Exosome-based targeted delivery of Proteolysis-Targeting Chimeras (PROTACs) is an innovative approach that provides a promising solution for addressing the complex issues of viral diseases. This strategy significantly mitigates the off-target effects associated with traditional therapeutics by facilitating targeted delivery of PROTACs, which in turn enhances the overall therapeutic outcomes. Challenges like poor pharmacokinetics and unintended side effects, commonly observed with conventional PROTACs usage, are effectively managed with this approach. Emerging evidence affirms the potential of this delivery mechanism in curbing viral replication. However, it is crucial to undertake more comprehensive investigations for optimizing exosome-based delivery systems and conducting stringent safety and efficacy assessments within preclinical and clinical settings. The advancements in this field could potentially redefine the therapeutic landscape for viral diseases, opening new vistas for their management and treatment.
To understand the mechanism of co-inoculation of Staphylococcus xylosus and Staphylococcus vitulinus (SX & SV) on structural protein degradation and taste enhancement of dry-cured bacon, protease activities, protein degradation, surface morphology of proteins and taste parameters of dry-cured bacon with Staphylococcus inoculation were investigated. The dry-cured bacon with co-inoculation of Staphylococcus xylosus and Staphylococcus vitulinus showed the best taste attributes. High residual activities in cathepsin B + L (more than 1.6-fold) and alanyl aminopeptidase (more than 1.4-fold) accelerated structural protein degradation in SX & SV. 32 down-regulated proteins were identified in SX & SV by TMT-labeled quantitative proteomic compared with control group; myosin and actin showed the most intense response to the accumulation of sweet and umami amino acids, and atomic force microscopy confirmed structural proteins breakdown by morphological changes. The accumulation of glutamic acid, alanine and lysine was mainly responsible for taste improvement of dry-cured bacon with Staphylococcus co-inoculation.
Four types of cheeses were prepared included plain- cheese (control), Codonopsis pilosula (CP)- cheese, plain- cheese with fish collagen (FC; control) and CP- cheese with FC. The effects of cheese samples on acidification, proteolysis of milk proteins using three methods (cadmium-ninhydrin method, O-phthaldialdehyde (OPA) assay, and electrophoresis assay), and angiotensin-converting enzyme (ACE)-inhibitory activity were investigated during 0, 2, & 4 weeks of ripening. In addition, the sensory evaluation was also investigated during 0, 2, 4, & 8 weeks of ripening. The presence of FC in CP- cheese increased the numbers of free amino acids (FAA) at 0 and 2 weeks. The addition of CP both in the presence and absence of FC affected positively (p
This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin- myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysin-myofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.
The objective of the present study was to develop a rapid, reliable and yet inexpensive protocol for genomic DNA extraction from frozen and ethanol-preserved Asian green-lipped mussels for random amplified microsatelite (RAM) analysis. The procedure comprised of three major steps: (1) Tissue degradation by boiling in 6% Chelex 100 resin in TE buffer; (2) Protein digestion by Proteinase K; and (3) DNA precipitation by adding 2 volumes of cold absolute ethanol. The entire procedure can be completed within two hours. The resulting RAM profiles were clear and reproducible. Our results demonstrate that the combined protocol of Chelex 100-Proteinase K-ethanol precipitation is a powerful yet economical DNA isolation method for population genetic studies involving a large sample size.
In spite of their excellent catalytic properties, enzymes should be improved before their implementation both in industrial and laboratorium scales. Immobilization of enzyme is one of the ways to improve their properties. Candida antarctica lipase B (Cal-B) has been reported in numerous publications to be a particularly useful enzyme catalizing in many type of reaction including regio- and enantio- synthesis. For this case, cross-linking of immobilized Cal-B with 1,2,7,8 diepoxy octane is one of methods that proved significantly more stable from denaturation by heat, organic solvents, and proteolysis than lyophilized powder or soluble enzymes. More over, the aim of this procedure is to improve the activity and reusability of lipase. Enzyme kinetics test was carried out by transesterification reaction between 4-nitrophenyl acetate (pNPA) and methanol by varying substrate concentrations, and the result is immobilized enzymes follows the Michaelis-Menten models and their activity is match with previous experiment. Based on the Vmax values, the immobilized enzymes showed higher activity than the free enzyme. Cross-linking of immobilized lipase indicate that cross-linking by lower concentration of cross-linker, FIC (immobilized lipase that was incubated for 24 h) gave the highest activity and cross-linking by higher concentration of cross-linker, PIC (immobilized lipase that was incubated for 2 h) gives the highest activity. However, pore size and saturation level influenced their activity.
Keratinous wastes have increasingly become a problem and accumulate in the environment mainly in the form of feathers, generated mainly from a large number of poultry industries. As keratins are very difficult to degrade by general proteases, they pose a major environmental problem. Therefore, microorganisms which would effectively degrade keratins are needed for recycling such wastes. A geophilic dermatophyte, Microsporum fulvum IBRL SD3 which was isolated from a soil sample collected from a chicken feather dumping site using a baiting technique, was capable to produce keratinase significantly. The crude keratinase was able to degrade whole chicken feathers effectively. The end product of the degradation was protein that contained essential amino acids and may have potential application in animal feed production. Thus, M. fulvum could be a novel organism to produce keratinase for chicken feathers degradation.
The stone fish (Actinopyga lecanora) ethanolic and methanolic tissue extracts were investigated for total phenolic contents (TPCs) as well as antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) radical scavenging activity and ferric reducing antioxidant power (FRAP) assays. Both extracts showed low amount of phenolics (20.33 to 17.03 mg of gallic acid equivalents/100 g dried sample) and moderate antioxidant activity (39% to 34% DPPH(•) radical scavenging activity and 23.95 to 22.30 mmol/100 mL FeSO4 FRAP value). Enzymatic proteolysis was carried out in order to improve the antioxidant activity using six commercially available proteases under their optimum conditions. The results revealed that the highest increase in antioxidant activity up to 85% was obtained for papain-generated proteolysate, followed by alcalase (77%), trypsin (75%), pepsin (68%), bromelain (68%), and flavourzyme (50%) as measured by DPPH(•) radical scavenging activity, whilst for the FRAP value, the highest increase in the antioxidant activity up to 39.2 mmol/100 mL FeSO4 was obtained for alcalase-generated proteolysate, followed by papain (29.5 mmol/100 mL FeSO4), trypsin (23.2 mmol/100 mL FeSO4), flavourzyme (24.7 mmol/100 mL FeSO4), bromelain (22.9 mmol/100 mL FeSO4), and pepsin (20.8 mmol/100 mL FeSO4). It is obvious that proteolysis of stone fish tissue by proteolytic enzymes can considerably enhance its antioxidant activity.
Plain and Lycium barbarum yogurt were made in the presence and absence of fish collagen. Yogurt samples were analyzed for acidification, milk protein proteolysis, angiotensin I-converting enzyme (ACE) inhibitory activity, and sensory evaluation during refrigerated storage for up to 21 days. The o-phthaldialdehyde peptides amount of L. barbarum yogurt both in the presence and absence of fish collagen were significantly increased during 14 days of storage. SDS-PAGE showed improvement in whey proteins degradation of L. barbarum yogurt with/without fish collagen after 3 weeks of storage. L. barbarum yogurt in absence of fish collagen was acting as a great ACE inhibitor reached up to 85% on day 7 of storage. The incorporation of L. barbarum and/or fish collagen affected to a small extent the overall sensory characteristics of yogurt. Yogurt supplemented with L. barbarum and/or fish collagen may lead to the improvement in the production and formulation of yogurt differing in their anti-ACE activity.
The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections.
The application of proteomic and peptidomic technologies for food-derived bioactive peptides is an emerging field in food sciences. These technologies include the use of separation tools coupled to a high-resolution spectrometric and bioinformatic tools for prediction, identification, sequencing, and characterization of peptides. To a large extent, one-dimensional separation technologies have been extensively used as a continuous tool under different optimized conditions for the identification and analysis of food peptides. However, most one-dimensional separation technologies are fraught with significant bottlenecks such as insufficient sensitivity and specificity limits for complex samples. To address this limitation, separation systems based on orthogonal, multidimensional principles, which allow for the coupling of more than one analytical separation tool with different operational principles, provide a higher separation power than one-dimensional separation tools. This review describes the structure-informed separation and purification of protein hydrolyzates to obtain peptides with desirable bioactivities. PRACTICAL APPLICATIONS: Application of bioactive peptides in the formulation of functional foods, nutraceuticals, and therapeutic agents have increasingly gained scholarly and industrial attention. The bioactive peptides exist originally in protein sources and are only active after hydrolysis of the parent protein. Currently, several tools can be configured in one-dimensional or multidimensional systems for the separation and purification of protein hydrolyzates. The separations are informed by the structural properties such as the molecular weight, charge, hydrophobicity or hydrophilicity, and the solubility of peptides. This review provides a concise discussion on the commonly used analytical tools, their configurations, advantages and challenges in peptide separation. Emphasis is placed on how the structural properties of peptides assist in the separation and purification processes and the concomitant effect of the separation on peptide bioactivity.
Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by
the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box
protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS.
A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat
(FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system
(Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins.
Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a
bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA)
and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before
the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter
genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared
to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands
of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique.
Those genes may code for potential interacting proteins that needs further identification and confirmation.
Cheddar cheese proteolysis were accelerated employing Penicillium candidum PCA1/TT031 protease into cheese curd. In the present study, several of the significant factors such as protease purification factor (PF), protease concentration and ripening time were optimized via the response surface methodology (RSM). The ideal accelerated Cheddar cheese environment consisted of 3.12 PF, 0.01% (v/v) protease concentration and 0.6/3 months ripening time at 10 °C. The RSM models was verified to be the most proper methodology for the maintain of chosen Cheddar cheese. Under this experimental environment, the pH, acid degree value (ADV), moisture, water activity (aw), soluble nitrogen (SN)%, fat and overall acceptability were found to be 5.4, 6.6, 35%, 0.9348, 18.8%, 34% and 13.6, respectively of ideal Cheddar cheese. Furthermore, the predicted and experimental results were in significant agreement, which confirmed the validity and reliability of the suggested method. In spite of the difference between the ideal and commercial Cheddar cheese in the concentration of some of amino acids and free fatty acids, the sensory evaluation did not show any significant difference in aroma profile between them.
Matrix metalloproteinases (MMPs) are enzymes that can degrade collagen in hybrid layer and reduce the longevity of adhesive restorations. As scientific understanding of the MMPs has advanced, useful strategies focusing on preventing these enzymes' actions by MMP inhibitors have quickly developed in many medical fields. However, in restorative dentistry, it is still not well established. This paper is an overview of the strategies to inhibit MMPs that can achieve a long-lasting material-tooth adhesion. Literature search was performed comprehensively using the electronic databases: PubMed, ScienceDirect and Scopus including articles from May 2007 to December 2019 and the main search terms were "matrix metalloproteinases", "collagen", and "dentin" and "hybrid layer". MMPs typical structure consists of several distinct domains. MMP inhibitors can be divided into 2 main groups: synthetic (synthetic-peptides, non-peptide molecules and compounds, tetracyclines, metallic ions, and others) and natural bioactive inhibitors mainly flavonoids. Selective inhibitors of MMPs promise to be the future for specific targeting of preventing dentin proteolysis. The knowledge about MMPs functionality should be considered to synthesize drugs capable to efficiently and selectively block MMPs chemical routes targeting their inactivation in order to overcome the current limitations of the therapeutic use of MMPs inhibitors, i.e., easy clinical application and long-lasting effect.
The NS2B/NS3 protease is crucial for the pathogenesis of the DENV. Therefore, the inhibition of this protease is considered to be the key strategy for the development of new antiviral drugs. In the present study, malabaricones C (3) and E (4), acylphenols from the fruits of Myristica cinnamomea King, have been respectively identified as moderate (27.33 ± 5.45 μM) and potent (7.55 ± 1.64 μM) DENV-2 NS2B/NS3 protease inhibitors, thus making this the first report on the DENV-2 NS2B/NS3 protease inhibitory activity of acylphenols. Based on the molecular docking studies, compounds 3 and 4 both have π-π interactions with Tyr161. While compound 3 has hydrogen bonding interactions with Gly151, Gly153 and Tyr161, compound 4 however, forms hydrogen bonds with Ser135, Asp129, Phe130 and Ile86 instead. The results from the present study suggests that malabaricones C (3) and E (4) could be employed as lead compounds for the development of new dengue antivirals from natural origin.
The study examined the protein profile of Pectoralis major muscle in broiler chickens subjected to different freezing and thawing methods. Pectoralis major muscle was excised from the carcasses of twenty broiler chickens and split into left and right halves. The left half was subjected to slow freezing (-20oC) while the right half was rapidly frozen (-80oC). The samples were stored at their respective temperature for 2 weeks and assigned to either of tap water (27oC, 30 min), room temperature (26oC, 60 min), microwave (750W, 10 min) or chiller (4oC, 6 h) thawing. Changes in myofibrillar proteins following the thawing methods were monitored through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile indicated differences (p < 0.05) in intensities of the components of myofibrillar proteins among the thawing methods in both slow and rapidly frozen samples. Chiller thawing had significantly higher (p < 0.05) protein concentration than other methods in rapidly frozen samples. However, in slow freezing, there were no significant differences in protein concentration among the thawing methods. In rapidly frozen samples, the protein optical densities at molecular weight of 21, 27, 55 and 151kDa in tap water, chiller and room temperature thawing did not differ (p < 0.05). Similarly, in slowly frozen samples, protein optical densities at molecular weight of 21, 27, 85 and 151 kDa were not significantly different among chill, tap water and room temperature thawing. Microwave thawing consistently caused higher protein degradation resulting in significantly lower (p < 0.05) protein quality and quantity in both freezing methods.
Amino acids are known as anabolic factors that are essential for formation of muscle by stimulating protein synthesis while inhibiting proteolysis, and they are significant component for the synthesis of various nitrogenous compounds. There are 20 amino acids are essential to require in cell for formation of body protein of which about 10 amino acids, which cannot be synthesized by the birds are termed essential. Among the essential amino acid arginine one of the essential amino acids for chickens because, like other birds, they are unable to obtain Arginine from endogenous sources due to the absence of most of the enzymes involved in the urea cycle. This amino acid involved in synthesis of proline, hydroxyl proline and polyamines which are essential for connective tissue synthesis as well as increased growth of chicken. Moreover, L-arginine (L-Arg) is effective for reducing fat deposition in broiler. Moreover, it decrease heat stress increase meat quality and increase immune response of broiler. This re-view presents the recent advances in the relevance of the inclusion of excess L-Arginine in broiler ration to growth, fat deposition and immune response in broiler.
The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.
Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging.
Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba.