Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

NMR characterization of the interaction between Bcl-xL and the BH3-like motif of hepatitis B virus X protein.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. Here we report the interaction between the HBx BH3-like motif and Bcl-xL, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-xL, with a binding affinity of 89 µM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-xL binding using three mutants. The W120A mutant showed weaker binding affinity (294 µM) to Bcl-xL, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-xL binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.

A novel neuropilin-1-binding sequence in the human T-cell lymphotropic virus type 1 envelope glycoprotein.

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 µM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

Structural characterization of the BH3-like motif of hepatitis B virus X protein.

Hepatitis B virus X protein (HBx) is a multifunctional protein, which is considered to be an essential molecule for viral replication and the development of liver diseases. Recently, it has been demonstrated that HBx can directly interact with Bcl-2 and Bcl-xL through a sequence (termed the BH3-like motif) that is related to the BH3 motif of pro-apoptotic BH3-only proteins. Here, we present the first structural characterization of the HBx BH3-like motif by circular dichroism and NMR spectroscopies. Our results demonstrated that the HBx BH3-like motif has the ability to form an α-helix, and the potential helical region involves residues L108-L134. This is a common characteristic among the BH3 peptides of pro-apoptotic BH3-only proteins, implying that HBx may interact with Bcl-2 and Bcl-xL, by forming an α-helix, similar to the interaction mode of other BH3 peptides with Bcl-2 and Bcl-xL.

Chemical shift assignments of a fusion protein comprising the C-terminal-deleted hepatitis B virus X protein BH3-like motif peptide and Bcl-xL.

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV has the multifunctional protein, HBV X protein (HBx, 154 residues), which plays key roles in HBV replication and liver disease development. Interaction of HBx through its BH3-like motif with the anti-apoptotic protein Bcl-xL leads to HBV replication and induction of apoptosis, resulting in HCC development. Our previous nuclear magnetic resonance (NMR) study revealed that the HBx BH3-like motif peptide (residues 101-136) binds to the common BH3-binding groove of Bcl-xL. Importantly, a C-terminal-truncated HBx, e.g., residues 1-120 of HBx, is strongly associated with the increased risk of HBV-related HCC development. However, the interaction mode between the C-terminal-truncated HBx and Bcl-xL remains unclear. To elucidate this interaction mode, the C-terminal-deleted HBx BH3-like motif peptide (residues 101-120) was used as a model peptide in this study. To facilitate the NMR analysis, we prepared a fusion protein of HBx (101-120) and Bcl-xL connected with five repeats of the glycine-serine dipeptide as a linker. Here, we report the 1H, 13C, and 15N resonance assignments of the fusion protein. This is the first step for the elucidation of the pathogenesis of liver diseases caused by the interaction between the C-terminal-truncated HBx and Bcl-xL.

The N93D mutation of the human T-cell leukemia virus type 1 envelope glycoprotein found in symptomatic patients enhances neuropilin-1 b1 domain binding.

Human T-cell leukemia virus type 1 (HTLV-1) infection of host cells is mainly mediated by interactions with the viral envelope glycoprotein surface unit (SU) and three host receptors: heparan sulfate proteoglycan, neuropilin-1 (Nrp1), and glucose transporter type 1. Residues 90-94 of SU are considered as a Nrp1 binding site, and our previous results show that an SU peptide consisting of residues 85-94 can bind directly to the Nrp1 b1 domain with a binding affinity of 7.4 µM. Therefore, the SU peptide is expected to be a good model to investigate the SU-Nrp1 interaction. Recently, the N93D mutation in the Nrp1 b1 binding region of the SU was identified in symptomatic patients with HTLV-1 infections in the Brazilian Amazon. However, it remains unclear how the SU-N93D mutation affects Nrp1 b1 binding. To elucidate the impact of the substituted Asp93 of SU on Nrp1 b1 binding, we analyzed the interaction between the SU-N93D peptide and Nrp1 b1 using isothermal titration calorimetry and nuclear magnetic resonance. The SU-N93D peptide binds directly to Nrp1 b1 with a binding affinity of 3.5 µM, which is approximately two-fold stronger than wild-type. This stronger binding is likely a result of the interaction between the substituted residue Asp93 of the N93D peptide and the four residues Trp301, Lys347, Glu348, and Thr349 of Nrp1 b1. Our results suggest that the interaction of SU Asp93 with the four residues of Nrp1 b1 renders the high affinity of the N93D mutant for Nrp1 b1 binding during HTLV-1 entry.

Validation of HPLC Method for Determination of Histamine in Human Immunoglobulin Formulations.

BACKGROUND: Histamine fixed-immunoglobulin formulations, which consisted of 0.15 µg of histamine dihydrochloride and 12 mg of human immunoglobulin in a vial, are used for anti-allergic treatments, and controlling the amounts of histamine in the formulations is essential to avoid histamine intoxication. OBJECTIVE: A high-performance liquid chromatography (HPLC) method for determination of histamine contents of the formulations was established and validated. METHODS: Histamine extracted from the formulation was labeled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and was analyzed by gradient elution HPLC with UV detection at 260 nm. RESULTS: The method showed linearity in the range 0.8-2.4 µM (R > 0.999), accuracy (100.1-105.8% recovery), and precision (relative standard deviation ≤ 1.93%). The validated method was applied for five lots of the pharmaceutical, and their histamine contents were determined to be 0.149-0.155 µg/vial. CONCLUSIONS: These results indicated that the validated method is useful to control amounts of histamine in biopharmaceutical products. HIGHLIGHTS: The HPLC method was developed for quantitative determination of histamine content of the histamine fixed-immunoglobulin formulations.

Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

Changes of urine metabolite profiles are induced by inactivated influenza vaccine inoculations in mice.

The safety evaluation of vaccines is critical to avoid the development of side effects in humans. To increase the sensitivity of detection for toxicity tests, it is important to capture not only pathological changes but also physiological changes. 1H nuclear magnetic resonance (NMR) spectroscopy analysis of biofluids produces profiles that show characteristic responses to changes in physiological status. In this study, mouse urine metabolomics analysis with 1H NMR was performed using different influenza vaccines of varying toxicity to assess the usefulness of 1H NMR in evaluating vaccine toxicity. Two types of influenza vaccines were used as model vaccines: a toxicity reference vaccine (RE) and a hemagglutinin split vaccine. According to the blood biochemical analyses, the plasma alanine transaminase levels were increased in RE-treated mice. Changes in metabolite levels between mice administered different types of influenza vaccines were observed in the 1H NMR spectra of urine, and a tendency toward dosage-dependent responses for some spectra was observed. Hierarchical clustering analyses and principal component analyses showed that the changes in various urine metabolite levels allowed for the classification of different types of vaccines. Among them, two liver-derived metabolites were shown to largely contribute to the formation of the cluster. These results demonstrate the possibility that urine metabolomics analysis could provide information about vaccine-induced toxicity and physiological changes.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies.

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Structural insights into the stability and flexibility of unusual erythroid spectrin repeats.

Erythroid spectrin, a major component of the cytoskeletal network of the red cell which contributes to both the stability and the elasticity of the red cell membrane, is composed of two subunits, alpha and beta, each formed by 16-20 tandem repeats. The properties of the repeats and their relative arrangement are thought to be key determinants of spectrin flexibility. Here we report a 2.4 A resolution crystal structure of human erythroid beta-spectrin repeats 8 and 9. This two-repeat fragment is unusual as it exhibits low stability of folding and one of its repeats lacks two tryptophans highly conserved among spectrin repeats. Two key factors responsible for the lower stability and, possibly, its flexibility, are revealed by the structure. A third novel feature of the structure is the relative orientation of the two repeats, which increases the range of possible conformations and provides new insights into atomic models of spectrin flexibility.

Independent movement, dimerization and stability of tandem repeats of chicken brain alpha-spectrin.

Previous X-ray crystal structures have shown that linkers of five amino acid residues connecting pairs of chicken brain alpha-spectrin and human erythroid beta-spectrin repeats can undergo bending without losing their alpha-helical structure. To test whether bending at one linker can influence bending at an adjacent linker, the structures of two and three repeat fragments of chicken brain alpha-spectrin have been determined by X-ray crystallography. The structure of the three-repeat fragment clearly shows that bending at one linker can occur independently of bending at an adjacent linker. This observation increases the possible trajectories of modeled chains of spectrin repeats. Furthermore, the three-repeat molecule crystallized as an antiparallel dimer with a significantly smaller buried interfacial area than that of alpha-actinin, a spectrin-related molecule, but large enough and of a type indicating biological specificity. Comparison of the structures of the spectrin and alpha-actinin dimers supports weak association of the former, which could not be detected by analytical ultracentrifugation, versus strong association of the latter, which has been observed by others. To correlate features of the structure with solution properties and to test a previous model of stable spectrin and dystrophin repeats, the number of inter-helical interactions in each repeat of several spectrin structures were counted and compared to their thermal stabilities. Inter-helical interactions, but not all interactions, increased in parallel with measured thermal stabilities of each repeat and in agreement with the thermal stabilities of two and three repeats and also partial repeats of spectrin.

Molecular Evolution of the Capsid Gene in Norovirus Genogroup I.

We studied the molecular evolution of the capsid gene in all genotypes (genotypes 1-9) of human norovirus (NoV) genogroup I. The evolutionary time scale and rate were estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also performed selective pressure analysis and B-cell linear epitope prediction in the deduced NoV GI capsid protein. Furthermore, we analysed the effective population size of the virus using Bayesian skyline plot (BSP) analysis. A phylogenetic tree by MCMC showed that NoV GI diverged from the common ancestor of NoV GII, GIII, and GIV approximately 2,800 years ago with rapid evolution (about 10(-3) substitutions/site/year). Some positive selection sites and over 400 negative selection sites were estimated in the deduced capsid protein. Many epitopes were estimated in the deduced virus capsid proteins. An epitope of GI.1 may be associated with histo-blood group antigen binding sites (Ser377, Pro378, and Ser380). Moreover, BSP suggested that the adaptation of NoV GI strains to humans was affected by natural selection. The results suggested that NoV GI strains evolved rapidly and date back to many years ago. Additionally, the virus may have undergone locally affected natural selection in the host resulting in its adaptation to humans.

¹H, ¹³C and ¹5N backbone resonance assignments of the monomeric human M-ficolin fibrinogen-like domain secreted by Brevibacillus choshinensis.

M-ficolin, which forms trimer-based multimers, is a pathogen-recognition protein in the innate immune system, and it binds to ligands through its fibrinogen-like (FBG) domain. As the first step toward the elucidation of the molecular basis for pathogen-recognition by the M-ficolin multimers, we assigned the backbone resonances of the monomeric mutant of the M-ficolin FBG domain, recombinantly expressed by Brevibacillus choshinensis. Like the wild-type trimeric FBG domain, the monomeric FBG domain also requires His251, His284 and His297 for the ligand-binding activity, as judged by mutational analyses using zonal affinity chromatography. The secondary structure predicted by the backbone resonance assignments is similar to that of the trimeric FBG domain in the crystal, indicating that the monomeric FBG domain is folded correctly to perform its function.

B-cell-intrinsic hepatitis C virus expression leads to B-cell-lymphomagenesis and induction of NF-κB signalling.

Hepatitis C virus (HCV) infection leads to the development of hepatic diseases, as well as extrahepatic disorders such as B-cell non-Hodgkin's lymphoma (B-NHL). To reveal the molecular signalling pathways responsible for HCV-associated B-NHL development, we utilised transgenic (Tg) mice that express the full-length HCV genome specifically in B cells and develop non-Hodgkin type B-cell lymphomas (BCLs). The gene expression profiles in B cells from BCL-developing HCV-Tg mice, from BCL-non-developing HCV-Tg mice, and from BCL-non-developing HCV-negative mice were analysed by genome-wide microarray. In BCLs from HCV-Tg mice, the expression of various genes was modified, and for some genes, expression was influenced by the gender of the animals. Markedly modified genes such as Fos, C3, LTßR, A20, NF-κB and miR-26b in BCLs were further characterised using specific assays. We propose that activation of both canonical and alternative NF-κB signalling pathways and down-regulation of miR-26b contribute to the development of HCV-associated B-NHL.

Estimation of lactose interference in vaccines and a proposal of methodological adjustment of total protein determination by the lowry method.

For national regulatory testing in Japan, the Lowry method is used for the determination of total protein content in vaccines. However, many substances are known to interfere with the Lowry method, rendering accurate estimation of protein content difficult. To accurately determine the total protein content in vaccines, it is necessary to identify the major interfering substances and improve the methodology for removing such substances. This study examined the effects of high levels of lactose with low levels of protein in freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated). Lactose was selected because it is a reducing sugar that is expected to interfere with the Lowry method. Our results revealed that concentrations of ≥ 0.1 mg/mL lactose interfered with the Lowry assays and resulted in overestimation of the protein content in a lactose concentration-dependent manner. On the other hand, our results demonstrated that it is important for the residual volume to be ≤ 0.05 mL after trichloroacetic acid precipitation in order to avoid the effects of lactose. Thus, the method presented here is useful for accurate protein determination by the Lowry method, even when it is used for determining low levels of protein in vaccines containing interfering substances. In this study, we have reported a methodological adjustment that allows accurate estimation of protein content for national regulatory testing, when the vaccine contains interfering substances.

HCV infection and B-cell lymphomagenesis.

Hepatitis C virus (HCV) has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma (NHL). Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.