Peptide Mixtures Research Articles

ECM derivatized alginate augmenting bio-functionalities of lyophilized mat for skin and liver wound treatment

Peptides and molecular residues sourced from the fragmentation of the extracellular matrix (ECM) can exacerbate a plethora of cellular functions. We selected a natural ECM-derived complex peptide mixture to functionalize sodium alginate. Three alginate derivatives (sodium alginate conjugated with ECM) SALE-1, SALE-2, and SALE-3 were synthesized using the lowest (10 % w/w), moderate (50 % w/w), and highest (100 % w/w) concentrations of ECM. Thereafter, they were used to fabricate three groups of mat scaffolds EMAT-1 (ECM derivatized alginate thrombin-mat), EMAT-2, and EMAT-3, respectively by the freeze-drying process. To enhance the hemostatic activity, thrombin was loaded onto the scaffolds. Another group, AT, without any derivatized alginate was additionally included in order to comparative analysis. Physical characteristics revealed that the porous mat scaffold showed enhancement in degradation and swelling ability with the increase in ECM content. The higher cell proliferation, migration, and cell viability were noticed in the higher ECM-containing samples EMAT-2 and EMAT-3. In vivo studies using rodent hepatic and rabbit ear models were carried out to ensure the hemostatic ability of the scaffolds. EMAT-2 and EMAT-3 demonstrate excellent liver regeneration ability in rat models. Moreover, the rat cutaneous wound model depicted that EMAT-3 dramatically elevated the skin's healing ability, thereby rendering it an excellent candidate for future clinical application in wound healing.

Atomistic insights into the reentrant phase-transitions in polyuracil and polylysine mixtures.

The phase separation of protein and RNA mixtures underpins the assembly and regulation of numerous membraneless organelles in cells. The ubiquity of protein-RNA condensates in cellular regulatory processes is in part due to their sensitivity to RNA concentration, which affects their physical properties and stability. Recent experiments with poly-cationic peptide-RNA mixtures have revealed closed-loop phase diagrams featuring lower and upper critical solution temperatures. These diagrams indicate reentrant phase transitions shaped by biomolecular interactions and entropic forces such as solvent and ion reorganization. We employed atomistic simulations to study mixtures with various RNA-polylysine stoichiometries and temperatures to elucidate the microscopic driving forces behind reentrant phase transitions in protein-RNA mixtures. Our findings reveal an intricate interplay between hydration, ion condensation, and specific RNA-polylysine hydrogen bonding, resulting in distinct stoichiometry-dependent phase equilibria governing stabilities and structures of the condensate phase. Our simulations show that reentrant transitions are accompanied by desolvation around the phosphate groups of RNA, with increased contacts between phosphate and lysine side chains. In RNA-rich systems at lower temperatures, RNA molecules can form an extensive pi-stacking and hydrogen bond network, leading to percolation. In protein-rich systems, no such percolation-induced transitions are observed. Furthermore, we assessed the performance of three prominent water force fields-Optimal Point Charge (OPC), TIP4P-2005, and TIP4P-D-in capturing reentrant phase transitions. OPC provided a superior balance of interactions, enabling effective capture of reentrant transitions and accurate characterization of changes in solvent reorganization. This study offers atomistic insights into the nature of reentrant phase transitions using simple model peptide and nucleotide mixtures. We believe that our results are broadly applicable to larger classes of peptide-RNA mixtures exhibiting reentrant phase transitions.

Enhancement of Tumor Antigen-specific T-cell Responses by Immune Checkpoint Blockade in Human Papillomavirus-related Head and Neck Squamous Cell Carcinoma.

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically and immunologically distinct from HPV-negative HNSCC. Herein, we investigated the presence of tumor antigens HPV E6/E7 and wild-type p53-specific T-cell responses, and the impact of immune checkpoint blockade in patients with HPV-positive HNSCC. Peripheral blood mononuclear cells (PBMCs) from patients with HPV-positive HNSCC were stimulated with HPV E6/E7 or wild-type p53-derived peptide mixture and evaluated using the interferon-γ enzyme-linked immunosorbent spot assay. Flow cytometry was performed to analyze the proportion of T-cell subsets and T cells expressing immune checkpoint molecules. HPV E6/E7-specific T cells were detected in 22 (95.7%) of 23 patients, whereas wild-type p53-specific T cells were detected in 3 (15.0%) of 20 patients. Seven (43.8%) of 16 patients exhibited wild-type p53-specific T-cell responses, as determined using whole proteins instead of peptides. Immune checkpoint blockade enhanced wild-type p53-specific T-cell responses in 9 (45.0%) of 20 patients. Flow cytometric analysis of PBMCs revealed that responders exhibiting enhanced wild-type p53-specific T-cell responses following immune checkpoint blockade had a significantly higher proportion of Ki-67+CD4+ T cells, Ki-67+CD8+ T cells, regulatory T cells, PD-1+CD4+ T cells, and TIM-3+CD4+ T cells than non-responders. Our findings indicate that tumor antigen-specific T cells are present in the peripheral blood of patients with HPV-positive HNSCC. Blockade of checkpoint pathways can enhance T-cell responses in certain patients, probably via activated T cells, Tregs, and/or exhausted CD4+ T cells.

Human Papillomavirus Type 16 Based L1, L2, E6, and E7 Peptide Microspheres Induce Encapsulated Peptide Mixture Specific Cytotoxic T Lymphocytes and Tumor Regression in a Murine Model of Cervical Cancer

Human Papillomavirus Type 16 Based L1, L2, E6, and E7 Peptide Microspheres Induce Encapsulated Peptide Mixture Specific Cytotoxic T Lymphocytes and Tumor Regression in a Murine Model of Cervical Cancer

On the utility of the extracted ion chromatograms for assigning the conjugation sites and side reactions in bioconjugates synthesized by the maleimide-thiol chemistry

Maleimide-thiol chemistry is widely used in the synthesis of protein–protein conjugate vaccines. In this reaction, thiol and maleimide groups, either in the carrier protein or in the antigen molecules, are linked via a thiosuccinimide linker. The assignment of the conjugation sites is key and is usually done by LC-MS/MS analysis of the conjugate proteolytic digestion, resulting in a very complex mixture of linear peptides and cross-linked peptide pairs. During proteolysis, the thiosuccinimide linker is converted to its hydrolyzed and transcyclized forms. The set of MS/MS spectra assigned to cross-linked peptide pairs contains valuable information about the conjugation sites that must be validated using objective criteria for more reliable assignment. The extracted ion chromatograms (XICs) have not previously been considered for these validation purposes. In the LC-MS/MS analysis, linear peptides eluted with a single-peak XIC pattern, while the cross-linked peptide pairs with a transcyclized linker (consisting of a mixture of diastereomers) eluted with a two-peak XIC pattern in 68–100 % of cases. Cross-linked peptide pairs with the hydrolyzed linker were detected with a multi-peak XIC pattern consisting of two, three and four peaks in 77 %, 15 % and 8 % of cases, respectively. The four-peak XIC pattern corresponds to cross-linked peptide pairs with two positional isomers of the hydrolyzed linker containing a mixture of diastereomers. Tandem digestions increased the analytical value of the XICs by converting the large and hydrophobic cross-linked peptide pairs, that show a single-peak XIC pattern, to more hydrophilic species eluting in multi-peak XIC patterns. XICs are easily obtained in any LC-MS/MS analysis, contain valuable information about conjugation sites and side reaction detection, and are worthy of routine inclusion in bioconjugate characterization.

Ion Mobility Separations Using Cocentric Architecture.

Ion mobility separations, especially using drift tube ion mobility spectrometers, are usually performed in linear channels, which can have a large footprint when extended to achieve higher resolving powers. In this work, we explored the performance of an ion mobility device with a curved architecture, which can have a more compact form. The cocentric ion mobility spectrometer (CoCIMS) manipulates ions between two cocentric surfaces containing a serpentine track. The mobility separation inside the CoCIMS is achieved using traveling waveforms (TWs). We initially evaluated the device using ion trajectory simulations using SIMION, which indicated that when ions traveled circularly inside the CoCIMS they resulted in similar resolving powers and transmitted m/z range as traveling in a straight path. We then performed experimental validation of the CoCIMS in conjunction with a TOF MS. The CoCIMS was made of two flexible printed circuit board materials folded into cocentric cylinders separated by a gap of 2.8 mm. The device was about 50 mm diameter ×152 mm long and provided 1.846 m of serpentine path length. Three sets of mixtures (Agilent tune mixture, tetraalkylammonium salts, and an eight-peptide mixture) and four traveling waveform profiles (square, sine, triangle, and sawtooth) were used. The sawtooth TW profile produced a slightly higher resolving power for the Agilent tuning mixture and tetraalkylammonium ions. The average resolving power for Agilent tune mixture ions ranged from 37 (using sawtooth TW) to 27 (using square TW). The average resolving powers ranged from 45 (sawtooth TW) to 31 (square TW) for tetraalkylammonium ions. The resolving power of the peptide mixture ions was similar among the four TW profiles and ranged from 51 to 56. The average percent error in TWCCS for the peptide mixture ions was about 0.4%. The new device showed promising results, but improvements are needed to further increase the resolving power.

Isolation of peptide mixtures with potential biological activities from the protein hydrolysate of Arsenura armida larval meal

Abstract This study explores using insects as a high-quality protein source rich in bioactive peptides for human nutrition. The objectives include establishing optimal conditions for peptide extraction, identifying their sequences, and assessing their potential as antioxidants and antimicrobial agents in Arsenura armida flour. The protein content in the A. armida flour extract was 54%. To obtain potential antioxidant and antimicrobial peptides, a 10:1 and 1:1 enzyme (pepsin)/substrate ratio was employed, with digestion times of 2 h (A2) and 1 h (A4), respectively. The IC50 value for the antioxidant assay DPPH (2,2-Difenil-1-Picrilhidrazilo) was 22.5 μg/μL for A2. In the antioxidant in vivo assay with Caenorhabditis elegans, A2 peptides at concentrations of 0.75 μg/μL and 1.5 μg/μL exhibited a survival percentage ranging from 32.6% to 35.3%. The A4 sample demonstrated the highest antimicrobial activity, inhibiting the growth of Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) with MIC (minimum inhibitory concentration) values of 0.11 μg/mL and 0.23 μg/mL and MBC (minimum bactericidal concentration) of 0.23 μg/mL for both. Peptide analysis revealed that A2 contained peptides with identified antihypertensive (37%) and antioxidant (58%) activities, while A4 comprised peptides with antihypertensive (62%), antioxidant (31%), and antimicrobial (2%) properties. The antimicrobial peptides identified through the BIOPEP database were LFGF and FLLF. This investigation underscores the potential of A. armida peptides as valuable components in functional foods or nutraceuticals, offering health benefits beyond basic nutrition.

Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Scorpion Peptides from Buthus martensii Karsch Venom with Dual Functions: Their Pharmaceutic Potential for Cancer Pain Therapy

Abstract: Buthus martensii Karsch (BmK) scorpion venom and its extracts are compounds found to contain a mixture of peptides with antitumor and analgesic activities. The extracts also contain polypeptides, lipids, nucleotides, mucoproteins, and other unknown substances showing potential therapeutic application. Finding medically relevant toxins, a range of peptides have been purified from BmK scorpion venom. These scorpion toxins are believed to modulate pain pathways to treat pain and cancer. This study aims to review the literature on BmK scorpion peptides with dual functions, their analgesic effects, and their mechanism of action in pain treat-ments and cancer. We reviewed the literature published between 1990 and 2023 from various databases and identified 59 articles suitable for our narrative review. In 1994, the first BmK scorpion peptide with an analgesic effect was purified. Since then, more analgesic peptides have been purified, including BmK AGAP, BmK AGAP-SYPU2, BmK AGP, BmK AGP-SYPU1, BmK AGP-SYPU2, BmK AS, and BmK iT2. Studies have demonstrated that these peptides bind to voltage sodium channels and inhibit the inactivation of the activated sodium channels to block neuron transmission. The analgesic activities of these peptides have been examined on different pain stimuli, resulting in strong analgesic and antitumor effects. Moreover, the antitumor activi-ties of these peptides have been investigated in vitro and in vivo. The findings showed that the analgesic peptides may have antitumor effects. Based on their dual functions, these peptides have the potential to be developed as practical therapeutic agents for the treatment of cancer-related pain.

Protective Effects of Polysaccharides on Antifungal Activity of Bread Waste-derived Bioactive Peptides During Freeze Drying

Bioactive peptides (BPs) derived from agri-food side-streams display great potential as functional ingredients and nutraceuticals. However, BPs can be susceptible to environmental stressors and food processing conditions and typically exist as an aqueous mixture of various peptides, making their storage and use in foods very challenging. Microencapsulation offers a solution, utilizing suitable wall materials to create a protective matrix and transform them into powders for improved stability and application versatility. Here, we investigated the possibility of creating freeze-dried microencapsulated BP powders from bread waste using maltodextrin as the wall material to maintain their antifungal properties against Penicillum albocoremium, Eurotium herbariorum and Penicillium roqueforti fungi. The results were compared to those obtained from galactoglucomannans (GGM) and glucuronoxylans (GX) sourced from forest industry side-streams, aiming to explore their potential suitability as innovative materials for microencapsulating BPs. Maltodextrin microcapsules exhibited the highest inhibition rate on the tested fungi (10 − 44%), followed by GX (26 − 39%) and then GGM (9 − 14%) counterparts. Interactions between wall materials and BPs, as illustrated by FTIR analyses, were found to be the cause of variations in antifungal activities among BP microcapsules. The use of wall materials, especially wood hemicelluloses, facilitated freeze drying of BPs. However, their application in food products is restricted by the decline in the BP bioactivity resulting from the freeze drying process. For this reason, additional research on alternative drying methods is needed. The results of this study are expected to open new ways of production of stable functional ingredients from side-streams of the forest and food industries.

Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties

Food-derived soybean peptides possess inflammation-preventive effects beyond basic nutrition due to their structural configuration. However, the relationship between sequence characteristics and their anti-inflammatory properties remains poorly understood. The lack of targeted peptide production methods limits their use in functional foods. Hydrophobic and basic amino acids enhance peptide anti-inflammatory activity. Alcalase and Papain facilitate hydrophobic and basic amino acid preparation, while Bromelain lacks specific cleavage sites. Endopeptidases selectively break peptide bonds within proteins. We subsequently used Flavourzyme, lacking specific cleavage sites, to sequentially shorten peptide chains from their terminal ends. This sequential use of endo- and exopeptidases enables efficient peptide preparation. However, the proteases' ability to selectively produce peptides with specific sequence characteristics and their contribution to the soybean peptide mixture remain unknown. This study examined three soybean peptide groups prepared using distinct methods (Alcalase + Flavourzyme: hydrophobic amino acid-rich peptides, Papain + Flavourzyme: basic amino acid-rich peptides, and Bromelain + Flavourzyme: balanced amino acid compositions) with different sequence characterizations linked to endonuclease cleavage sites. All groups inhibited nitric oxide (NO) and inducible nitric oxide synthase (iNOS) secretion, suppressing protein phosphorylation in the Phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (Akt)/Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (PI3K/Akt/NF-κB) signaling pathway. PF peptides showed the strongest anti-inflammatory effect, targeting iNOS, inhibiting TLR4, and reducing Akt and IκB phosphorylation. Mass spectrometry revealed correlations between regulatory mechanisms and the abundance of branched amino acids at PF peptides' N-terminals and significant Arg presence at their C-terminals. This study validates optimal enzymatic processes, introduces sequence characterization analysis, and identifies key features influencing anti-inflammatory properties, providing a theoretical basis for soybean peptide applications in inflammation-preventing functional foods.

Evaluation of PAC and FASP Performance: DIA-Based Quantitative Proteomic Analysis.

The aim of this study was to compare filter-aided sample preparation (FASP) and protein aggregation capture (PAC) starting from a three-species protein mix (Human, Soybean and Pisum sativum) and two different starting amounts (1 and 10 µg). Peptide mixtures were analyzed by data-independent acquisition (DIA) and raw files were processed by three commonly used software: Spectronaut, MaxDIA and DIA-NN. Overall, the highest number of proteins (mean value of 5491) were identified by PAC (10 µg), while the lowest number (4855) was identified by FASP (1 µg). The latter experiment displayed the worst performance in terms of both specificity (0.73) and precision (0.24). Other tested conditions showed better diagnostic accuracy, with specificity values of 0.95-0.99 and precision values between 0.61 and 0.86. In order to provide guidance on the data analysis pipeline, the accuracy diagnostic of three software was investigated: (i) the highest sensitivity was obtained with Spectronaut (median of 0.67) highlighting the ability of Spectronaut to quantify low-abundance proteins, (ii) the best precision value was obtained by MaxDIA (median of 0.84), but with a reduced number of identifications compared to Spectronaut and DIA-NN data, and (iii) the specificity values were similar (between 0.93 and 0.99). The data are available on ProteomeXchange with the identifier PXD044349.

A Fully Automated Online Enrichment and Separation System for Highly Reproducible and In-Depth Analysis of Intact Glycopeptide.

A fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-μg sample amount, while a 10-μg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

Improvement of sleep duration and quality through GABAA receptor by whey protein hydrolysate containing DIQK as the main active compound

This study characterized the sleep activity, sleep mechanism, and active peptides of whey protein hydrolysates selected through behavioral analysis of fruit flies (Drosophila melanogaster). Sleep-inducing whey protein (WP) hydrolysate was selected through fruit fly behavior analysis, and sleep activity was measured using a pentobarbital model and electroencephalographic analysis. The mechanism of action was confirmed using a γ-aminobutyric acid (GABA) receptor antagonist, and the active peptide was identified using liquid chromatography-mass spectrometry. Whey protein hydrolysate, prepared using Alcalase and Prozyme (WP-AP), increased sleep time in a dose-dependent manner. The WP-AP significantly increased not only sleep time but also slow-wave sleep and showed an insomnia-alleviating effect in a caffeine-induced insomnia mouse model. In addition, the gene and protein expression levels of GABA subtype A receptors increased in the brains of mice orally administered with WP-AP. Through peptide analysis, the mixture of DIQK, VPPF peptide, and GABA contained in WP-AP was estimated to exhibit sleep activity, and due to its high content, DIQK was speculated to be the main sleep-inducing ingredient. These results indicate that WP-AP has the potential to be used as a new ingredient to improve sleep quality.

Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry by Matrix-Assisted Laser Desorption Ionization.

Two-dimensional Fourier transform ion cyclotron resonance (2D FTICR) mass spectrometry is a developing form of data-independent acquisition that allows for the simultaneous fragmentation and correlation of fragment ions to their precursors across a range of m/z values. The modern usage of 2D FTICR is performed using electrospray ionization (ESI) as the dried droplet preparation for matrix-assisted laser desorption ionization (MALDI) does not produce a consistent packet of ions over a number of scans. This work uses pneumatic spray techniques from mass spectrometry imaging to create a homogeneous surface for use with MALDI as an ionization source for 2D FTICR. A mixture of peptides and matrix was deposited onto a glass slide using an HTX pneumatic sprayer. MALDI was then used to ionize the peptide mixture for use with a standard 2D FTICR pulse sequence. The generated 2D spectrum reveals comparable structural information to spectra collected in a 1D experiment. Artifacts observed in the collected 2D MALDI spectra do not significantly differ from those expected from 2D ESI spectra.

Preharvest calcium application boosts the biostimulatory effect of protein hydrolysate by improving the postharvest life of gerbera cut flower

Among the various categories of plant biostimulants currently available on the market, protein hydrolysates, primarily composed of a heterogeneous mixture of peptides and amino acids, have gradually gained prominence. Nevertheless, the positive effects of these products on the production and longevity of cut flowers are still not well-explored. The purpose of this research was thus to fill this gap by assessing the impacts of a plant-derived protein hydrolysate, with and without the addition of calcium (PH and PH+Ca), on the growth, quality, and vaselife of two cultivars of gerbera with different pigmentation (Brunello and Tommy, red and yellow, respectively). Regardless of cultivar, foliar application of PH improved growth, flower production, absorption of key nutrients, and photosynthetic performance, compared to the control. On the other hand, the PH+Ca treatment exhibited the best results during vaselife. For both cultivars, the application of PH+Ca increased the durability of gerbera flowers, reducing the weight loss by an average of 19 % on day 10 compared to untreated flowers. Although cultivar-dependent, the positive effects of the PH+Ca biostimulant were also observed on petal pigmentation during vase life. For the Tommy cultivar, for instance, the Chroma values of treated flowers remained unchanged during the post-harvest period, unlike what was observed for untreated flowers. Therefore, considering the positive effects resulting from the use of PH and PH+Ca on gerbera, it would be interesting to further investigate the use of biostimulants in floriculture to increase the sustainability of the sector and especially with regard to cut flower production, without reducing or even improving the performance that is conventionally achieved.

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8±1.8mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6±0.6mg calcium/g protein with a high association constant of 420M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation.

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.

Molecular mechanism analysis of apoptosis induced by silk fibroin peptides

Silk fibroin derived from silkworm cocoons exhibits excellent mechanical properties, good biocompatibility, and low immunogenicity. Previous studies showed that silk fibroin had an inhibitory effect on cells, suppressing proliferation and inducing apoptosis. However, the source of the toxicity and the mechanism of apoptosis induction are still unclear. In this study, we hypothesized that the toxicity of silk fibroin might originate from the crystalline region of the heavy chain of silk fibroin. We then verified the hypothesis and the specific induction mechanism. A target peptide segment was obtained from α-chymotrypsin. The potentially toxic mixture of silk fibroin peptides (SFPs) was separated by ion exchange, and the toxicity was tested by an MTT assay. The results showed that SFPs obtained after 4 h of enzymatic hydrolysis had significant cytotoxicity, and SFPs with isoelectric points of 4.0–6.8 (SFPα II) had a significant inhibitory effect on cell growth. LC-MS/MS analysis showed that SFPα II contained a large number of glycine-rich and alanine-rich repetitive sequence polypeptides from the heavy-chain crystallization region. A series of experiments showed that SFPα II mediated cell death through the apoptotic pathway by decreasing the expression of Bcl-2 protein and increasing the expression of Bax protein. SFPα II mainly affected the p53 pathway and the AMPK signaling pathway in HepG2 cells. SFPα II may indirectly increase the expression of Cers2 by inhibiting the phosphorylation of EGFR, which activated apoptotic signaling in the cellular mitochondrial pathway and inhibited the Akt/NF-κB pathway by increasing the expression of PPP2R2A.