Protein Peptide Research Articles

Dihydroergotamine and Bromocriptine: Potential Drugs for the Treatment of Major Depressive Disorder and Alzheimer's Disease Comorbidity.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease that is characterized by memory loss and cognitive impairment. Evidence shows that depression is a common co-occurrence in AD patients, and major depressive disorder (MDD) is considered a risk factor for AD. The crosstalk between the biological procedures related to the two disorders makes it very difficult to treat the comorbid conditions caused by them. Considering the common pathophysiological mechanisms underlying AD and MDD, antidepressant drugs may have beneficial therapeutic effects against their concurrence. In this study, we aimed to explore the potential drug candidates for the prevention and treatment of the comorbidity of AD and MDD. First, we screened the potential drugs for treating MDD by evaluating the distances of drug targets to MDD-related genes on the human protein-protein interaction network (PPIN) via a network-based algorithm. Then, the drugs were further screened to identify those that may be effective for AD treatment by analyzing their affinities with tau protein and Aβ42 peptide via molecular docking. Furthermore, the most stable binding modes were identified via molecular dynamics simulations, and the regulatory effects of drug candidates on genes involved in the pathogenesis of AD and MDD were analyzed. A total of 506 MDD-related genes were retrieved, and 831 drug candidates for MDD treatment were screened via the network-based approach. The results from molecular docking and molecular dynamics simulations indicated dihydroergotamine had the lowest binding affinity with tau protein and bromocriptine could form the most stable binding mode with Aβ42 peptide. Further analyses found that both dihydroergotamine and bromocriptine could regulate the expression of genes involved in the pathogenesis of AD and/or MDD in the brain. The exact mechanisms of the two drugs in treating AD and MDD, as well as their comorbidity, are still unclear, and further exploration is needed to evaluate their roles and mechanisms, both in vitro and in vivo. This study revealed that dihydroergotamine and bromocriptine may be the potential drug candidates for the treatment of the comorbidity of AD and MDD, and the therapeutic effects may be achieved by inhibiting the accumulation and aggregation of Aβ42 and tau protein and regulating the expression of disease-related genes in the brain.

Abstract We007: High Throughput 3D-Printed Human Engineered Heart Tissues for Cardiac Disease Modeling

While cell models have been used to study cardiac diseases, there is currently no standardization or fully mature in vitro system. Obtaining a mature cardiomyocyte (CM) phenotype is necessary to create a physiologically relevant environment and accurately model and study cardiac diseases. Increased success in maturation of human induced pluripotent stem cell derived CMs (hiPS-CMs) has been achieved in 3D in vitro models in the form of engineered heart tissues (EHTs). Digital light processing (DLP) offers a high throughput and efficient method to fabricate hydrogels that recapitulate properties of native tissues. In this study, we use DLP-printed EHTs to seed and mature differentiated hiPS-CMs and test their potential for modeling pathological cardiac hypertrophy. To further encourage maturity of CMs, we utilized a low glucose maturation medium supplemented with fatty acids. DLP-printed EHTs displayed high reproducibility and increased CM maturity. To determine degree of maturity of our system, we performed RT-qPCR, western blot analysis, and immunofluorescence (IF) microscopy. We found increased expression of fatty-acid transport and beta oxidation genes and improved sarcomere alignment compared to 2D hiPS-CMs. To model pathological cardiac hypertrophy, we used two different approaches: 1. acute adrenergic agonism with isoproterenol and phenylephrine; 2. chronic culturing of EHTs in pathologically stiff conditions. To assess pathology, we conducted RT-qPCR, western blot, ELISA, IF microscopy, and functional analyses. We found that agonist-treated EHTs displayed increased pathology-associated gene expression compared to standard 2D hiPS-CMs. Both agonist-treated and stiff EHTs had increased secreted B-Type Natriuretic Peptide (BNP) and phosphorylation of proteins involved in pathological remodeling, including ERK and P38. Additionally, we observed increased nuclear area, increased calcium handling, and increased contractile force in the treatment groups. In conclusion, we have established a high throughput, reproducible, mature EHT platform that can be used to model pathological cardiac hypertrophy. This platform can be easily adopted by other laboratories to study a wide array of cardiac diseases.

Electromembrane extraction of human chorionic gonadotropin – A case study on mass transfer of intact protein versus signature peptide

For the first time, we present targeted protein detection by tryptic digestion of human chorionic gonadotropin (hCG) followed by electromembrane extraction (EME). Operational parameters were optimized, and urine and serum samples spiked with hCG underwent tryptic digestion followed by EME of the βT5 signature peptide. The liquid membrane comprised nitrophenyl octyl ether (NPOE), carvacrol, and di(2-ethyl hexyl) phosphate (DEHP) at ratios of 49:49:2 (w/w/w). Extractions were performed in a conductive vial format for 45 min at 5 V. Even from highly complex digested samples of serum and urine, the signature peptide βT5 was extracted by EME and detected by LC-MS/MS. While attempts to extract intact hCG protein were unsuccessful, the extraction of the signature peptide was efficient. The extraction recovery from undigested and digested urine was 71 % (RSD = 17 %) and 116 % (RSD = 17 %), respectively. For serum, the extraction recoveries were 11 % (RSD = 23 %) for undigested samples and 110 % (RSD = 14 %) for digested samples. This study demonstrates both the potential and challenges of EME for protein analysis. Experiments regarding EME of intact proteins provided new insights into protein phase distribution. This fundamental case study underscores the potential of EME as a sample preparation technique for the targeted determination of protein biomarkers and drugs.

Valorization of animal waste proteins for agricultural, food production, and medicinal applications

IntroductionAnimal waste proteins have been increasing in the past decade, along with consumer demands. Their huge volume and the environmental issues caused by improper treatment probably pose a massive threat to human health. These animal waste proteins contain many valuable bioactive peptides and can be used not only as nutrient substances but also as primary functional ingredients in many industries, including agriculture, food, and pharmaceuticals. However, the advancement of the value-added application of animal waste proteins within the past 10 years has not been elucidated yet. In this regard, this paper scrutinized the studies on the applications of hydrolysates and peptides from animal waste proteins throughout the last decade, hoping to display a whole picture of their value-adding applications.MethodsThe Web of Science and Google Scholar were searched from January 1, 2013, to December 12, 2023. This review included field trials, in vitro and in vivo assays, and in silico analysis based on literature surveys or proteolysis simulation. The quality of the included studies was evaluated by Journal Citation Reports, and the rationality of the discussion of studies included.ResultsNumerous studies were performed on the application potential of hydrolysates and peptides of animal waste proteins in agricultural, food, and medicinal industries. Particularly, due to the nutritional value, safety, and especially competitive effects, the peptide with antioxidant, antimicrobial, antihypertensive, antidiabetic, or antithrombotic activities can be used as a primary functional ingredient in food and pharmaceuticals.DiscussionThese value-added applications of animal waste proteins could be a step towards sustainable animal by-products management, and simultaneously, open new avenues in the rapid development of nutraceuticals and pharmaceuticals. However, further studies on the bioavailability and structure-activity relationship are required to verify their therapeutic effects.

Preparation of wheat protein peptides-calcium chelate by the ultrasound method: Structural characteristics and bioactivity

Calcium-chelating peptides are a type of calcium dietary supplement with significant development potential, offering advantages in structural stability, absorption efficiency, safety, and various other aspects. This study investigated the biological activity and structural characteristics of wheat protein peptide (WPP)–calcium (Ca2+) chelates. The structures of the WPP, ultrasound-treated WPP (UWPP), and UWPP-Ca were analyzed using scanning electron microscopy. Ultraviolet spectroscopy and Fourier transform infrared spectroscopy revealed that oxygen and nitrogen atoms were the chelation sites. Quadrupole time-of-flight mass spectrometry revealed that Ca ions chelated with UWPP, and the chelation sites were identified as follows: Ca ions bound to amino acids D (Asp), K (Lys), R (Arg), W (Trp), E (Glu), H (His), Q (Gln), and N (Asn) and H (His) in the peptides. Notably, among the WPP, UWPP and UWPP-Ca, UWPP-Ca exhibited the strongest reducing power. Furthermore, UWPP-Ca exhibited excellent pH stability and gastrointestinal digestibility. Under the influence of phytic acid and oxalic acid, the solubility and permeability of the UWPP-Ca were greater than those of the other calcium supplements, which is attributed to its stable structure. This study provides essential theoretical confirmation for the application of Ca–peptide chelates as functional foods.

Autoantibodies to joint-related peptides as predictive markers in early rheumatoid arthritis.

For better management of rheumatoid arthritis (RA), new biomarkers are needed to predict the development of different disease courses. This study aims to identify autoantibodies against epitopes on proteins in the joints and to predict disease outcome in patients with new onset RA. Sera from new onset RA patients from the Swedish BARFOT and TIRA-2 cohorts (n = 1986) were screened for autoantibodies to selected peptides (JointIDs) in a bead-based multiplex flow immunoassay. Disease outcomes included Boolean remission 1.0, swollen joint count and radiographic destruction. Multivariate logistic regression and zero-inflated negative binomial models that accounted for clinical factors were used to identify JointIDs with the strongest potential to predict prognosis. Boolean remission was predicted with 42% sensitivity and 75% specificity in male patients positive for antibodies to a non-modified collagen type II (COL2) peptide at 12 months. When antibodies to a specific citrullinated cartilage oligomeric protein (COMP) peptide were absent and the patient was in Boolean remission at 6 months, the sensitivity was 13% and the specificity 99%. Positivity for the non-modified COL2 peptide also reduced the frequency of swollen joints by 41% and 33% at 6 and 12 months, respectively. Antibodies to cyclic citrullinated peptides (aCCP) predicted joint destruction with low specificity (58%). Positivity for a COL2 and a glucose-6-phosphate dehydrogenase peptide in citrullinated forms increased specificity (86%) at the expense of sensitivity (39%). Autoantibodies against joint-related proteins at RA diagnosis predict remission with high specificity and, in combination with clinical factors, may guide future treatment decisions.

Protein extracts from microalgae and cyanobacteria biomass. Techno-functional properties and bioactivity: A review

Microalgae and cyanobacteria are photosynthetic and unicellular organisms that contain considerable amounts of proteins, lipids, carbohydrates, and polyunsaturated fatty acids, among others, with applications in the cosmetic, pharmaceutical, and food industries. These microorganisms can accumulate protein up to 70 % of total biomass depending on the microalgal strain, hence they have been regarded as an alternative protein source for the future. Microalgal proteins have important applications such as emulsifying, foaming, and gelation properties, which are important for the determination of quality and texture of foods. Some microalgal peptides possess important bioactivity with many health-benefit effects. Therefore, to maximize the production of proteins from microalgae and cyanobacteria, many protein extraction procedures have been studied to increase the economic return. They have been tested towards higher protein yields at low energy cost, the preservation of protein native properties, and lower cell debris. This later is fundamental to facilitate the subsequent purification processes so that the overall cost can be reduced. The aim of this work is to review some cell disruption processes for the extraction of protein from microalgae and cyanobacteria, considering that this step is crucial for the overall process due to the high rigidness of microalgal cell covering, which can hamper the release of proteins. It also aims at reviewing the purification techniques after cellular disruption, from conventional to more recent approaches, and finally addresses the antioxidant, antidiabetic, antihypertensive, antibacterial and other bioactive properties of microalgal protein hydrolysates and peptides.

Development and Evaluation of a Novel Fish-Derived Protein Peptide Conditioner Formulated with Pneumatophorus japonicus Heads Peptides

Given the unique advantage of hair conditioners formulated with marine-derived proteins and their hydrolysates in terms of green, effective, sustainable, and environmentally friendly. This study presents a novel fish-derived protein peptide-based hair conditioner that featuring Pneumatophorus japonicus heads peptides (PHP). Results demonstrate that the PHP conditioner exhibits favorable sensory properties, improves the surface morphology and structure of damaged hair, reduce the degree of fragmentation and roughness, improve the gloss and make hair smoother. PHP penetrates into the hair shaft, interacting with hair fiber keratin to enhance the integrity and stability of the α-conformation of hair keratin. Furthermore, the PHP conditioner treatment increased total amino acid content by 4.98% and hydrophobic amino acid content by 2.67%, approaching the levels observed in virgin hair. Therefore, the PHP conditioner effectively permeates the surface and interior of hair fibers, filling voids and ameliorating changes in both the microstructural and compositional aspects of the hair. These improvements surpass those observed with a commercially available wheat protein conditioner. Overall, the PHP conditioner effectively repairs damaged hair, offering a new strategy for the development of innovative natural hair care products.

Bioactivities of black soldier fly larvae protein hydrolysate

One of the potential insects for feed and food sources is the Black Soldier Fly (BSF) (Hermetia illucens). The BSF originated in South America and has now spread to many tropical countries. The BSF larvae (BSFL) had attracted much attention due to their good nutrient content and were recently reported as having useful bioactivities. BSFL has been widely used as feed and has been found to reduce bad smells associated with the growth of pathogenic microbes. The high protein content in BSFL is advantageous in the production of bioactive peptides. This research aimed to extract and hydrolyze the protein from BSFL with protease enzyme to produce protein hydrolysate or peptides with antioxidant and antibacterial activities. The antioxidant activity was measured using DPPH and FRAP, while the antibacterial was analyzed using the agar well diffusion method. BSFL was hydrolyzed with bromelain extracted from pineapple or commercial enzyme (Enzyplex). The IC50 shown by protein hydrolysate was 9.43±0.84% (v/v) or 0.72 mg/mL. The FRAP value was 521.21±264.53 µM for BSFL protein without enzyme addition, 792.14±291.02 for BSFL with the addition of bromelain, and 5,352.114±861.99 µM for BSFL with the addition of Enzyplex. The addition of bromelain increased the FRAP value 1.2 times, while the addition of Enzyplex increased the FRAP value 10.2 times. The antibacterial activity indicated that protein hydrolysate with phosphate buffer pH 8 at 50℃ and 24 hrs incubation showed the highest antibacterial activity to the lowest are Vibrio cholerae, Bacillus cereus and Staphylococcus aureus, respectively. The results suggest that BSFL has antibacterial and antioxidant bioactivities using bromelain extracted from pineapple and Enzyplex, a commercial enzyme that contains protease, lipase and amylase. Using Enzyplex as an enzyme to hydrolyze BFSL shows a very high antioxidant capacity compared to bromelain as an enzyme. This suggests that more protein and other macromolecules contribute to the antioxidant capacity. Using bromelain as an enzyme to hydrolyze BSFL, shows that BSFL peptide has higher antibacterial activity in gramnegative bacteria.

Integrating Mechanics and Bioactivity: A Detailed Assessment of Elasticity and Viscoelasticity at Different Scales in 2D Biofunctionalized PEGDA Hydrogels for Targeted Bone Regeneration.

Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

The Mechanism of Action of L-Tyrosine Derivatives against Chikungunya Virus Infection In Vitro Depends on Structural Changes.

Although the disease caused by chikungunya virus (CHIKV) is of great interest to public health organizations around the world, there are still no authorized antivirals for its treatment. Previously, dihalogenated anti-CHIKV compounds derived from L-tyrosine (dH-Y) were identified as being effective against in vitro infection by this virus, so the objective of this study was to determine the mechanisms of its antiviral action. Six dH-Y compounds (C1 to C6) dihalogenated with bromine or chlorine and modified in their amino groups were evaluated by different in vitro antiviral strategies and in silico tools. When the cells were exposed before infection, all compounds decreased the expression of viral proteins; only C4, C5 and C6 inhibited the genome; and C1, C2 and C3 inhibited infectious viral particles (IVPs). Furthermore, C1 and C3 reduce adhesion, while C2 and C3 reduce internalization, which could be related to the in silico interaction with the fusion peptide of the E1 viral protein. Only C3, C4, C5 and C6 inhibited IVPs when the cells were exposed after infection, and their effect occurred in late stages after viral translation and replication, such as assembly, and not during budding. In summary, the structural changes of these compounds determine their mechanism of action. Additionally, C3 was the only compound that inhibited CHIKV infection at different stages of the replicative cycle, making it a compound of interest for conversion as a potential drug.

Diagnostic Value of Interleukin 6, Interleukin 12P70, Serum Amyloid A, and Procalcitonin for Rheumatoid Arthritis and Their Relationship With the Disease Activity

To observe the diagnostic value of four serum inflammatory biomarkers, including interleukin 6 (IL-6), interleukin 12P70 (IL-12P70), serum amyloid A (SAA), and procalcitonin (PCT), in rheumatoid arthritis (RA) and to analyze their relationship with the disease activity. The study included 60 RA patients admitted to the Department of Rheumatology at Anhui Provincial Hospital of Traditional Chinese Medicine between December 2022 and December 2023. Thirty healthy individuals from the hospital's physical examination center served as the control group. Serum levels of IL-6 and IL-12P70 were detected using flow cytometry. SAA levels were determined by immunoturbidimetry, and PCT levels were assessed by chemiluminescence. Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), and anticyclic citrullinated peptide (ACCP) were detected using an automated biochemical analyzer. The 28-joint disease activity scores (DAS28-ESR) based on ESR were observed. Statistical analysis included t-tests, rank-sum tests, and Kruskal-Wallis H tests to compare the expression differences of the biomarkers among different groups. The diagnostic value of these biomarkers for RA was analyzed by ROC curve analysis. Spearman correlation analysis was performed to assess the relationships between the four inflammatory biomarkers and CRP, ESR, RF, ACCP, and DAS28-ESR. 1) The expression levels of SAA, IL-6, and IL-12P70 in the RA group were significantly higher than those in the control group (P<0.01). 2) ROC curve analysis showed that the area under the curve (AUC) for PCT was 0.611 (95% confidence interval [CI]: 0.488-0.735, P>0.05), for SAA, it was 0.819 (95% CI: 0.733-0.906, P<0.01), for IL-6, it was 0.875 (95% CI: 0.803-0.946, P<0.01), and for IL-12P70, it was 0.832 (95% CI: 0.746-0.917, P<0.01). The combined index of IL-6, IL-12P70, SAA, and PCT had an AUC of 0.973 (95% CI: 0.942-1.000, P<0.01). This indicates that the four inflammatory biomarkers can assist in the diagnosis of rheumatoid arthritis. 3) The expression levels of PCT and SAA varied significantly among the high, moderate, and low activity RA groups (P<0.01). 4) In RA patients, CRP was positively correlated with SAA (rs =0.75, P<0.01), and IL-6 (rs =0.52, P<0.01). ESR was positively correlated with SAA (rs =0.36, P<0.01). DAS28-ESR was positively correlated with PCT (rs =0.34, P=0.01), SAA (rs =0.51, P<0.01) and IL-6 (rs =0.33, P=0.01). The four inflammatory biomarkers (PCT, SAA, IL-6, and IL-12P70) are closely related to rheumatoid arthritis disease activity and can serve as serum indicators to assist in the diagnosis and assessment of RA.

Tau Protein and Tauopathies: Exploring Tau Protein-Protein and Microtubule Interactions, Cross-Interactions and Therapeutic Strategies.

Tau, a microtubule-associated protein (MAP), is essential to maintaining neuronal stability and function in the healthy brain. However, aberrant modifications and pathological aggregations of Tau are implicated in various neurodegenerative disorders, collectively known as tauopathies. The most common Tauopathy is Alzheimer's Disease (AD) counting nowadays more than 60 million patients worldwide. This comprehensive review delves into the multifaceted realm of Tau protein, puzzling out its intricate involvement in both physiological and pathological roles. Emphasis is put on Tau Protein-Protein Interactions (PPIs), depicting its interaction with tubulin, microtubules and its cross-interaction with other proteins such as Aβ1-42, α-synuclein, and the chaperone machinery. In the realm of therapeutic strategies, an overview of diverse possibilities is presented with their relative clinical progresses. The focus is mostly addressed to Tau protein aggregation inhibitors including recent small molecules, short peptides and peptidomimetics with specific focus on compounds that showed a double anti aggregative activity on both Tau protein and Aβ amyloid peptide. This review amalgamates current knowledge on Tau protein and evolving therapeutic strategies, providing a comprehensive resource for researchers seeking to deepen their understanding of the Tau protein and for scientists involved in the development of new peptide-based anti-aggregative Tau compounds.

Fabrication of resveratrol-loaded soy peptide nanogels with transglutaminase and in vitro gastrointestinal delivery and cholesterol-lowering effect

To improve the intestinal delivery of resveratrol, soy protein peptides (SPP) and transglutaminase (TGase) were used to prepare nanogels (NGs). The results demonstrated that, compared with native soy protein isolate (SPI) NG, SPP-NG showed a lower encapsulation efficiency (EE, 70.26%), a higher release efficiency (RE, 95.83%), and a smaller particle size (194.43 nm). However, the addition of TGase increased the EE and particle size to 74.37% and 217.73 nm, respectively. The RE of SPP-TGNG was also maintained at as high as 93.28%. Interestingly, SPP-TGNG exhibited optimal bile acid–binding capacity and cholesterol-lowering digests. The differences in delivery effects and morphology were attributed to structural changes in NGs. The unfolding and exposure of hydrophobic residues and weakened hydrogen bonds by hydrolysis resulted in a disordered and loose network structure, but it was beneficial to the production of cholesterol-lowering peptides and amino acids after digestion. The presence of TGase strengthened the crosslinking network by increasing β-sheet contents, hydrogen bonds, hydrophobic interactions, and isopeptide bonds. The alteration in multilevel structure would further affect the digestive products. The exposure of more hydrophobic residues and isopeptide crosslinking may be the intrinsic reason for the production of more hypocholesterolemic peptides and amino acids. This study indicated that the combination of hydrolysis and TGase treatment enhanced the delivery efficiency and bioactive properties of peptide-based NGs to resveratrol and revealed the internal mechanism between the structural changes and delivery efficiency. This work offers a theoretical foundation and technical suggestions for the production of peptide-based cargo systems and hydrophobic active substances.

Sequence-specific targeting of intrinsically disordered protein regions.

A general approach to design proteins that bind tightly and specifically to intrinsically disordered regions (IDRs) of proteins and flexible peptides would have wide application in biological research, therapeutics, and diagnosis. However, the lack of defined structures and the high variability in sequence and conformational preferences has complicated such efforts. We sought to develop a method combining biophysical principles with deep learning to readily generate binders for any disordered sequence. Instead of assuming a fixed regular structure for the target, general recognition is achieved by threading the query sequence through diverse extended binding modes in hundreds of templates with varying pocket depths and spacings, followed by RFdiffusion refinement to optimize the binder-target fit. We tested the method by designing binders to 39 highly diverse unstructured targets. Experimental testing of ∼36 designs per target yielded binders with affinities better than 100 nM in 34 cases, and in the pM range in four cases. The co-crystal structure of a designed binder in complex with dynorphin A is closely consistent with the design model. All by all binding experiments for 20 designs binding diverse targets show they are highly specific for the intended targets, with no crosstalk even for the closely related dynorphin A and dynorphin B. Our approach thus could provide a general solution to the intrinsically disordered protein and peptide recognition problem.

Modifications of the Structural, Nutritional, and Allergenic Properties of Atlantic Cod Induced by Novel Thermal Glycation Treatments.

This study aimed to assess the effect of novel thermal glycation, utilizing microwave processing (100-150 °C) combined with sugars (glucose and lactose), on the in vitro protein digestibility, peptides, secondary structures, microstructures, and allergenic properties of Atlantic cod. The research demonstrated that microwave heating at 150 °C with glucose significantly reduced cod allergenicity by up to 16.16%, while also enhancing in vitro protein digestibility to 69.05%. Glucose was found to be more effective than lactose in conjunction with microwave heating in reducing the allergenicity of Atlantic cod. Moreover, treatments conducted at 150 °C were more effective in increasing in vitro protein digestibility and peptide content compared to those at 100 °C. This study revealed that the novel processing technique of thermal glycation effectively reduced the allergenicity of Atlantic cod. It also offered fresh insights into the potential benefits of combining microwave heating with sugars.

Identification of Immunodominant Epitopes of Dengue Virus 2 Envelope and NS1 Proteins: Evaluating the Diagnostic Potential of a Synthetic Peptide.

Dengue is a major infectious disease with potential for outbreaks and epidemics. A specific and sensitive diagnosis is a prerequisite for clinical management of the disease. We designed our study to identify epitopes on theDengue virus (DENV) envelope (E) and non-structural protein 1 (NS1) with potential for diagnosis. Serology and immunoinformatic approaches were employed. We collected DENV-positive, DENV-negative and Japanese encephalitis virus-positive samples from collaborating hospitals in 2019 and 2022-2023. Seropositive peptides in 15-18 mer peptide arrays of E and NS1 proteins of DENV2 were determined by an indirect enzyme-linked immunosorbent assay. B-cell linear and conformational epitopes were predicted using BepiPred2.0 and ElliPro, respectively. A consensus recombinant peptide was designed, synthesised and evaluated for its diagnostic potential using patient sera. Eight peptides of E protein and six peptides of NS1 protein were identified to be the most frequently recognised by Dengue-positive patients. These peptide sequences were compared with B-cell epitope regions and found to be overlapped with predicted B-cell linear and conformational epitopes. EP11 and NSP15 showed a 100% amino acid sequence overlap with B-cell epitopes. EP1 and NSP15 had 14 whereas EP28, EP31, EP60 16, NSP12 and NSP32 had more than 15 interacting interface residues with a neutralising antibody, suggesting a strength of interaction. Interestingly, potential epitopes identified were localised on the surface of proteins as visualised by PyMOL. Validation with a recombined synthetic peptide yielded 92.3% sensitivity and 91.42% specificity. Immunodominant regions identified by serology and computationally predicted epitopes overlapped, thereby showing the robustness of the methodology and the peptide designedfor diagnosis.

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Ordered immobilization of serine proteases enabled by a linchpin directed modification platform.

We report a chemoselective and site-selective precision engineering of lysine in proteases. The mild and physiological reaction conditions keep their auto-degradation under control. Furthermore, it enables single-site ordered immobilization, enhancing protein digestion and peptide mapping efficiency.