Intramolecular Disulfide Bonds Research Articles

Biomimetic Remineralization of Dental Hard Tissues via Amyloid‐Like Protein Matrix Composite with Amorphous Calcium Phosphate

AbstractNumerous remineralizing coatings aim to prevent or treat early enamel lesions and occlude exposed dentinal tubules (DTs). Nevertheless, the pace of remineralization is inadequate, and the mechanical robustness of the newly established mineral layer fails to match the inherent strength. In this study, a biomimetic mineralization strategy aimed at replicating key events in biological mineralization, specifically focusing on the organic–inorganic composite matrix, is proposed. The material utilizes Tris(2‐carboxyethyl)phosphine (TCEP), which serves a dual role: stabilized amorphous calcium phosphate (ACP) (ACP@TCEP) nanoparticles as its inorganic component, and catalyzing the cleavage of intramolecular disulfide bonds in poly(ethylene glycol) (PEG) grafted lysozyme (lyso‐PEG) to facilitate the formation of an amyloid‐like protein matrix composite with ACP (ACP@lyso‐PEG nanocomplexes). ACP@lyso‐PEG nanocomplexes can rapidly and efficiently form an enamel‐like remineralization layer on the surface of damaged dental hard tissue, reaching ≈4.205 µm thickness after 3 days of acid‐etched enamel. Furthermore, achieving a depth of DTs occlusion exceeding 60 µm after 5 days, using a simple immersion process. The resulting mineralized layer exhibits mechanical strength comparable to natural teeth. This study introduces a conceptual biomimetic mineralization strategy for effective enamel repair or DTs occlusion in clinical practices, and offers potential insights into the mechanisms of biomineral formation.

A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders.

Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely Momordica charantia 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of de novo binders to drug targets of therapeutic relevance.

Abstract 4066: Generation of tumor targeted self-assembling split IL-12 subunits for the treatment of cancer

Abstract A major milestone in immuno-oncology was the development of immune checkpoint inhibitors, resulting in significant benefits for numerous patients. However, many patients are not responsive, become refractory, or cannot tolerate the treatment due to toxicity or side effects, demonstrating the need for additional immunotherapies. Cytokines are a class of promising immunomodulatory proteins being explored as therapeutics, but their success has been limited due to their rapid clearance or pleiotropic properties. Classically known as ‘signal 3’ of an endogenous immune response, IL-12p70 is a heterodimeric cytokine comprising p35 and p40 subunits. When administered exogenously, IL-12 is a potent stimulator of the immune system and can have profound anti-tumor activity; however, the clinical use of IL-12 has been limited due to poor systemic tolerability. Therefore, developing an immunotherapy that can be systemically administered and improve the therapeutic index could capitalize on the potential clinical benefit of IL-12. Mural Oncology has developed an innovative approach to mitigate the toxicity of IL-12 by splitting the heterodimer into inactive p35 and p40 monomers. The individual subunits are separately fused to two non-competitive antibody fragments targeting a highly expressed tumor-associated antigen. The goal of this approach is to conditionally activate IL-12 preferentially in the tumor microenvironment (TME). This is achieved by sequential administration of the targeted subunits, which will drive assembly and activity of the IL-12 heterodimer primarily at the tumor site, reducing systemic exposure and thereby potentially reducing associated toxicities. To engineer the split IL-12 subunits, a structure-based rational design approach was used to remove the natural intra-molecular disulfide bond and generate variants that maintained affinity between the p35 and p40 monomers. The engineered subunits were characterized using analytical methods and screened for the pair with the strongest affinity. Additionally, the candidate pair was assessed for receptor binding and functional activity on human T cells through phosphorylation of STAT4 and secretion of IFN-γ. Pharmacokinetics and pharmacodynamics were evaluated in mouse models with and without tumors demonstrating that dual targeting led to an increase in tumor retention, rapid clearance from circulation, and dose dependent release of IFN-γ. The results thus far demonstrate that the innovative approach of tumor targeted self-assembling split IL-12 has the potential to enhance the overall therapeutic potential of this cytokine. A development candidate will be identified and moved into clinical testing in cancer patients to enable us to fully understand if this design approach holds true in the clinical setting. Citation Format: Joshua Heiber, Pinar Gurel, Robert G. Newman, Kristiana Dreaden, Yanchun Zhao, Chunhua Wang, Su-Ping Pearson, Jean Chamoun. Generation of tumor targeted self-assembling split IL-12 subunits for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4066.

Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet β cells, which modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single-nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ∼8-fold and ∼3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Cyclization of Peptides Enhances the Inhibitory Activity against Ganglioside-Induced Aβ Fibril Formation.

Alzheimer's disease is a progressive neurodegenerative disease and is the most common cause of dementia. It has been reported that the assembly of amyloid β-protein (Aβ) on the cell membrane is induced by the interaction of the Aβ monomer with gangliosides such as GM1. The ganglioside-bound Aβ (GAβ) complex acts as a seed to promote the toxic assembly of the Aβ fibrils. In a previous study, we found that a GM1 cluster-binding peptide (GCBP) specifically recognizes Aβ-sensitive ganglioside nanoclusters and inhibits the assembly of Aβ on a GM1-containing lipid membrane. In this study, cysteine-substituted double mutants of GCBP were designed and cyclized by intramolecular disulfide bond formation. Affinity assays indicated that one of the cyclic peptides had a higher affinity to a GM1-containing membrane compared to that of GCBP. Furthermore, surface topography analysis indicated that this peptide recognizes GM1 nanoclusters on the lipid membrane. An evaluation of the inhibitory kinetics indicated that the cyclic peptide could inhibit the formation of Aβ fibrils with an IC50 value of 1.2 fM, which is 10,000-fold higher than that of GCBP. The cyclic peptide was also shown to have a clearance effect on Aβ fibrils deposited on the lipid membrane and suppressed the formation of toxic Aβ assemblies. Our results indicate that the cyclic peptide that binds to the Aβ-sensitive ganglioside nanocluster is a potential novel inhibitor of ganglioside-induced Aβ assembly.

Effects of electrostatic field-assisted freezing on the functional properties and aggregation behavior of gluten

Changes in gluten during the freezing process could cause a deterioration in dough quality, which would be solved by electrostatic field treatment. This paper investigated the effects of electrostatic field assisted freezing (EFAF) on the functional properties, structural characteristics and aggregation behavior of gluten. The results showed that EFAF improved the functional properties of gluten with enhanced water holding, emulsifying and foaming properties. When the electrostatic field was applied, the α-helix content and g-g-g conformation of gluten proteins increased with a maximum of 27.50 % and 40.05 %, respectively, which were significantly different from the control (P < 0.05). The degree of depolymerization of gluten macromolecules decreased when the electrostatic field was applied, and was obtained to be the lowest at 600 V with 5.71 %, which was significantly different from that without applied electrostatic field (P < 0.05). After EFAF, AFM analysis showed a more intact glutenin chain structure and smaller particle size of the gliadin. These results indicated that EFAF improved the functional properties of gluten proteins by the mechanism of slowing down the depolymerization of gluten macromolecules, maintaining the chain structure of gluten, stabilizing the intramolecular disulfide bonds of gliadin, and stabilizing the microstructure of gluten.

SAT023 The Role Of Cysteine Residues In The Binding Of Inhibitors Of The N-Terminal Domain Of The Androgen Receptor

Abstract Disclosure: L. Deneault: None. A. Tien: None. A. Banuelos: Stock Owner; Self; ESSA. T. Tam: None. R. Andersen: Stock Owner; Self; ESSA. M.D. Sadar: Stock Owner; Self; ESSA. Background: Therapies for prostate cancer (PC) involve pharmaceutical castration by targeting the androgen receptor (AR) ligand-binding domain (LBD). These therapies are not curative with the malignancy progressing to castration-resistant PC (CRPC). Most CRPC remains driven by AR through multiple resistances, e.g. constitutively active AR splice variants that lack LBD (AR-Vs), or gain-of-function mutations in LBD. This has led to clinical development of EPI analogues (“EPI”) which target the AR N-terminal domain (NTD) to block the transcriptional activities of, AR-Vs and mutated ARs. The EPI-binding pocket involves region 341-446 amino acid residues of the NTD. AR NTD is enriched in cysteine residues with C404 suggested to be important in the binding mechanism of EPI. Free cysteine residues are able interact forming disulfide bonds, which are important in intra- and intermolecular protein-protein interactions. Here we begin to elucidate the role of cysteines in the inhibition of AR activity by EPI. Methods: Binding assays assess the role of cysteines in the covalent binding mechanism used a fluorescein-labelled EPI with fragments of recombinant AR-NTD (rNTD) with or without the addition of iodoacetamide (caps cysteines). Protein-protein interactions requiring disulfide bonds with AR examined by non-reducing SDS-PAGE and diagonal SDS-PAGE of cells treated with inhibitors. A role of specific cysteines in the activity of EPI to block AR transcriptional activity was assessed by cysteine mutants using a reporter gene construct driven by AR. Results: Capping cysteine residues on rNTD with iodoacetamide reduced the amount of covalent binding of EPI. This data suggests that cysteines are essential for covalent binding of EPI to AR-NTD. EPI bound preferentially to the oxidized form of rNTD suggesting the importance of disulfide bridges. Diagonal SDS-PAGE revealed differential alterations in intramolecular and intermolecular disulfide bonds in response to EPI compared to AR LBD inhibitors. These gels also revealed unique differences in protein-protein interactions with AR depending on the inhibitor. AR-driven reporter gene assays revealed mutations of cysteine residues in the AR- NTD did not adversely affect AR transcriptional activity in response to androgen. Conclusions: Although cysteines residing in the AR-NTD were required for covalent binding of EPI, mutation of C404 in the EPI-binding site was not essential for the efficacy of EPI to block AR transcriptional activity. There were differential patterns in protein-protein interactions with AR depending upon which domain the inhibitor targeted. Our future goal is to identify these unique proteins to provide insight into the mechanisms and develop more potent and more specific therapeutics for the treatment of CRPC. Presentation: Saturday, June 17, 2023

Oxidized and Reduced Dimeric Protein Complexes Illustrate Contrasting CID and SID Charge Partitioning.

Charge partitioning during the dissociation of protein complexes in the gas phase is influenced by many factors, such as interfacial interactions, protein flexibility, protein conformation, and dissociation methods. In the present work, two cysteine-containing homodimer proteins, β-lactoglobulin and α-lactalbumin, with the disulfide bonds intact and reduced, were used to gain insight into the charge partitioning behaviors of collision-induced dissociation (CID) and surface-induced dissociation (SID) processes. For these proteins, we find that restructuring dominates with CID and dissociation with symmetric charge partitioning dominates with SID, regardless of whether intramolecular disulfide bonds are oxidized or reduced. CID of the charge-reduced dimeric protein complex leads to a precursor with a slightly smaller collision cross section (CCS), greater stability, and more symmetrically distributed charges than the significantly expanded form produced by CID of the higher charged dimer. Collision-induced unfolding plots demonstrate that the unfolding-restructuring of the protein complexes initiates the charge migration of higher charge-state precursors. Overall, gas collisions reveal the charge-dependent restructuring/unfolding properties of the protein precursor, while surface collisions lead predominantly to more charge-symmetric monomer separation. CID's multiple low-energy collisions sequentially reorganize intra- and intermolecular bonds, while SID's large-step energy jump cleaves intermolecular interfacial bonds in preference to reorganizing intramolecular bonds. The activated population of precursors that have taken on energy without dissociating (populated in CID over a wide range of collision energies, populated in SID for only a narrow distribution of collision energies near the onset of dissociation) is expected to be restructured, regardless of the activation method.

Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticles for the Treatment of Hepatitis.

Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.

A Hairpin Motif in the Amyloid-β Peptide Is Important for Formation of Disease-Related Oligomers.

The amyloid-β (Aβ) peptide is associated with the development of Alzheimer's disease and is known to form highly neurotoxic prefibrillar oligomeric aggregates, which are difficult to study due to their transient, low-abundance, and heterogeneous nature. To obtain high-resolution information about oligomer structure and dynamics as well as relative populations of assembly states, we here employ a combination of native ion mobility mass spectrometry and molecular dynamics simulations. We find that the formation of Aβ oligomers is dependent on the presence of a specific β-hairpin motif in the peptide sequence. Oligomers initially grow spherically but start to form extended linear aggregates at oligomeric states larger than those of the tetramer. The population of the extended oligomers could be notably increased by introducing an intramolecular disulfide bond, which prearranges the peptide in the hairpin conformation, thereby promoting oligomeric structures but preventing conversion into mature fibrils. Conversely, truncating one of the β-strand-forming segments of Aβ decreased the hairpin propensity of the peptide and thus decreased the oligomer population, removed the formation of extended oligomers entirely, and decreased the aggregation propensity of the peptide. We thus propose that the observed extended oligomer state is related to the formation of an antiparallel sheet state, which then nucleates into the amyloid state. These studies provide increased mechanistic understanding of the earliest steps in Aβ aggregation and suggest that inhibition of Aβ folding into the hairpin conformation could be a viable strategy for reducing the amount of toxic oligomers.

The role of disulfide bonds in the formation of the spatial structure of the human epidermal growth factor

The epidermal growth factor (EGF) is a peptide of the EGF-like growth factor family with a common conserved EGF-like domain formed by three intramolecular disulfide bonds. This article describes changes in the spatial structure of EGF and its mutant form with the D46G substitution in its C-terminal fragment observed upon disulfide bonds reduction in the corresponding synthetic peptides in a 0.01 M phosphate buffer (pH = 7.4). The structure was analyzed using circular dichroism spectroscopy, spectrofluorimetry, native polyacrylamide gel electrophoresis, and centrifugal ultrafiltration. It was shown that disulfide bonds reduction changes the geometry of the EGF-like domain towards an increase in the content of the beta-structure, while these peptides remain in dimeric form. According to the molecular modeling results, this can lead to the elongation of the main beta-hairpin of the EGF-like domain, to the elongation of the intermolecular beta-structure, or to the formation of a new beta-structure between the N- and C-terminal fragments of each molecule, which will change the intermolecular interface in dimeric form. Disulfide bonds reduction prevents EGF dimer dissociation to monomers. Under physiological conditions, this can lead to the inability of EGF to form binding sites for EGFR (epidermal growth factor receptor) and to cause its activation.

Characterization of a novel cysteine-less Cu/Zn-superoxide dismutase in Paenibacillus lautus missing a conserved disulfide bond

Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.

Identification of the SARS-unique domain of SARS-CoV-2 as an antiviral target

SARS-CoV-2 nsp3 is essential for viral replication and host responses. The SARS-unique domain (SUD) of nsp3 exerts its function through binding to viral and host proteins and RNAs. Herein, we show that SARS-CoV-2 SUD is highly flexible in solution. The intramolecular disulfide bond of SARS-CoV SUD is absent in SARS-CoV-2 SUD. Incorporating this bond in SARS-CoV-2 SUD allowed crystal structure determination to 1.35 Å resolution. However, introducing this bond in SARS-CoV-2 genome was lethal for the virus. Using biolayer interferometry, we screened compounds directly binding to SARS-CoV-2 SUD and identified theaflavin 3,3’-digallate (TF3) as a potent binder, Kd 2.8 µM. TF3 disrupted the SUD-guanine quadruplex interactions and exhibited anti-SARS-CoV-2 activity in Vero E6-TMPRSS2 cells with an EC50 of 5.9 µM and CC50 of 98.5 µM. In this work, we provide evidence that SARS-CoV-2 SUD harbors druggable sites for antiviral development.

Stable Mammalian Serum Albumins Designed for Bacterial Expression

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.

Amyloid-Like Assembly to Form Film at Interfaces: Structural Transformation and Application.

Protein-based biomaterials are attracting broad interest for their remarkable structural and functional properties. Disturbing the native protein's three-dimensional structural stability in vitro and controlling subsequent aggregation is an effective strategy to design and construct protein-based biomaterials. One of the recent developments in regulating protein structural transformation to ordered aggregation is amyloid assembly, which generates fibril-based 1D to 3D nanostructures as functional materials. Especially, the amyloid-like assembly to form films at interfaces has been reported, which is induced by the effective reduction of the intramolecular disulfide bond. The main contribution of this amyloid-like assembly is the large-scale formation of protein films at interfaces and excellent adhesion to target substrates. This review presents the research progress of the amyloid-like assembly to form films and related applications and thereby provides a guide to exploiting protein-based biomaterials. This article is protected by copyright. All rights reserved.

Characterization and mechanism of action of amphibian-derived wound-healing-promoting peptides.

Wound-healing-promoting peptides are excellent candidates for developing wound-healing agents due to their small size and low production cost. Amphibians are one of the major sources of bioactive peptides, including wound-healing-promoting peptides. So far, a series of wound-healing-promoting peptides have been characterized from amphibians. We hereby summarized the amphibian-derived wound-healing-promoting peptides and their mechanism of action. Among these peptides, two peptides (tylotoin and TK-CATH) were characterized from salamanders, and twenty five peptides were characterized from frogs. These peptides generally have small sizes with 5-80 amino acid residues, nine peptides (tiger17, cathelicidin-NV, cathelicidin-DM, OM-LV20, brevinin-2Ta, brevinin-2PN, tylotoin, Bv8-AJ, and RL-QN15) have intramolecular disulfide bonds, seven peptides (temporin A, temporin B, esculentin-1a, tiger17, Pse-T2, DMS-PS2, FW-1, and FW-2) are amidated at the C-terminus, and the others are linear peptides without modifications. They all efficiently accelerated the healing of skin wounds or photodamage in mice or rats. They selectively promoted the proliferation and migration of keratinocytes and fibroblasts, recruited neutrophils and macrophages to wounds, and regulated the immune response of neutrophils and macrophages in wounds, which were essential for wound healing. Interestingly, MSI-1, Pse-T2, cathelicidin-DM, brevinin-2Ta, brevinin-2PN, and DMS-PS2 were just antimicrobial peptides, but they also significantly promoted the healing of infected wounds by clearing off bacteria. Considering the small size, high efficiency, and definite mechanism, amphibian-derived wound-healing-promoting peptides might be excellent candidates for developing novel wound-healing-promoting agents in future.

Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.

The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH2 , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1-linear is a prodrug of EMP1, which is a performance-enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1-linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1-linear mainly existed in its cyclic form as EMP1, and four N-terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG-NH2 (M1), SCHFGPLTWVCKPQGG-NH2 (M2), WVCKPQGG-NH2 (M3) and VCKPQGG-NH2 (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1-linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1-linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively.

Identification of antibacterial activity of LEAP2 from Antarctic icefish Chionodraco hamatus.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a small peptide, which is consisted of signal peptide, pro-peptide and the bioactive mature peptide. Mature LEAP2 is an antibacterial peptide with four highly conserved cysteines forming two intramolecular disulfide bonds. Chionodraco hamatus, an Antarctic notothenioid fish that lives in the coldest water, has white blood unlike most fish of the world. In this study, the LEAP2 coding sequence was cloned from C. hamatus, including a 29 amino acids signal peptide and mature peptide of 46 amino acids. High levels of LEAP2 mRNA were detected in the skin and liver. Mature peptide was obtained by chemical synthesis in vitro, displayed selective antimicrobial activities against Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus and Streptococcus agalactiae. Liver-expressed antimicrobial peptide 2 showed bactericidal activity by destroying the cell membrane integrity and robustly combined with bacterial genomic DNA. In addition, overexpression of the Tol-LEAP2-EGFP in zebrafish larva showed stronger antimicrobial activity in C. hamatus than in zebrafish, accompanied by lower bacterial load and expression of pro-inflammatory factors. This is the first demonstration of the antimicrobial activity of LEAP2 from C. hamatus, which is of useful value in improving resistance to pathogens.