Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Immunological evaluation of a novel HLA-A2 restricted phosphopeptide of tumor associated Antigen, TRAP1, on cancer therapy.

The tumor necrosis factor receptor associated protein 1 (TRAP1) is a mitochondria chaperon protein that has been previously implicated as a target for cancer therapy due to its expression level is linked to tumor progression. In this study, an immunodominant phosphopeptide of TRAP1 was identified from an HLA-A2 gene transfected mouse cancer cell line using mass spectrometry, and a synthetic phosphopeptide was generated to evaluate the potency on cancer immunotherapy. In the transporter associated with antigen processing (TAP) deficient cell, the conjugated phosphate group plays a critical role to enhance the binding affinity of phosphopeptide with HLA-A2 molecule. On the basis of immunological assay, immunization of synthetic phosphopeptide could induce a high frequency of IFN-γ-secreting CD8+ T cells in HLA-A2 transgenic mice, and the stimulated cytotoxic T lymphocytes showed a high target specificity to lysis the epitope-pulsed splenocytes in vivo and the human lung cancer cell in vitro. In a tumor challenge assay, vaccination of the HLA-A2 restricted phosphopeptide appeared to suppress the tumor growth and prolong the survival period of tumor-bearing mice. These results suggest that novel phosphopeptide is naturally presented as a HLA-A2-restricted CTL epitope and capable of being a potential candidate for the development of therapeutic vaccine against high TRAP1-expressing cancers.

Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology.

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a ß-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom.

Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA) simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV) and Vietnamese snake antivenom-Naja (SAV-Naja) exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX) and cytotoxicity of cardiotoxin/cytotoxin (CTX), respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

A biphasic response pattern of lipid metabolomics in the stage progression of hepatitis B virus X tumorigenesis.

Metabolic syndrome has closely linked to the development of human hepatocellular carcinoma (HCC). By using the hepatitis B virus (HBV) X (HBx) transgenic mouse model, we studied the dynamic evolution of serum and liver profiles of lipids and global cDNA expression at different stages of HBx tumorigenesis. We observed that the lipid (triglycerides, cholesterol, and fatty acids) profiles revealed a biphasic response pattern during the progression of HBx tumorigenesis: a small peak at early phase and a large peak or terminal switch at the tumor phase. By analyzing cDNA microarray data, the early peak correlated to the oxidative stress and pro-inflammatory response, which then resolved at the middle phase and were followed by the terminal metabolic switch in the tumor tissues. Five lipid metabolism-related genes, the arachidonate 5-lipoxygenase, lipoprotein lipase, fatty acid binding protein 4, 1-acylglycerol-3-phosphate O-acyltransferase 9, and apolipoprotein A-IV were identified to be significantly activated in HBx transgenic HCCs and further validated in human HBV-related HCCs. Inhibition of these lipid genes could reverse the effect of HBx on lipid biosynthesis and suppress HBx-induced cell proliferation in vitro. Our results support the concept that metabolic syndrome plays an important role in HBV tumorigenesis. The dysregulation of lipid metabolic genes may predict the disease progression to HCC in chronic hepatitis B patients.

Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins.

In this study, new angiotensin-I converting enzyme (ACE) inhibitory peptides were comprehensively identified from a thermolysin digest of bitter melon (Momordica charantia) seed proteins. The hydrolysate was fractionated by reversed-phase high performance liquid chromatography (RP-HPLC), and the inhibitory activities of the resulting fractions were evaluated using ACE inhibitory assay. Two novel ACE inhibitory peptides (VY-7 and VG-8) were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database-assisted peptide sequencing. VY-7 and VG-8 were derived from momordin A and MAP30, respectively, and their IC50 values were as low as 8.64±0.60 and 13.30±0.62 µM, respectively. Lineweaver-Burk plots further indicated that VY-7, which showed the best IC50 value, acts as a competitive inhibitor. Notably, the content of VY-7 in crude thermolysin digest was determined to be as high as 14.89±0.88 µg/mg using LC-MS/MS quantification. In the spontaneously hypertensive rat (SHR) model, oral administration of VY-7 at 2mg/kg body weight significantly decreased the systolic blood pressure. The interaction between VY-7 and ACE was examined using molecular docking calculations and the results suggested that certain residues of VY-7 can fit perfectly into the S1, S1' and S2' regions of the binding pocket of ACE. BIOLOGICAL SIGNIFICANCE: One of the most common supportive therapies for treating hypertension is the use of synthetic drugs to inhibit ACE activity. Synthetic ACE inhibitors possess good antihypertensive effects, but come with accompanying side effects. Therefore, food-derived ACE inhibitory peptides are regarded as safer alternatives and are attracting much attention for hypertension treatment. In this study, we comprehensively identified peptides derived from bitter melon (Momordica charantia) seed proteins (BMSPs) using a shotgun proteomics approach. Based on results from an in vitro ACE inhibitory assay, two peptides (VY-7 and VG-8) derived from momordin A and MAP30 proteins, respectively, showed good ACE inhibitory activities. For VY-7, which showed the best IC50 value (8.64±0.60 µM), the inhibition type was determined to be competitive inhibition, as found using a Lineweaver-Burk plot. The novel ACE inhibitory peptide VY-7 (at 2mg/kg body weight) as well as the crude hydrolysate of BMSPs (at 10 mg/kg body weight) showed significant and moderate antihypertensive effects, respectively, in an animal model of hypertension, spontaneously hypertensive rats (SHRs). The present work demonstrated the screening of ACE inhibitory peptides from BMSPs and, as far as we know, VY-7 is the first well-characterized antihypertensive peptide derived from bitter melon seeds.

Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation.

Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D ß-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis.

The structural analysis of post-translational modifications (PTMs) of lipoproteins is difficult due to the hydrophobic properties of their fatty acid moieties. At the present time, the relative positions of fatty acid components on the N-acyl-S-diacylglycerylcysteine core structure has not been specifically identified in any natural or bacterial expressed recombinant lipoproteins. In this study, we describe a rapid solid-phase extraction using acetonitrile and isopropanol method that can be performed manually to isolate large amounts of relatively pure lipopeptides generated by the limited tryptic-digestion of recombinant lipoproteins. Using these lipopeptides and LC/MS mass spectra analysis, two groups of N-terminal lipidated (diacyl or triacyl) molecules that differ by one fatty acid unit were successfully identified. This LC/MS method also provided the separation of lipopeptides differing by 14 Da for the on-line MS identification. Multiple-stage fragmentation analyses of the di- and triacyl lipopeptides using both the positive and negative ion modes enabled to identify the putative structure of the N-acyl-S-diacylglycerylcysteine containing an amide bond to palmitic acid at the N-terminal cysteine, a palmitic acid at sn1 position, and an unsaturated fatty acid of either hexadecenoic acid, cyclopropaneoctanoic acid, oleic acid and nonadecenoic acid at sn2 position of diacylglycerol residue through ester bonding. For diacyl lipoprotein, the saturated palmitoyl fatty acid group is absent at sn1 position of glycerol-derived lipid residue of lipopeptide.

Functional fluorinated modifications on a polyelectrolyte coated polydimethylsiloxane substrate for fabricating antibody microarrays.

Fluorinated compounds exhibit hydrophobic, nonstick, and self-cleaning properties, making them attractive for use as the coating material for biochips. In this study, we copolymerized the fluorinated compound 1H,1H,2H-perfluoro-1-decene (FD) with acrylic acid (AA) and bonded the resulting copolymer with protein G on the surface of polyelectrolyte coated polydimethylsiloxane (PDMS) to form a functional surface that captures antibodies. We demonstrated that the modified PDMS surface remained hydrophobic while becoming resistant to nonspecific protein binding. Thus, aqueous sample solutions formed the droplets (4 µL) on the surface without spreading and drying during the sample printing. Contact angle measurements showed that this functionalized surface was as hydrophobic as the native PDMS with a virtually constant contact angle over seven days of the study under dried condition at 4 °C. Spectroscopic measurements revealed that FD/AA copolymerization formed a homogeneous and highly dense multilayer (50 mg/mm(2)) with a fluorine coverage of 35.4%. Moreover, protein G was shown to covalently bind to AA molecules on the surface at a binding density of 0.24 µg/mm(2). We demonstrated that the fluorinated coating withstood nonspecific binding with extremely low background emission, leading to bioassays that, without the need of blocking agents, exhibited six times more sensitivity than PEG coatings. The fluorinated PDMS antibody microarrays were further applied to accurately determine the absolute concentration of ERα in MCF-7 cells. In conclusion, the unique properties of fluorinated compounds, such as withstanding wetting, nonspecific binding and contamination, make them an excellent coating material for use in sensitive and simple on-chip assays.

Functionalized 3D-hydrogel plugs covalently patterned inside hydrophilic poly(dimethylsiloxane) microchannels for flow-through immunoassays.

Integration of a hydrogel and polydimethylsiloxane (PDMS)-based microfluidic device can greatly reduce the cost of developing channel-based devices. However, there are technical difficulties including the hydrophobic and inert surface properties associated with PDMS as well as back pressure and fragile material associated with the use of hydrogel in microchannels. In this study, a strategy to covalently photopattern 3-D hydrogel plugs with functionalized protein G inside microfluidic channels on a hydrophilic PDMS substrate coated with polyelectrolyte multilayers (PEMS) is presented. In this process, a UV-light microscope is applied to initiate the protein G-poly(acryl amide) copolymerization from the bulk substrate to solution areas via the deeply implanted photoinitiator (PI), resulting in sturdy 3D plugs covalently bonded to the upper and lower channel wall, while leaving open spaces in the channel width for the fluid to flow through. In addition, the long-term hydrophilicity and low nonspecific binding property associated with PEMS surface can be conserved for the nonpatterned area, leading to hydrogel plugs in extremely hydrophilic and permeable environment in a restricted channel space for bubble-free fluid transport and affinity interaction. By immobilization of well-oriented antibodies via protein G on the hydrogel plugs in the channel, estrogen receptor alpha (ERalpha) is demonstrated to be captured quantitatively with high loading capacity and high specificity.

Heme protein assisted dispersion of gold nanoparticle multilayers on chips: from stabilization to high-density double-stranded DNAs fabricated in situ for protein/DNA binding.

Heme proteins in general are shown to be an effective linking agent in stabilizing gold nanoparticles (AuNPs) and thus facilitate the fabrication of three-dimensional (3D) AuNP multilayers on a chip, resulting in a higher coating density than that on polymer linker anchored surfaces for analytical applications. With the use of electron spectroscopy for chemical analysis (ESCA) measurements, a lower oxidation state of Au(0) and dramatic changes among multiple chemical states of N1s are detected upon coating AuNPs with heme proteins but not detected upon coating AuNPs with non-heme proteins. Thus, we propose that the stabilization power arises from pi-conjugation between AuNPs and the heme group. We also propose that such conjugation must be facilitated by the exposure of the heme group through a conformational change of the protein as well as interactions of other functional groups with AuNPs to bring the heme moiety to a close face-to-face distance with the AuNPs. A high-density double-stranded DNA (dsDNA) composed of a sequence of estrogen response element (ERE) is then fabricated on heme protein anchored chips. An in situ hybridization and tracking method is developed based on hybridization-induced fluorescence restoration associated with AuNPs and assists in the subsequent detection of DNA/protein binding on the same chip. The AuNP ERE chips are shown to have high sensitivity and specificity for quantitative detection of ERE binding with its two transcription factor isoforms, estrogen receptors alpha and beta (ERalpha and ERbeta), in cell lysates with reduced reagents and reaction time.

Poly(dimethylsiloxane)-based microfluidic device with electrospray ionization-mass spectrometry interface for protein identification.

An easy method to fabricate poly(dimethylsiloxane) (PDMS)-based microfluidic chips for protein identification by tandem mass spectrometry is presented. This microchip has typical electrophoretic microchannels, a flow-through sampling inlet, and a sheathless nanoelectrospray ionization (ESI) interface. The surface of the microchannel was modified with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and the generated electroosmotic flow under acidic buffer condition used for the separation was found to be more stable compared to that generated by the microchannel without modification. The feasibility of the device for flow-through sampling, separation, and ESI-MS/MS analysis was demonstrated by the analysis of a standard mixture composed of three tryptic peptides. Results show that four peaks corresponding to three peptide standards and acetylated products of the standard peptide were well resolved and the deduced sequences were consistent with those expected. Furthermore, the compatibility of this device with other miniaturized devices to integrate the whole process was also explored by connecting a miniaturized enzymatic digestion cartridge and a desalting cartridge in series to the sampling inlet of the microchip for the identification of a model protein, beta-casein.