CRM1-spike-mediated nuclear export of hepatitis B virus encapsidated viral RNA.

Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NESCRM1) cluster at the conformationally flexible spike tips of HBc particles. Mutant NESCRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 in vivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking.

Expression and epitope identification of myosin light chain isoform 1, an allergen in Procambarus clarkii.

Myosin light chain isoform 1 (MLC1) is reported to be a novel allergen in crayfish (Procambarus clarkii). However, little information is available about its allergic epitopes. In this study, recombinant crayfish MLC1 (rMLC1) was expressed and confirmed by mass spectrometry. Circular dichroic analysis and serological test were performed for the measuring of structural and immunological properties of rMLC1. Specific-protein-A-enriched IgG raised in rabbits against purified rMLC1 was used to screen a phage display random peptide library. Nine MLC1 mimotope clones were identified among 16 random clones after biopanning. Five conformational epitopes were identified with the program LocaPep, and mapped into 3 epitope regions at the antibody-binding interface of MLC1. MLC1 of crayfish showed high primary and secondary structure identity to MLC of other allergenic species, its epitopes were located in the structure conserved regions, and its cross-reactivity among related species was indicated by immunological assays.

Adding a C-terminal Cysteine (CTC) Can Enhance the Bactericidal Activity of Three Different Antimicrobial Peptides.

The emergence of antibiotic-resistant bacteria has threatened our health worldwide. There is an urgent need for novel antibiotics. Previously, we identified a novel 37-mer antimicrobial peptide (AMP), HBcARD, with broad spectrum antimicrobial activity. Here, we improved the efficacy of HBcARD, by re-engineering the peptide, including the addition of a new cysteine to its C-terminus (CTC). The new 28-mer derivative, D-150-177C, contains all D-form arginines, in addition to a C-terminal cycteine. This peptide can kill antibiotic-resistant clinical isolates of Gram-negative bacteria, and is more potent than the parental HBcARD peptide in a mouse sepsis model. In another lung infection mouse model, D-150-177C showed protection efficacy against colistin-resistant Acinetobacter baumannii. Unlike colistin, we observed no acute toxicity of D-150-177C in vivo. Interestingly, we found that CTC modification could enhance the antibacterial activity of several other AMPs, such as buforinII and lysin. The potential application and mechanism of this CTC method as a general approach to improving drug efficacy, warrants further investigation in the future.

Improvement of in vivo antimicrobial activity of HBcARD peptides by D-arginine replacement.

We previously identified a novel antimicrobial peptide with a broad spectrum bactericidal activity from human hepatitis B virus (HBV) core protein (HBc) arginine-rich domain (ARD). We compared the antimicrobial activities of HBcARD peptides from different hepadnaviruses which share similar amino acid sequences. In general, mammalian HBcARD peptides exhibited stronger antimicrobial activity than avian peptides. Using the strategy of D-amino acid substitutions, we improved the antimicrobial efficacy of human HBcARD peptide. This D-HBcARD peptide was much more resistant than L-HBcARD peptide to proteolytic degradation in vitro. Moreover, this D-HBcARD peptide maintained similar minimal bactericidal concentrations (MBC) against tested bacteria, and showed very low hemolytic activity. In the Staphylococcus aureus-infected mouse model, this D-HBcARD peptide was more protective than the L-HBcARD peptide. Repeated treatments with either L- or D-HBcARD peptides induced no significant immunogenicity. New derivatives of HBcARD peptides could serve as alternatives to the conventional antibiotics in clinical medicine in the future.

Selection and Characterization of DNA Aptamers Targeting All Four Serotypes of Dengue Viruses.

Dengue viruses (DENVs) are members of Flaviviridae family, which are associated with human disease. The envelope (E) protein plays an important role in viral infection. However, there is no effective antibody for clinical treatment due to antibody dependent enhancement of infection. In this study, using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we demonstrated the first aptamer (S15) that can bind to DENV-2 envelop protein domain III (ED3) with a high binding affinity. S15 was found to form a parallel quadruplex based on Quadfinder prediction, gel mobility assay and circular dichroism studies. Both the quadruplex structure and the sequence on 5'-end were necessary for the binding activity of S15. NMR titration experiments indicated that S15 bound to a highly conserved loop between ßA and ßB strands of ED3. Moreover, S15 can neutralize the infections by all four serotypes of DENVs. Our result provides a new opportunity in the development of DNA aptamers against DENVs in the future.

The serologic decoy receptor 3 (DcR3) levels are associated with slower disease progression in HIV-1/AIDS patients.

BACKGROUND/PURPOSE: The decoy receptor 3 (DcR3) is a member of the tumor necrosis factor receptor (TNFR) super-family. It counteracts the biological effects of Fas ligands and inhibits apoptosis. The goals of this study were to understand the associations between serologic DcR3 (sDcR3) levels and different human immunodeficiency virus type 1 (HIV-1) subtypes, as well as the AIDS disease progression. METHODS: Serum samples from 61 HIV/AIDS patients, who had been followed up every 6 months for 3 years, were collected. sDcR3 levels were quantified using an enzyme immunoassay (EIA). RESULTS: The sDcR3 levels in patients with HIV-1 subtype B were significantly higher than those in patients infected with subtype CRF01_AE (p < 0.001). In addition, multivariable linear mixed model analysis demonstrated that HIV-1 subtype B and slow disease progression were associated with higher levels of sDcR3, adjusting for potential predictors (p = 0.0008 and 0.0455, respectively). CONCLUSION: HIV-1-infected cells may gain a survival advantage by activating DcR3, which prevents infected cell detection by the host immune system. These data indicate that the sDcR3 level is a biomarker for AIDS disease progression.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Purification, physicochemical and immunological characterization of arginine kinase, an allergen of crayfish (Procambarus clarkii).

Arginine kinase (AK) has attracted considerable attention because it has been identified as a shellfish allergen. However, little information is available about AK in crayfish (Procambarus clarkii). In this study, crayfish AK was purified and cloned. Its physicochemical properties, processing stability, and immunological characteristics were analyzed. Crayfish AK was purified by column chromatography, which revealed a single band with molecular mass of 40 kDa; this result was further confirmed by mass spectrometry. The full-length gene sequence of crayfish AK was 1462 bp and encoded a protein of 357 amino acid residues. The results of this study revealed that crayfish AK is a glycoprotein with an isoelectric point of approximately 6.5. Thermal stability assays revealed that crayfish AK easily forms aggregates at temperatures >44°C and was stable at pH 4.0-8.0. SDS-PAGE and dot blotting were used to assess processing stability of purified AK. The results revealed that the IgE-binding activity of crayfish AK is reduced after boiling.

Correlations between membrane immersion depth, orientation, and salt-resistance of tryptophan-rich antimicrobial peptides.

The efficacies of many antimicrobial peptides are greatly reduced in the presence of high salt concentrations therefore limiting their development as pharmaceutical compounds. PEM-2-W5K/A9W, a short Trp-rich antimicrobial peptide developed based on the structural studies of PEM-2, has been shown to be highly active against various bacterial strains with less hemolytic activity. Here, correlations between membrane immersion depth, orientation, and salt-resistance of PEM-2 and PEM-2-W5K/A9W have been investigated via solution structure and paramagnetic resonance enhancement studies. The antimicrobial activities of PEM-2-W5K/A9W and PEM-2 against various bacterial and fungal strains including multidrug-resistant and clinical isolates under high salt conditions were tested. The activities of the salt-sensitive peptide PEM-2 were reduced and diminished at high salt concentrations, whereas the activities of PEM-2-W5K/A9W were less affected. The results indicated that the strong salt-resistance of PEM-2-W5K/A9W may arise from the peptide positioning itself deeply into microbial cell membranes and thus able to disrupt the membranes more efficiently.

Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD).

The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176) and ARD I-III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I-IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.

Purification and characterisation of sarcoplasmic calcium-binding protein, a novel allergen of red swamp crayfish (Procambarus clarkii).

Crayfish sarcoplasmic calcium-binding protein (SCP) was purified. The physicochemical and polymorphic characterisations were also analysed. SCP was purified by column chromatography to reveal a single band with molecular mass of 22 kDa and further confirmed by mass spectrometry. The results of physicochemical characterisation showed that SCP was stable in the processes of thermal or acid/alkali treatment, and could be digested by simulate gastrointestinal fluid. Importantly, the comparison of SCP polymorphism using sera from crustacean-allergic patients demonstrated SCP-II had a weaker IgE-binding activity. The isoelectric points of SCP subunits a, b and c were 4.6, 4.7, and 4.8, respectively, as determined by two-dimensional electrophoresis and IgE immunoblotting analysis showed that patients' sera reacted to three subunits of SCP. Finally, it can be concluded that SCP is a stable polymorphic allergen in crayfish, and all of its isotypes and subunits have allergenicity.

The multi-functional roles of GNMT in toxicology and cancer.

Although glycine N-methyltransferase (GNMT) has been discovered for five decades, its function was not elucidated until recently. In this review, we discuss the multiple roles of GNMT in toxicology and cancer. Besides catalyzing the production of methylglycine (sarcosine) in one carbon metabolism pathway, GNMT was found to be able to bind a number of polycyclic aromatic hydrocarbons and inhibit DNA adducts formation. Moreover, GNMT exerts protective effects against the cytotoxicity and carcinogenicity of benzo(a)pyrene and aflatoxin B(1) in vitro and in vivo. Occupational study showed that workers who had genotypes with higher GNMT promoter activity may have lower content of oxidative damaged DNA products in their urine. In terms of cancer, recent studies using GNMT knockout mouse models demonstrated that GNMT deficiency has high penetrance in inducing the development of steatohepatitis and hepatocellular carcinoma. In terms of the mechanism, besides dysregulation of epigenetic modification, insights have been provided by recent identification of two novel proteins interacting with GNMT-DEPTOR and NPC2. These studies suggest that GNMT not only is involved in mTOR signaling pathway, but also plays an important role in the intracellular trafficking of cholesterol. The implication of these findings to the preventive medicine and translational research will be discussed.

Easy strategy to increase salt resistance of antimicrobial peptides.

The efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations, limiting their development as pharmaceutical compounds. Here, we describe an easy strategy to increase salt resistance of antimicrobial peptides by replacing tryptophan or histidine residues with the bulky amino acids ß-naphthylalanine and ß-(4,4'-biphenyl)alanine. The activities of the salt-sensitive peptide P-113 were diminished at high salt concentrations, whereas the activities of its ß-naphthylalanine and ß-(4,4'-biphenyl)alanine-substituted variant were less affected.

Rational design of tryptophan-rich antimicrobial peptides with enhanced antimicrobial activities and specificities.

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp-rich peptides derived from the C-terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A(2) (PLA(2)), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK-designated PEM-2-was found to display improved activity against both Gram-positive and Gram-negative bacteria. To improve the antimicrobial activity of PEM-2 for potential clinical applications further, we determined the solution structure of PEM-2 bound to membrane-mimetic dodecylphosphocholine (DPC) micelles by two-dimensional NMR methods. The DPC micelle-bound structure of PEM-2 adopts an α-helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM-2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM-2 could be due to interactions with bacterial membranes. Potent analogues of PEM-2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.

Identification of a heparin binding peptide from the Japanese encephalitis virus envelope protein.

The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. It has been shown that highly sulfated forms of heparin sulfate can bind to the envelope protein and are involved in flavivirus infection. Among the three structural domains, domain III is the major antigenic domain of the envelope protein. We have prepared an extended form of the JEV domain III protein with residues ranging from 261 to 402 and determined its heparin binding sites. Based on NMR, fluorescence spectroscopy, and site-directed mutagenesis studies, we have identified that only the N-terminal region (residues 279-293) and some spatially adjacent residues of JEV domain III are involved in heparin binding. Moreover, a synthetic peptide corresponding to this region also demonstrates strong affinity to heparin. Our results provide a basis for further understanding the interactions of flaviviruses and glycosaminoglycans on the host cell surfaces.

Increased potency of a novel D-beta-naphthylalanine-substituted antimicrobial peptide against fluconazole-resistant fungal pathogens.

The antifungal activities of the known antimicrobial peptide, P-113, as well as a new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), Pac-525, and its modified peptide, D-Nal-Pac-525, were determined using the broth microdilution method in three different media. All peptides had similar activities against yeast pathogens in low-salt LYM media. However, only D-Nal-Pac-525 retained its antifungal activity in the media containing high concentrations of salt. Hence, D-Nal-Pac-525 has the potential of becoming a promising antifungal agent, especially for fungal pathogens with intrinsic resistance to fluconazole.

Solution structure of a novel D-naphthylalanine substituted peptide with potential antibacterial and antifungal activities.

A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH2, designated as Pac-525, was found to possess improved activity against both gram-positive and negative bacteria. We have synthesized two Pac-525 analogues, D-Pac-525 containing all D-amino acids and D-Nal-Pac-525, the D-Pac-525 analogue with tryptophan replaced by D-beta-naphthylalanine. We have determined the solution structure of D-Nal-Pac-525 bound to membrane-mimetic DPC micelles by two-dimensional NMR methods. The DPC micelle-bound structure of D-Nal-Pac-525 adopts a left-hand alpha-helical segment and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates the three D-beta-naphthylalanines are packed against the peptide backbone and form an amphipathic structure. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that D-Nal-Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the strong antimicrobial activity of D-Nal-Pac-525 may be due to interactions with bacterial and fungus membranes.

Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity.

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.