Preparation Of Peptides Research Articles

MYC-Targeting Inhibitors Generated from a Stereodiversified Bicyclic Peptide Library.

Here, we present the second generation of our bicyclic peptide library (NTB), featuring a stereodiversified structure and a simplified construction strategy. We utilized a tandem ring-opening metathesis and ring-closing metathesis reaction (ROM-RCM) to cyclize the linear peptide library in a single step, representing the first reported instance of this reaction being applied to the preparation of macrocyclic peptides. Moreover, the resulting bicyclic peptide can be easily linearized for MS/MS sequencing with a one-step deallylation process. We employed this library to screen against the E363-R378 epitope of MYC and identified several MYC-targeting bicyclic peptides. Subsequent in vitro cell studies demonstrated that one candidate, NT-B2R, effectively suppressed MYC transcription activities and cell proliferation.

Comparing the biological activity and composition of Cerebrolysin with other peptide preparations.

Neurological disorders, ranging from acute forms such as stroke and traumatic brain injury to neurodegenerative diseases like dementia, are the leading cause of disability-adjusted life years (DALYs) worldwide. A promising approach to address these conditions and promote nervous system regeneration is the use of the neuropeptide preparation Cerebrolysin, which has been shown to be effective in both clinical and preclinical studies. Despite claims of similar clinical efficacy and safety by several peptide preparations, concerns regarding their generic composition and efficacy have been previously raised. Based on these reports, we analyzed the peptide composition and neurotrophic activity of several peptide preparations allegedly similar to Cerebrolysin and approved in some countries for treating neurological diseases. Our results demonstrate that these preparations lack relevant biological activity and that the peptide composition is significantly different from Cerebrolysin. peptide.

Quantifying mitochondrial protein import by mePRODmt proteomics.

Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.

In silico prediction aided preparation of antioxidant soybean peptides by enzymatic hydrolysis for ameliorating lead exposure-induced toxicity.

Food derived bioactive peptides are prominent dietary supplements to protect against oxidative stress induced by lead (Pb) exposure. This study aimed to develop a new strategy for rapid preparation of highly active antioxidant soybean polypeptides (ASPs) against Pb toxicity. In silico enzymatic hydrolysis simulation and antioxidant activity prediction showed that pepsin, chymotrypsin and bromelain can produce peptides with the highest activity. The preparation process was then optimized, and the obtained ASP showed good antioxidant and metal-chelating activities in vitro. An in vivo study showed that ASP exerted prominent protective effects against Pb-induced cognitive impairment and tissue damage in mice by reducing Pb deposition and enhancing the antioxidant capacity in tissues and was superior to Vc, DMSA or their combination in some aspects. ASP composition analysis demonstrated that its prominent antioxidant activity might be attributed to the high proportion of amino acid residues E, L, P and V in the peptide sequence and L, V and A at the C- and N-termini. In conclusion, in silico prediction could facilitate the preparation of ASP. And the ASP prepared with the new strategy exerted prominent protective effects against Pb toxicity.

Bioactive peptides as a novel strategy to prevent alcoholic liver injury.

Alcohol intake has become one of the leading risks to human health and wellness, among which acute and/or chronic alcohol-induced liver injury is a leading threaten, with few therapeutic options other than abstinence. In recent years, studies suggested that certain bioactive peptides from food sources could represent natural and safe alternatives for the prevention of alcoholic liver injury. Hence, this chapter focus on the advanced research on bioactive peptides exerting hepatoprotective activity against alcoholic liver injury. The main sources of protein, strategies for the preparation of hepatoprotective hydrolysates and peptides, underlying mechanisms of peptides on hepatoprotection, and possible structure-activity relationship between peptides and hepatoprotective activity were summarized and discussed, aiming to give a systematic insight into the research progress of hepatoprotective peptides. However, more efforts would be needed to give a clearer insight into the underlying mechanisms and structure-activity relationship before using hepatoprotective peptides as functional food ingredients or dietary supplements.

Preparation of antioxidant peptides from yak skin gelatin and their protective effect on myocardial ischemia reperfusion injury.

We herein report a study on the antioxidant peptides that show potential in alleviating myocardial ischemia reperfusion injury (MI/RI). Yak skin gelatin fraction Ac (YSG-Ac), obtained through ultrafiltration and gel filtration with Sephadex G-15, exhibits a favorable nutrient composition, high foaming capacity and stability, and resistance against gastrointestinal digestion. LC-MS/MS analysis reveals that YSG-Ac contains 26 peptide segments with sequence lengths of 8 to 12 amino acids. Online screening suggests that the antioxidant capacity of YSG-Ac is mainly attributed to the presence of hydrophobic and antioxidant amino acids. In vitro, our results demonstrate the MI/RI protective effects of YSG-Ac by effectively repairing H2O2-induced oxidative damage in H9c2 cells, which is achieved by inhibiting malondialdehyde (MDA) levels, and increasing glutathione peroxidase (GSH-pX) and superoxide dismutase (SOD) activity. In vivo, our results further confirm the effectiveness of YSG-Ac in narrowing the area of myocardial infarction, decreasing MDA levels, increasing SOD activity, and reducing the content of lactate dehydrogenase (LDH) in a mouse MI/RI model. Molecular docking analysis indicates that PGADGQPGAK with xanthine dehydrogenase (XDH) and GAAGPTGPIGS with tumor necrosis factor-alpha (TNF-α) exhibit strong bonding capability, and other related targets also show certain binding ability toward YSG-Ac. This suggests that YSG-Ac can regulate MI/RI through multiple targets and pathways. Overall, our findings highlight the potential of YSG-Ac as a functional food ingredient with antioxidant and MI/RI protective characteristics.

Effect of short peptides on seed germination and morphometric parameters of flax seedlings

The use of peptide preparations is one of the current trends in modern agriculture. These preparations can be used to increase the efficiency of sowing treatment of agricultural crops seeds. The article presents the results of a laboratory experiment aimed at studying the effect of a complex of short peptides AC-3 (glutamic acid, aspartic acid, leucine) on seed germination and morphometric parameters of flax seedlings (Linum usitatissimum L.) - Tverskoy variety. The maximum germination energy (68, 68 and 74%, 50% at the control) and seed germination values (95, 95 and 100%, 75% at the control) were obtained in variants with seed treatment with a complex of short peptides at concentrations of 1*10-12, 1*10-13, and 1*10-15 g/l, respectively. According to the totality of the maximum values of seed quality indicators and morphometric characteristics of flax seedlings, the Tverskoy variety (the length of roots and sprouts, the yield of raw and dry biomass of seedlings), the optimal concentration of a complex of short peptides for pre-sowing seed treatment is 1*10-15 g/l.

In silico and in vivo discovery of antioxidant sea cucumber peptides with antineurodegenerative properties.

Since oxidative stress is often associated with neurodegenerative diseases, antioxidants are likely to confer protection against neurodegeneration. Despite an increasing number of food-derived peptides being identified as antioxidants, their antineurodegenerative potentials remain largely unexplored. Here, a sea cucumber peptide preparation - the peptide-rich fraction of <3 kDa (UF<3K) obtained by ultrafiltration from Apostichopus japonicus protein hydrolyzate - was found to protect PC12 cells and Caenorhabditis elegans from neurodegeneration by reducing oxidative stress and apoptosis, demonstrating its in vitro and in vivo neuroprotective effects. As many food-originated peptides are cryptides (cryptic peptides - short amino acid sequences encrypted in parent proteins) released in quantities by protein hydrolysis, UF<3K was subjected to sequencing analysis. As expected, a large repertoire of peptides were identified in UF<3K, establishing a sea cucumber cryptome (1238 peptides in total). Then 134 peptides were randomly selected from the cryptome (>10%) and analyzed for their antioxidant activities using a number of in silico bioinformatic programs as well as in vivo experimental assays in C. elegans. From these results, a novel antioxidant peptide - HoloPep#362 (FETLMPLWGNK) - was shown to not only inhibit aggregation of neurodegeneration-associated polygluatmine proteins but also ameliorate behavioral deficits in proteotoxicity nematodes. Proteomic analysis revealed an increased expression of several lysosomal proteases by HoloPep#362, suggesting proteostasis maintenance as a mechanism for its antineurodegenerative action. These findings provide an insight into the health-promoting potential of sea cucumber peptides as neuroprotective nutraceuticals and also into the importance of training in silico peptide bioactivity prediction programs with in vivo experimental data.

Nephroprotective peptides of Laennec® in the context of pharmacotherapy for nephro-hepato-metabolic disorders

Background. Renal-hepatic dysfunction, which often occurs in liver dysfunction, requires the use of effective and safe nephroprotective agents. Human placenta hydrolysates (HPH) are hepatoprotectors, but little is known about HPH nephroprotective properties and the molecular mechanisms of their implementation.Objective: identification of potential molecular mechanisms of Laennec® HPH neuroprotective action based on bioinformatic analysis of collected mass spectrometric data.Material and methods. Methods of proteomic analysis of peptide preparations were used. The analysis of Laennec® HPH peptide composition included four stages: drug purification, chromatographic separation of peptides, determination of the multidimensional mass spectrum of peptide fraction and de novo sequencing of the isolated peptides.Results. The study of Laennec® HPH peptide composition allowed to identify 48 peptides that can exhibit nephroprotective effects. It was shown that HPH contains biologically active fragments of nephroprotective adrenomedullins, inhibitor peptides of a number of kinases (FYN, SHH, WNK1/4, SGK1, IRAK4, ROCK1/2) and fibrogenic receptors (PDGFR, TGFB1I1).Conclusion. By inhibiting the listed target proteins, HPH peptides provide nephroprotection through reducing inflammation, anti-stress effects and preventing fibrotic changes in kidney tissue

Compatibility Study of Peptide and Glycerol Using Chromatographic and Spectroscopic Techniques: Application to a Novel Antimicrobial Peptide Cbf-14 Gel.

The interactions between active pharmaceutical ingredients (APIs) and excipients may lead to API degradation, thereby affecting the safety and efficacy of drug products. Cbf-14 is a synthetic peptide derived from Cathelicidin-BF, showing potential for bacterial and fungal infections. In order to assess impurities in Cbf-14 gel, we developed a two-dimensional liquid chromatography coupled with quadrupole/time-of-flight mass spectrometric method. A total of eleven peptide degradation impurities were identified and characterized. Furthermore, the compatibility tests were conducted to evaluate the interactions of Cbf-14 with glycerol and methylcellulose, respectively. The results revealed that the impurities originated from condensation reactions between Cbf-14 and aldehydes caused by glycerol degradation. Several aldehydes were employed to validate this hypothesis. The formation mechanisms were elucidated as Maillard reactions between primary amino groups of Cbf-14 and aldehydes derived from glycerol degradation. Additionally, the compatibility of Cbf-14 with glycerol from different sources and with varying storage times was investigated. Notably, the interaction products in the gel increased with extended storage time, even when fresh glycerol for injection was added. This study offers unique insights into the compatibility study of peptides and glycerol, contributing to the ongoing quality study of Cbf-14 gel. It also serves as a reference for the design of other peptide preparations and excipients selections.

Preparation of Novel Sea Cucumber Intestinal Peptides to Promote Tibial Fracture Healing in Mice by Inducing Differentiation of Hypertrophic Chondrocytes to the Osteoblast Lineage.

Hypertrophic chondrocytes have a decisive regulatory role in the process of fracture healing, and the fate of hypertrophic chondrocytes is not only apoptosis. However, the mechanism of sea cucumber (Stichopus japonicus) intestinal peptide (SCIP) on fracture promotion is still unclear. This study aims to investigate the effect of sea cucumber intestinal peptide on the differentiation fate of hypertrophic chondrocytes in a mouse tibial fracture model. Mice are subjected to open fractures of the right tibia to establish a tibial fracture model. The results exhibit that the SCIP intervention significantly promotes the mineralization of cartilage callus, decreases the expression of the hypertrophic chondrocyte marker Col X, and increases the expression of the osteoblast marker Col I. Mechanically, SCIP promotes tibial fracture healing by promoting histone acetylation and inhibiting histone methylation, thereby upregulating pluripotent transcription factors induced the differentiation of hypertrophic chondrocytes to the osteoblast lineage in a manner distinct from classical endochondral ossification. This study is the first to report that SCIP can promote tibial fracture healing in mice by inducing the differentiation of hypertrophic chondrocytes to the osteoblast lineage. SCIP may be considered raw material for developing nutraceuticals to promote fracture healing.

Preparation and identification of hypoglycemic bioactive peptide from Amygdalus communis L. by multienzyme hydrolysis

Amygdalus communis L. is rich in high-quality protein and its natural peptides have potential in controlling and preventing diseases. The objective of this study was to evaluate the hypoglycemic activity of bioactive peptides obtained from Amygdalus communis L. protein by synergistic hydrolysis of trypsin and protamex. After ultrafiltration and purification, active peptide B4 with small molecular weight in four hypoglycemic active peptides showed the best inhibitory activities on α-amylase and α-glucosidase. Moreover, insulin resistance model was established with HepG2 cells to study the hypoglycemic mechanism of active peptide B4. The experimental results confirmed that the active peptide B4 was able to up-regulate GYS1 gene mRNA expression and down-regulate PEPCK, G6Pase and GP gene mRNA expression. Meanwhile, active peptide B4 significantly increased the glucose consumption and glycogen synthesis in IR-HepG2. This research enhances the additional value of nut proteins and contributes to the development of potential functional products to expand their application in the food field.

Native Peptide Cyclization, Sequential Chemoselective Amidation in Water.

Chemical synthesis offers robust tactics for structural alterations of peptides and proteins. It remains a labor-intensive and complex process due to the challenges in selectively modifying diverse amino acid side chains and termini. Direct α-peptide ligation without premodification is a significant hurdle, especially when aiming to include all proteinogenic amino acids at the ligation site. We introduce Native Peptide Cyclization (NPC), a chemoselective method enabling intramolecular peptidyl ligation without the need for premodification. NPC cyclizes unprotected linear peptides through controlled, sequential C- and N-terminal activation via pH modulation. Water-based NPC simplifies peptide ligation, easing the labor-intensive nature of peptide synthesis, aiding efficient cyclic peptide preparation and enabling cost-effective macrocycle-based therapeutics.

Functional development of porcine spleen as a by-product of pig slaughterhouse: Preparation, identification and bioactive activities of a novel peptide

Porcine spleen, by-product of pig slaughterhouses, was often threw away due to underutilization, resulting in the neglect of the development of high-quality active materials. In this study, water-soluble porcine spleen peptide (w-PSP) was prepared by enzymatic hydrolysis. It was a mixed peptide with a molecular weight of less than 3000 Da and contained 7097 short peptides consisting of 5–15 amino acids residues. The w-PSP contained 65.33% hydrophilic amino acids. β-sheet was the main secondary structure conformation in w-PSP. The activity verification experiments confirmed that w-PSP presented no antitumor activity in vitro and in vivo. But it exhibited conspicuous antioxidant properties. The maximum scavenging rates of w-PSP to 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were 78.19% and 75.53%, respectively. Anti-superoxide anion (O2-) radical activity and anti-hydroxyl (OH) radical activity of w-PSP reached 94.19 U/L and 396.40 U/mL. Besides, the activities of glutathione peroxidase (GSH-PX) in liver, spleen, kidney and serum of w-PSP treated mice were significantly increased to 266.82 mgGSH/gprot, 226.37 mgGSH/gprot, 239.03 mgGSH/gprot and 1376.38 mgGSH/L. The activities of superoxide dismutase (SOD) in liver, spleen, kidney and serum of w-PSP treated mice were increased to 1614.66 U/mgprot, 1614.51 U/mgprot, 677.11 U/mgprot and 156.91 U/mL. The malondialdehyde (MDA) contents in w-PSP treatment groups were obviously decreased. De novo sequencing and bioinformatics prediction were used to identify and analyze peptides in w-PSP. Good molecular docking results suggested the antioxidant mechanism of w-PSP might be related to the Keap1-Nrf2 signaling pathway.

Tea (Camellia sinensis (L.) Kuntze) as an emerging source of protein and bioactive peptides: A narrative review.

Tea residues represent one of the major agricultural wastes that are generated after the processing of tea. They account for 21-28% of crude protein and are often discarded without the extraction of valuable proteins. Due to various bioactivity and functional properties, tea proteins are an excellent alternative to other plant-based proteins for usage as food supplements at a higher dosage. Moreover, their good gelation capacity is ideal for the manufacturing of dairy products, jellies, condensation protein, gelatin gel, bread, etc. The current study is the first to comprehend various tea protein extraction methods and their amino acid profile. The preparation of tea protein bioactive peptides and hydrolysates are summarized. Several functional properties (solubility, foaming capacity, emulsification, water/oil absorption capacity) and bioactivities (antioxidant, antihypertensive, antidiabetic) of tea proteins are emphasized.

Virtual screening and structure optimization of xanthine oxidase inhibitory peptides from whole protein sequences of Pacific white shrimp via molecular docking.

Xanthine oxidase (XO) inhibitory peptides are safer than conventional pharmacological therapy in relieving hyperuricemia. However, traditional enzymatic hydrolysis, separation, and purification techniques for bio-active peptide preparation are time-consuming, inefficient, and labor-intensive. In this study, molecular docking and BLAST were used to virtually screen XO inhibitory peptides from whole protein sequences of Pacific white shrimp according to the bio-active peptides database, and the structure of peptides was optimized based on the structure-effective relationship. Seven new XO inhibitory peptides were virtual screened rapidly from Pacific white shrimp, and YNITGW (IC50=9.78±0.13mM) showed the strongest activity. The results of YNITGW optimization showed that the insertion of Trp residue in the middle position of peptides could effectively enhance the activity. This study revealed that screening and optimizing peptides by molecular docking were a novel and feasible method to obtain bio-active peptides.

Global Emergence of Infectious Bronchitis Virus Variants: Evolution, Immunity, and Vaccination Challenges

Infectious bronchitis is an acute, extremely contagious viral disease affecting chickens of all ages, leading to devastating economic losses in the poultry industry worldwide. Affected chickens show respiratory distress and/or nephritis, in addition to decrease of egg production and quality in layers. The avian coronavirus, infectious bronchitis virus (IBV), is a rapidly evolving virus due to the high frequency of mutations and recombination events that are common in coronaviruses. This leads to the continual emergence of novel genotypes that show variable or poor crossprotection. The immune response against IBV is complex. Passive, innate and adaptive humoral and cellular immunity play distinct roles in protection against IBV. Despite intensive vaccination using the currently available live-attenuated and inactivated IBV vaccines, IBV continues to circulate, evolve, and trigger outbreaks worldwide, indicating the urgent need to update the current vaccines to control the emerging variants. Different approaches for preparation of IBV vaccines, including DNA, subunit, peptides, virus-like particles, vectored and recombinant vaccines, have been tested in many studies to combat the disease. This review focuses on several key aspects related to IBV, including its clinical significance, the functional structure of the virus, the factors that contribute to its evolution and diversity, the types of immune responses against IBV, and the characteristics of both current and emerging IBV vaccines. The goal is to provide a comprehensive understanding of IBV and explore the emergence of variants, their dissemination around the world, and the challenges to define the efficient vaccination strategies.

Ultrasound-assisted fermentation for antioxidant peptides preparation from okara: Optimization, stability, and functional analyses

This study investigated the effects of ultrasound-assisted fermentation (UAF) on the preparation of antioxidant peptides (UAFP) from okara and examined their content, chemical structures, and antioxidant activity. After the optimal ultrasonic processing (time, 20 min; frequency, 45 KHz; power, 120 W/L), the peptide content yield reached the maximum of 12.36 ± 0.02 mg/mL, and their DPPH free radical scavenging rate was 65.15 ± 0.32 %. UAF increased the number of globular aggregates with deeper gullies, a looser structure, and higher porosity. The experiments conducted using the oxidative stress injury model of HepG2 cells showed that okara UAFP promoted cell growth and exerted a protective effect. Moreover, ultrasonic treatment remarkably improved the environmental stability (NaCl, glucose, sodium benzoate, temperature, pH, metal ions) and antioxidant activity of UAFP. Concisely, optimal ultrasonic processing can aid the fermentation of agroindustrial by-products to prepare antioxidant peptides, such as natural food antioxidant peptides from soybean waste.

Preparation, structural characterization and functional properties of a novel selenium chelating peptide derived from the hydrolyzate of wheat protein

ABSTRACT Organic selenium has been widely studied for its ability to better fulfill the physiological functions of selenium. In this study, a novel organic selenium (wheat protein hydrolyzate chelated selenium, WPH-Se) was prepared by chelating the hydrolyzate of wheat protein hydrolyzed by alkaline protease (WPH) with selenium, its preparation process was optimized; and structural and functional properties were investigated. The results showed that the highest selenium content of 10.97 mg/g was obtained in the chelate under the optimal conditions (peptide/selenium mass ratio 2:1, temperature 80°C, pH 8, time 60 min). UV-Vis, FTIR, DSC and SEM provided more information for the characterization of WPH-Se. During the chelating process, selenium ions might be effectively bound to WPH through carboxyl, carbonyl and amino ligands, and the C=O bond and -NH, -OH groups might be the sites. At different pHs, the solubility of WPH-Se was generally lower than that of WPH, which reached a maximum of 32.69% at pH 6. The EAI of WPH-Se was higher than that of WPH, which reached a maximum of 0.84 m2/g, and the ESI reached a maximum of 47.3 min at pH 10. The free sulfhydryl content of WPH-Se was greater than that of WPH, which reached a maximum of 12.78 μmol/g at pH 8. WPH-Se was superior to WPH in terms of foaming ability and emulsification properties. WPH-Se contained a certain amount of aromatic amino acids (7.962%) and a relatively high amount of hydrophobic amino acids (38.490%), and had high nutritional value. Wheat protein peptide chelated selenium would be a useful functional additive, this study would provide data to support further research and application of selenium dietary supplements.

Anticancer effect of hUC-MSC-derived exosome-mediated delivery of PMO-miR-146b-5p in colorectal cancer

Antisense oligonucleotide (ASO) is a novel therapeutic platform for targeted cancer therapy. Previously, we have demonstrated that miR-146b-5p plays an important role in colorectal cancer progression. However, a safe and effective strategy for delivery of an ASO to its targeted RNA remains as a major hurdle in translational advances. Human umbilical cord mesenchymal cell (hUC-MSC)–derived exosomes were used as vehicles to deliver an anti-miR-146b-5p ASO (PMO-146b). PMO-146b was assembled onto the surface of exosomes (e) through covalent conjugation to an anchor peptide CP05 (P) that recognized an exosomal surface marker, CD63, forming a complex named ePPMO-146b. After ePPMO-146b treatment, cell proliferation, uptake ability, and migration assays were performed, and epithelial-mesenchymal transition progression was evaluated in vitro. A mouse xenograft model was used to determine the antitumor effect and distribution of ePPMO-146b in vivo. ePPMO-146b was taken up by SW620 cells and effectively inhibited cell proliferation and migration. The conjugate also exerted antitumor efficacy in a xenograft mouse model of colon cancer by systematic administration, where PPMO-146b was enriched in tumor tissue. Our study highlights the potential of hUC-MSC-derived exosomes anchored with PPMO-146b as a novel safe and effective approach for PMO backboned ASO delivery.Graphical Schematic illustration of the preparation of an exosomal anchor peptide (CP05)-PMO that conjugately binds to exosomes from hUC-MSCs (ePPMO-146b) and the antitumor effect of ePPMO-146b in CRC, which occurs through the inhibition of Smad signaling and epithelial–mesenchymal transition.