Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product. 相似文献
Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN. 相似文献
Vibrio cholerae hemolysin (HlyA) is a 65?kDa pore-forming toxin which causes lysis of target eukaryotic cells by forming heptameric channels in the plasma membrane. Deletion of the 15?kDa C-terminus β-prism carbohydrate-binding domain generates a 50?kDa truncated variant (HlyA50) with 1000-fold-reduced pore-forming activity. Previously, we showed by cryo-electron microscopy that the two toxin oligomers have central channels, but the 65?kDa toxin oligomer is a seven-fold symmetric structure with bowl-, ring-, and arm-like domains, whereas the 50?kDa oligomer is an asymmetric jar-like heptamer. In the present study, we determined three-dimensional(3D) structures of HlyA and HlyA50 in presence of erythrocyte stroma and observed that interaction of the 65?kDa toxin with the stroma induced a significant decrease in the height of the β-barrel oligomer with a change in conformation of the ring- and arm-like domains of HlyA. These features were absent in interaction of HlyA50 with stroma. We propose that this conformational transition is critical for membrane-insertion of the toxin. 相似文献
Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers (HBF) and their children. In this study, we used nest polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells (PBMC) from 74 cases of HBFs and their children who were born after their fathers' HBV infection (caHBF). We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B. 相似文献
Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 Å. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 Å and a single shell thickness of 15 Å. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The “Rossmann canyon” is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed. 相似文献
Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (KD = 7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all 7 mice, which was sustained for the observation period of 144 d in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection. 相似文献
The present study was designed to investigate possible relationships between the genotypes of hepa-titis B virus (HBV) and the HBV-specific cytotoxic T lymphocyte (CTL) responses. HBV genotypes, HBV specific CTL HBV DNA and other markers of HBV infection were determined in 138 patients with chronic hepatitis B. The results showed that the patients infected with genotype C (n=62) had a significantly lower HBV-specific CTL response than those who were infected with HBV genotype B (P<0.01). HBV DNA titer was higher in patients infected with HBV genotype C than in those infected with HBV geno-type B (P<0.01). Both alanine aminotransferase (ALT) and total bilirubin (TBIL) were higher in HBV genotype C infected patients than in those infected with genotype B (P<0.01 and <0.05, respectively). These results suggest that compared with CHB patients infected with HBV genotype B, the higher HBV DNA level and more severe liver damages in the patients infected with genotype C of HBV may be as-sociated with genotype C of the virus. 相似文献
Hepatitis B virus(HBV) is a major cause of hepatocellular carcinoma(HCC). Its chronic infection can lead to chronic liver inflammation and the accumulation of genetic alterations to result in the oncogenic transformation of hepatocytes. HBV can also sensitize hepatocytes to oncogenic transformation by causing genetic and epigenetic changes of the host chromosomes. HBV DNA can insert into host chromosomes and recent large-scale whole-genome sequencing studies revealed recurrent HBV DNA integrations sites that may play important roles in the initiation of hepatocellular carcinogenesis. HBV can also cause epigenetic changes by altering the methylation status of cellular DNA, the post-translational modification of histones, and the expression of micro RNAs. These changes can also lead to the eventual hepatocellular transformation. These recent findings on the genetic and epigenetic alterations of the host chromosomes induced by HBV opened a new avenue for the development of novel diagnosis and treatments for HBV-induced HCC. 相似文献
The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection. 相似文献
Chronic hepatitis B infection is caused by hepatitis B virus (HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA (cccDNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing cccDNA reservoir. Therefore, the study of the molecular mechanism of cccDNA formation is becoming a major focus of HBV research. This review summarizes the current advances in cccDNA molecular biology and the latest studies on the elimination or inactivation of cccDNA, including three major areas: (1) epigenetic regulation of cccDNA by HBV X protein, (2) immune-mediated degradation, and (3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.
We used cryo-electron tomography to visualize Rous sarcoma virus, the prototypic alpharetrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix, capsid protein (CA), and nucleocapsid moieties. CA reassembles into cores housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity. The virions in our data set range from ∼ 105 to ∼ 175 nm in diameter. Their cores are highly polymorphic. We observe angular cores, including some that are distinctively “coffin-shaped” for which we propose a novel fullerene geometry; cores with continuous curvature including, rarely, fullerene cones; and tubular cores. Angular cores are the most voluminous and densely packed; tubes and some curved cores contain less material, suggesting incomplete packaging. From the tomograms, we measured the surface areas of cores and, hence, their contents of CA subunits. From the virion diameters, we estimated their original complements of Gag. We find that Rous sarcoma virus virions, like the human immunodeficiency virus, contain unassembled CA subunits and that the fraction of CA that is assembled correlates with core type; angular cores incorporate ∼ 80% of the available subunits, and open-ended tubes, ∼ 30%. The number of glycoprotein spikes is variable (∼ 0 to 118) and also correlates with core type; virions with angular cores average 82 spikes, whereas those with tubular cores average 14 spikes. These observations imply that initiation of CA assembly, in which interactions of spike endodomains with the Gag layer play a role, is a critical determinant of core morphology. 相似文献