate TLR7 function. We have previously showed that TLR7-mediated activation was not affected by volatile anesthetics. However, we found that propofol attenuated TLR7 activation among intravenous sedatives in the reporter assay. TLR7 agonist R837 stimulation increased TNF-α, IL-1ß, IL-6, IL-10, and IFN-ß mRNA levels in bone marrow-derived dendritic cells, while these levels were attenuated by propofol. Our murine lung slice experiments showed that propofol attenuated IFN production. R837 increased IFN-ß expression in the lungs, and propofol attenuated IFN-ß expression in an in vivo model of R837 intranasal instillation. We also found that propofol directly bound to and hindered its association of TLR7 with MyD88. Our analysis using fropofol, propofol derivative showed that the hydroxyl group in propofol was important for propofol-TLR7 interaction.
Assuntos
COVID-19 , Propofol , Animais , Células Dendríticas , Humanos , Hipnóticos e Sedativos/farmacologia , Imiquimode , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Camundongos , Propofol/análogos & derivados , Propofol/farmacologia , RNA Viral/metabolismo , SARS-CoV-2 , Receptor 7 Toll-LikeRESUMO
Colloidal quantum dots (QDs) are promising luminescent materials for display and lighting, but their stability has long been an issue. Here, we designed a passivation strategy of doping Ti ions into the shell of alloyed CdZnSeS@ZnS QDs. The results showed that Ti ions were successfully doped into the ZnS shell and the stability of QDs was improved. In the aging test, the Ti ions doped QDs maintained 51.4% of the initial performance after 90 h of aging, while the pristine QDs decreased to less than 25% of the initial value. In addition, we discuss the reasons why Ti ions doping improves the stability of QDs. Ti ions are found to form Ti-S bonds in the ZnS shell, which has high binding energy and strong oxidation resistance. Most importantly, since there is no external physical insulating coating, the optimized QDs can also be directly used in electroluminescent devices, showing great potential in electroluminescence applications.
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Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.
Assuntos
Vírus da Hepatite B , Simportadores , Humanos , Vírus da Hepatite B/genética , Hepatócitos/metabolismo , Ligação Proteica , Ligação Viral , Peptídeos/metabolismo , Simportadores/metabolismo , Internalização do VírusRESUMO
The sensing of self RNA by the endosomal Toll-like receptors (TLRs) 7 and 8 initiates pathogenic mechanisms underlying the autoimmune disease lupus. A blockade of the TLR7/8 signals may, therefore, be a novel therapeutic intervention for lupus. To test the hypothesis, a novel compound E6742 that blocks TLR7/8 activation was identified. The mode of action of E6742 was investigated by analysis of the tertiary structure of TLR7 and 8 in complex with E6742. The in vitro activities of the compound were examined in cellular systems and its therapeutic potential was evaluated in murine lupus models. Tertiary structures of the extracellular domain of TLR7 and 8 in complex with E6742 showed that E6742 binds specifically and non-covalently to the hydrophobic pocket located at the interface of TLR7 or TLR8 homodimers. E6742 potently and selectively inhibited several TLR7/8-mediated cytokine responses in human PBMC. In two mouse models of lupus, oral dosing of E6742 after the onset of disease suppressed increase in autoantibodies and blocked the advance of organ damage. Collectively, the data show that TLR7/8 activation contributes to disease progression and its blocking by E6742 has potential as a therapeutic intervention for lupus.
Assuntos
Leucócitos Mononucleares , Receptor 7 Toll-Like , Camundongos , Humanos , Animais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Modelos Animais de Doenças , Leucócitos Mononucleares/metabolismo , Receptor 8 Toll-Like/metabolismo , Autoanticorpos , Receptor Toll-Like 9RESUMO
BACKGROUND: Common treatments for metastatic/unresectable HER2-negative gastric cancer include chemotherapy, immune checkpoint inhibitor monotherapy and chemotherapy plus immune checkpoint inhibitor. However, significant drug resistance exists regardless of the treatment regimen. METHODS: Patients with metastatic/unresectable HER2-negative gastric/gastroesophageal junction adenocarcinoma were enrolled. All patients were divided into three groups according to the treatment regimen and were further divided into responders and non-responders according to efficacy evaluation. Metagenomics sequencing were performed to analyze gut microbiome signature of patients receiving different treatments at baseline and throughout treatment. RESULTS: One hundred seventeen patients with HER2-negative advanced gastric or gastroesophageal junction adenocarcinoma receiving chemotherapy alone, anti PD-1/PD-L1 immunotherapy alone or combined regimen were included in this study. Microbiome signatures related to clinical response are distinct among the three treatment groups. Among which, 14, 8 and 13 species were significantly different between responders and non-responders in immunotherapy, immunotherapy plus chemotherapy and chemotherapy group, respectively. Patients with higher relative abundance of Lactobacillus possessed higher microbiome diversity and significantly better response to anti-PD-1/PD-L1 immunotherapy and had a trend to achieve better progression-free survival. Another cohort of 101 patients has been used as an external validation set to confirm the stability and reliability of these findings. CONCLUSIONS: Gut microbiome affects response of treatments in HER2-negative advanced gastric cancer in a treatment-specific way, immunotherapy plus chemotherapy did not equal to a simple superposition of immunotherapy and chemotherapy. Lactobacillus is expected to become a novel choice as an adjuvant agent in promoting the efficacy of immunotherapy in gastric cancer.
Assuntos
Adenocarcinoma , Microbioma Gastrointestinal , Neoplasias Gástricas , Humanos , Microbioma Gastrointestinal/genética , Antígeno B7-H1/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Inibidores de Checkpoint Imunológico , Reprodutibilidade dos Testes , LactobacillusRESUMO
Objective: This paper aims to explore the relationship between the syndrome differentiation of traditional Chinese medicine (TCM) in colorectal cancer and the clinical laboratory indicators of patients, and to further seek the laboratory indicators to assist TCM syndrome differentiation. Methods: From May 2020 to June 2021, 122 colorectal cancer patients with a clear pathological diagnosis who had not undergone surgery or chemotherapy were classified according to the TCM syndrome classification. The clinical laboratory indicators of 122 patients with preoperative colorectal cancer were collected, and the correlation between preoperative colorectal cancer TCM syndromes and Karnofsky score and clinical laboratory indicators was analyzed. The indicators affecting TCM syndromes were included in the disordered multivariate logistic regression analysis model to analyze the relative risk of the influencing factors. Results: The syndromes of colorectal cancer patients were classified into excess syndrome, deficiency syndrome, and syndrome of intermingled deficiency & excess. The differences in total bilirubin (TBIL), hemoglobin (HB), uric acid (UA), and hematocrit (HCT) between the three groups were statistically significant (P < 0.05). The indexes such as TBIL, HB, UA, and HCT in preoperative patients with excess syndrome of colorectal cancer were higher than those in patients with syndrome of intermingled deficiency & excess and deficiency syndrome, and the comparison between groups using the LSD method showed that UA and HCT were different between the excess syndrome and deficiency syndrome groups (P < 0.05). Multivariate logistic regression analysis indicated that Gender, Tumor location, TNM stage, Total protein (TP), Red blood cell (RBC), HB, HCT, Platelet (PLT) and Fibrinogen (FIB) were all risk factors affecting TCM syndromes of preoperative colorectal cancer (P < 0.05). Conclusion: There is a correlation between the TCM syndromes of colorectal cancer and the clinical laboratory indicators of the patients. Gender, Tumor location, TNM stage, TP, RBC, HB, HCT, PLT and FIB are the risk factors of TCM syndrome differentiation in preoperative patients with colorectal cancer. TBIL, UA, HB, and HCT may be the four relevant indicators of TCM syndrome differentiation in colorectal cancer.
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The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.
Assuntos
COVID-19 , SARS-CoV-2 , Microscopia Crioeletrônica , Humanos , Proteínas de Membrana , Montagem de VírusRESUMO
Quantum dots (QDs) have attracted much attention as one of the most promising candidates for next-generation display materials. However, stability is still a big challenge for QDs. Herein, we encapsulated QDs in a thermoplastic polypropylene (PP) matrix by thermal processing technology to prepare a stabler color conversion film for the first time. Thermal processing technology expands the packaging materials of QDs from traditional soluble polymers to thermoplastic polymers such as PP with easy processing and a low cost. We showed that the QDs in the PP film exhibited longer-lasting stability than the traditional PMMA film. After 216 h of blue light accelerated aging test, the QDs maintained more than 90% of the initial performance in the PP film but dropped to less than 25% in the PMMA film. Moreover, the reasons for the improved stability have been further discussed. It was found that the PP-H film not only possessed better barriers to moisture and oxygen, but the absence of ester groups also led to a milder environment around the QDs. The results show that ester groups have stronger electronegativity and easily cause the ligands on the surface of QDs to fall off, which lead to performance degradation.
RESUMO
Cryo-electron microscopy (cryo-EM) is widely used for structural biology studies and has been developed extensively in recent years. However, its sample vitrification process is a major limitation because it causes severe particle aggregation and/or denaturation. This effect is thought to occur because particles tend to stick to the "deadly" air-water interface during vitrification. Here, we report a method for PEGylation of proteins that can efficiently protect particles against such problems during vitrification. This method alleviates the laborious process of fine-tuning the vitrification conditions, allowing for analysis of samples that would otherwise be discarded.
Assuntos
Microscopia Crioeletrônica/métodos , Animais , Microscopia Crioeletrônica/normas , Humanos , Proteína Adaptadora de Sinalização NOD2/química , Polietilenoglicóis/química , Desnaturação Proteica , Proteínas de Saccharomyces cerevisiae/química , Células Sf9 , Spodoptera , VitrificaçãoRESUMO
Nucleic acid-sensing Toll-like receptors (TLRs) play a pivotal role in innate immunity by recognizing foreign DNA and RNA. Compartmentalization of these TLRs in the endosome limits their activation by self-derived nucleic acids and reduces the possibility of autoimmune reactions. Although chaperone Unc-93 homolog B1, TLR signaling regulator (UNC93B1) is indispensable for the trafficking of TLRs from the endoplasmic reticulum to the endosome, mechanisms of UNC93B1-mediated TLR regulation remain largely unknown. Here, we report two cryo-EM structures of human and mouse TLR3-UNC93B1 complexes and a human TLR7-UNC93B1 complex. UNC93B1 exhibits structural similarity to the major facilitator superfamily transporters. Both TLRs interact with the UNC93B1 amino-terminal six-helix bundle through their transmembrane and luminal juxtamembrane regions, but the complexes of TLR3 and TLR7 with UNC93B1 differ in their oligomerization state. The structural information provided here should aid in designing compounds to combat autoimmune diseases.
Assuntos
Proteínas de Membrana Transportadoras , Receptor 3 Toll-Like , Receptor 7 Toll-Like , Animais , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Camundongos , Ligação Proteica , Multimerização Proteica , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/ultraestrutura , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/ultraestruturaRESUMO
Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. These compounds protect mice against lethal autoimmunity. Combining crystallography and cryo-electron microscopy, we identify the open conformation of the receptor and reveal the structural equilibrium between open and closed conformations that underlies TLR7 antagonism, as well as the detailed mechanism by which TLR7-specific antagonists bind to their binding pocket in TLR7. Our work provides small-molecule TLR7-specific antagonists and suggests the TLR7-targeting strategy for treating autoimmune diseases.
Assuntos
Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/química , Animais , Doenças Autoimunes , Autoimunidade , Sítios de Ligação , Microscopia Crioeletrônica , Feminino , Ligantes , Lúpus Eritematoso Sistêmico , Camundongos , Camundongos Endogâmicos NZB , Modelos Moleculares , Conformação Proteica , Taxa de SobrevidaRESUMO
Toll-like receptor 7 (TLR7) is an innate immune receptor for single-stranded RNA (ssRNA) and has important roles in infectious diseases. We previously reported that TLR7 shows synergistic activation in response to two ligands, guanosine and ssRNA. However, the specific ssRNA sequence preference, detailed recognition mode of TLR7 and its ligand, and molecular determinants of TLR7 and TLR8 selectivity remain unknown. Here, we report on TLR7 from a large-scale crystallographic study combined with a multifaceted approach. We reveal that successive uridine-containing ssRNAs fully or moderately bind TLR7, whereas single uridine-containing ssRNAs have reduced affinities. We also reveal the detailed relationships between the chemical structures of ligands and their binding to TLR7. We demonstrate that an engineered TLR8 mutant alters its responsiveness to TLR7-specific ligands. Finally, we identify guanosine 2',3'-cyclic phosphate (2',3'-cGMP) as a possible endogenous ligand for TLR7 with greater affinity than guanosine. The abundant structural information will facilitate future development of treatments targeting TLR7.
Assuntos
RNA/metabolismo , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/química , Sequência de Aminoácidos , Animais , Sequência de Bases , GMP Cíclico/metabolismo , Drosophila , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Mutagênese , Ligação Proteica , Quinolinas/farmacologia , Relação Estrutura-Atividade , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
The history of mankind has been plagued by the tug of war with viral infections. Toll-like receptors (TLRs) and other receptors of the innate immune system constitute an early defense system against invading viruses by recognizing the viral genetic material, the nucleic acids (NAs). Agonistic ligands of NA-sensing TLRs play an emerging role in the treatment of viral diseases, demonstrating a crucial role of these receptors. Recently, crystal structures have afforded new insights into TLR recognition of NAs. An aberrant activation by self-NAs, which leads to the inflammation and autoimmunity, is avoided by strict regulation of NA-TLR interaction at multiple check-points. This Review summarizes the novel structural understanding of NA-sensing by TLRs and regulatory mechanisms of these receptors.