Synthetic Antibiotic Derived from Sequences Encrypted in a Protein from Human Plasma
Encrypted peptides have been recently found in the human proteome and represent a potential class of antibiotics. Here we report three peptides derived from the human apolipoprotein B (residues 887-922) that exhibited potent antimicrobial activity against drug-resistant Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococci both in vitro and in an animal model. The peptides had excellent cytotoxicity profiles, targeted bacteria by depolarizing and permeabilizing their cytoplasmic membrane, inhibited biofilms, and displayed anti-inflammatory properties.
Importantly, the peptides, when used in combination, potentiated the activity of conventional antibiotics against bacteria and did not select for bacterial resistance. To ensure translatability of these molecules, a protease resistant retro-inverso variant of the lead encrypted peptide was synthesized and demonstrated anti-infective activity in a preclinical mouse model. Our results provide a link between human plasma and innate immunity and point to the blood as a source of much-needed antimicrobials.
Protein Chemical Modification Using Highly Reactive Species and Spatial Control of Catalytic Reactions
Protein bioconjugation has become an increasingly important research method for introducing artificial functions in to protein with various applications, including therapeutics and biomaterials. Due to its amphiphilic nature, only a few tyrosine residues are exposed on the protein surface. Therefore, tyrosine residue has attracted attention as suitable targets for site-specific modification, and it is the most studied amino acid residue for modification reactions other than lysine and cysteine residues. In this review, we present the progress of our tyrosine chemical modification studies over the past decade. We have developed several different catalytic approaches to selectively modify tyrosine residues using peroxidase, laccase, hemin, and ruthenium photocatalysts.
In addition to modifying tyrosine residues by generating radical species through single-electron transfer, we have developed a histidine modification method that utilizes singlet oxygen generated by photosensitizers. These highly reactive chemical species selectively modify proteins in close proximity to the enzyme/catalyst. Taking advantage of the spatially controllable reaction fields, we have developed novel methods for site-specific antibody modification, detecting hotspots of oxidative stress, and target identification of bioactive molecules.
Determination of low molecular thiols and protein sulfhydryl groups using heterocyclic disulfides
A promising area in the analytical chemistry of thiol-containing compounds is the use of heterocyclic disulfides as analytical agents, but now only a few of them are widely used. In this paper, we evaluate the possibility of using three different heterocyclic disulfides 2,2′-dithiobis[5-phenyl-1,3,4-oxadiazole] (I), 2,2′-dithiobis[benzoxazole] (II) and 8,8′-dithiobis-quinoline (III) as analytical reagents for the low-mass aminothiols cysteine and glutathione determination. The optimal analysis conditions were found. Spectrophotometric, kinetic, CE, and HPLC methods using I, II, III for the determination of cysteine and glutathione were developed.
The obtained methods are characterized by accuracy and sensitivity (detection limits in the range of 10-5-10-6 M) sufficient to quantify cysteine and glutathione in their physiological concentrations. Finally, the proposed disulfides were used to determine the SH-content in the bovine serum albumin (BSA). Considering a number of criteria (applicable pH range, absorption properties, susceptibility to hydrolysis) it was concluded that the proposed reagents have advantages over the commonly used ones (such as the Ellman reagent).
Expression of chimeric proteins based on a backbone of the GII.4 norovirus VP1 and their application in the study of a GII.6 norovirus-specific blockade epitope
- The surface-exposed loop regions of the protruding domain of the norovirus (NoV) major capsid protein VP1 can tolerate the insertion of foreign antigens without affecting its assembly into subviral particles. In this study, we investigated the tolerance of the surface-exposed loop region of the GII.4 NoV VP1 by replacing it with homologous or heterologous sequences. We designed a panel of constructs in which the amino acid sequence from position 298-305 of the GII.4 NoV VP1 was replaced by sequences derived from the same region of GI.3, GII.3, GII.6, and GII.17 NoVs as well as neutralizing epitopes of enterovirus type 71 and varicella-zoster virus.
- The constructs were synthesized and expressed using a recombinant baculovirus expression system. The expression of target proteins was measured by indirect enzyme-linked immunosorbent assay (ELISA), and the assembly of virus-like particles (VLPs) was confirmed by electron microscopy. Our results showed that all of the constructs expressed high levels of target chimeric proteins, and all of the chimeric proteins successfully assembled into VLPs or subviral particles. An in vitro VLP-histo-blood group antigen (HBGA) binding assay revealed that chimeric-protein-containing VLPs did not bind or showed reduced binding to salivary HBGAs, a ligand for NoV particles.
- The results of an in vitro VLP-HBGA binding blockade assay indicated that the predicted surface-exposed loop region of the GII.6 NoV VP1 may comprise a blockade epitope. In summary, the surface-exposed loop region of the GII.4 NoV VP1 can be replaced by foreign sequences of a certain length. Using this strategy, we found that the predicted surface-exposed loop region of GII.6 NoV VP1 might contain a blockade epitope
Protein-Export Protein SecB (SecB) Protein |
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| 20-abx261442 | Abbexa |
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Protein-Export Protein SecB (SecB) Protein |
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| abx261442-100g | Abbexa | 100 µg | 3550 EUR |
Protein-Export Protein SecB (SecB) Protein |
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| abx261442-10g | Abbexa | 10 µg | 325 EUR |
Protein-Export Protein SecB (SecB) Protein |
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| abx261442-2g | Abbexa | 2 µg | 225 EUR |
Protein A Protein |
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| abx069907-100mg | Abbexa | 100 mg | 560.4 EUR |
Protein A Protein |
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| abx061461-10mg | Abbexa | 10 mg | 526.8 EUR |
Protein A protein |
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| 30R-1304 | Fitzgerald | 100 ug | 275 EUR |
Protein G Protein |
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| 20-abx262181 | Abbexa |
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Protein L Protein |
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| 20-abx262993 | Abbexa |
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Protein L Protein |
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| 20-abx262994 | Abbexa |
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Protein G Protein |
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| 20-abx260044 | Abbexa |
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Protein A Protein |
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| abx061461-100g | Abbexa | 100 µg | Ask for price |
Protein A Protein |
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| abx061461-10g | Abbexa | 10 µg | 825 EUR |
Protein A Protein |
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| abx061461-50g | Abbexa | 50 µg | Ask for price |
Protein G Protein |
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| abx262181-25mg | Abbexa | 2.5 mg | 325 EUR |
Protein G Protein |
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| abx260044-20g | Abbexa | 20 µg | 487.5 EUR |
Protein G Protein |
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| abx260044-5g | Abbexa | 5 µg | 225 EUR |
Protein L Protein |
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| abx262993-25mg | Abbexa | 25 mg | 1350 EUR |
Protein L Protein |
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| abx262993-5mg | Abbexa | 5 mg | 225 EUR |
Protein L Protein |
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| abx262994-25mg | Abbexa | 25 mg | 1350 EUR |
Protein L Protein |
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| abx262994-5mg | Abbexa | 5 mg | 225 EUR |
pLDH Protein Protein |
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| abx061407-1mg | Abbexa | 1 mg | 3166.8 EUR |
pLDH Protein Protein |
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| abx061408-1mg | Abbexa | 1 mg | 3166.8 EUR |
pLDH Protein Protein |
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| abx061407-100g | Abbexa | 100 µg | Ask for price |
pLDH Protein Protein |
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| abx061407-10g | Abbexa | 10 µg | 3175 EUR |
pLDH Protein Protein |
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| abx061407-50g | Abbexa | 50 µg | Ask for price |
pLDH Protein Protein |
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| abx061408-100g | Abbexa | 100 µg | Ask for price |
pLDH Protein Protein |
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| abx061408-10g | Abbexa | 10 µg | 3175 EUR |
Roles of high mobility group box 1 protein released from endothelial cells with hypoxic injury on neuronal amyloidogenesis
Background: Hypoxia-reperfusion injury is one of the major risk factors for neurodegeneration. However, it is unclear whether ischaemic damage in brain microvascular endothelial cells plays roles in neurodegeneration, particularly in the amyloidogenic changes contributing to the development of Alzheimer’s disease (AD) pathologies. Therefore, we investigated the roles of hypoxia-reoxygenation (H/R)-induced release of high mobility group box protein 1 (HMGB1), a risk molecule for AD pathogenesis in the ischaemic damaged brain, from human brain microvascular endothelial cells (HBMVECs) in neuronal amyloid beta (Aβ) production.
Method: H/R experiments were carried out in a purpose-built hypoxia glove-box chamber with continuous monitoring of O2 concentration. HBMVECs were treated with hypoxia (0.5% O2 ) and glucose deprivation for 1 h, and then the cells were restored to 37 °C with fresh M199 medium in a humidified atmosphere of 95% air and 5% CO2 . Endothelial permeability was assessed by measuring FITC-tagged dextran flux across monolayers of cultured HBMVECs. H4swe or SH-SY5Y cells were exposed to CM from HBMVECs with or without H/R for 24 h. Aβ40 and Aβ42 levels in the CM of H4swe cells were determined with commercial ELISA kits. Transfections of HMGB1 or Sirt1 siRNAs were performed using RNAi Max. Plasmids encoding Flag-tagged WT or dominant negative (DN) mutant H363Y SIRT1 were transfected using Lipofectamine.
Result: H/R increased nuclear-cytosolic translocation and secretion of HMGB1 in HBMVECs, along with increased permeability and HMGB1-dependent p-c-Jun activation. In addition, H/R increased the expression of Sirtuin 1 (Sirt1), coincident with an increase of intracellular Sirt1-HMGB1 binding in HBMVECs. H/R increased the acetylation of HMGB1 and extracellular secretion, which was significantly inhibited by Sirt1 overexpression. Furthermore, Sirt1 contributed to autophagy-mediated endogenous HMGB1 degradation. More importantly, treatment of neuronal cells with conditioned medium from H/R-stimulated HBMVECs (H/R-CM) activated their amyloidogenic pathways. The neuronal amyloidogenic changes (i.e., increased levels of extracellular Aβ40 and Aβ42) by H/R-CM from HBMVECs were further increased by Sirt1 inhibition, which was significantly suppressed by neutralization of the HMGB1 in H/R-CM.
Conclusion: Our results suggest that HMGB1 derived from H/R-stimulated HBMVECs contributes to amyloidogenic pathways in neurons playing roles in the pathogenesis of AD, which are regulated by endothelial Sirt1.