Isoelectric Point Research Articles

Microwave-Functionalized Natural Tannic Acid as an Anticorrosive UV-Curable Coating

Corrosion causes serious steel deterioration with consequent negative impacts on the environment and economy. Organic coatings are widely exploited to provide corrosion protection on low-carbon steel. However, the raw materials and preparation methods for common anticorrosive coatings are not sustainable. In this framework, the efficient microwave-assisted methacrylation of a natural polyphenolic compound, tannic acid (TA), provided a UV-curable monomer with a high degree of substitution. The produced methacrylated tannic acid (MTA) was characterized by means of 31P NMR and FTIR spectroscopies. The UV-curing of MTA by radical photopolymerization was deeply investigated via the real-time FTIR, photo-DSC, and photo-rheological analyses, confirming the high photo-reactivity of MTA with a conversion of 80% and a gel point at 2.5 s. The UV-cured MTA showed good thermal stability and a glass transition temperature (Tg) of 133 °C. Furthermore, UV-cured MTA coating exhibited high hardness and hydrophobicity. The zeta potential measurement indicated a negatively charged surface with an isoelectric point (IEP) at pH 2.7. Finally, the good corrosion protection performance of UV-cured MTA coating on plasma pre-treated steel surface was assessed through electrochemical impedance spectroscopy.

Concentrating Cocoa Polyphenols—Clarification of an Aqueous Cocoa Extract by Protein Precipitation and Filtration

The seeds of Theobroma cacao L. are rich in antioxidant flavonoids such as flavan-3-ols, which are valued for their health benefits. In this context, it is of interest to improve flavanol content in cocoa extracts. The present study aimed at improving the clarification process of an aqueous cocoa extract using protein precipitation and filtration. Five pH modifications and two bentonite amounts were tested for their effects on protein precipitation and flavanol content. Micro- and ultrafiltration as a subsequent step was done by testing three different ceramic membranes (30, 80, and 200 nm). Lower pH in pre-treatment reduced protein content and kept flavanols constant, while at higher pH, flavanols were reduced up to 40%. Larger membrane pores enhanced polyphenol permeation, while smaller pores limited protein permeation. Adjusting pH to the isoelectric point increased protein adsorption, improving filtration quality despite decreased permeate flux. However, membrane fouling results in higher permeate quality due to increased selectivity. Furthermore, the addition of bentonite during filtration reduced both protein and flavanol content in the permeate, similar to the effects seen in the pre-treatment of the supernatant. Optimizing pH and membrane pore size enhances the recovery and quality of polyphenols during filtration, balancing protein removal and flavanol retention.

A new paradigm for optimized experimental design in cIEF platforms aimed at an accurate robust and reliable mAbs charge-variant assessment.

The success of therapeutic monoclonal antibodies (mAbs) and their biosimilars, highlights the challenge to control their purity, identity, and stability. For this purpose, among several orthogonal methodologies, imaged capillary Iso Electric Focusing (icIEF) is one of the leading techniques. Despite the isoelectric point (pI) being a univocal parameter relying on the protein's primary sequence and its post-translational modifications (PTMs), the current Charge Variants Profile Assessment (CVPA) carried out by cIEF relies on relative comparisons with corresponding reference standards, this is because the inconsistent outputs for the same sample across different instruments preclude the uniqueness of the measured parameters. We demonstrate that refining the current calibration approach in the iciEF method allows for obtaining more reliable and objective pIs, and a deeper understanding of the pH gradients along the capillaries. We are confident our advancements will enhance CVPA, by exploring the concept of univocal charge identity. This is crucial for constructing biobanks and developing algorithms to quickly identify divergences from the originators, thus ensuring drug quality, efficacy, and safety. Moreover, our method allows an experimental design optimized to minimize bias ("Unbiased" Experimental Design-UED) to study resolution as a multivariate function of different input variables. This endeavor aims to develop optimal methods tailored to specific pH ranges.

Structural diversity and distribution of NMCP-class nuclear lamina proteins in streptophytic algae.

Nuclear Matrix Constituent Proteins (NMCPs) in plants function like animal lamins, providing the structural foundation of the nuclear lamina and regulating nuclear organization and morphology. Although they are well-characterized in angiosperms, the presence and structure of NMCPs in more distantly related species, such as streptophytic algae, are relatively unknown. The rapid evolution of NMCPs throughout the plant lineage has caused a divergence in protein sequence that makes similarity-based searches less effective. Structural features are more likely to be conserved compared to primary amino acid sequence; therefore, we developed a filtration protocol to search for diverged NMCPs based on four physical characteristics: intrinsically disordered content, isoelectric point, number of amino acids, and the presence of a central coiled-coil domain. By setting parameters to recognize the properties of bona fide NMCP proteins in angiosperms, we filtered eight complete proteomes from streptophytic algae species and identified strong NMCP candidates in six taxa in the Classes Zygnematophyceae, Charophyceae, and Klebsormidophyceae. Through analysis of these proteins, we observed structural variance in domain size between NMCPs in algae and land plants, as well as a single block of amino acid conservation. Our analysis indicates that NMCPs are absent in the Mesostigmatophyceae. The presence versus absence of NMCP proteins does not correlate with the distribution of different forms of mitosis (e.g., closed/semi-closed/open) but does correspond to the transition from unicellularity to multicellularity in the streptophytic algae, suggesting that an NMCP-based nucleoskeleton plays important roles in supporting cell-to-cell interactions.

Physical Isolation of Single Protein Molecules within Well‐Defined Coordination Cages to Enhance Their Stability

Encapsulation of a single protein within a confined space can lead to distinct properties compared to bulk solutions, but controlling the number of encapsulated proteins and their environment remains challenging. This study demonstrates the encapsulation of single proteins within well‐defined, tunable cavities of self‐assembled coordination cages, thereby enhancing protein stability. Within uniform cavities of size‐tunable coordination cages, 15 different proteins of varying sizes (3‐6 nm in diameter) and properties (e.g., isoelectric points and hydrophobicity) were successfully confined. Various analytical techniques confirmed that the proteins maintained their secondary structures and enzymatic activities under denaturing conditions such as exposure to organic solvents, heat, and buffers. These findings suggest that such coordination cages have the potential to serve as synthetic hosts for precisely controlling protein functions within their customizable cavities.

Physical Isolation of Single Protein Molecules within Well-Defined Coordination Cages to Enhance Their Stability.

Encapsulation of a single protein within a confined space can lead to distinct properties compared to bulk solutions, but controlling the number of encapsulated proteins and their environment remains challenging. This study demonstrates the encapsulation of single proteins within well-defined, tunable cavities of self-assembled coordination cages, thereby enhancing protein stability. Within uniform cavities of size-tunable coordination cages, 15 different proteins of varying sizes (3-6 nm in diameter) and properties (e.g., isoelectric points and hydrophobicity) were successfully confined. Various analytical techniques confirmed that the proteins maintained their secondary structures and enzymatic activities under denaturing conditions such as exposure to organic solvents, heat, and buffers. These findings suggest that such coordination cages have the potential to serve as synthetic hosts for precisely controlling protein functions within their customizable cavities.

Unraveling the physiochemical characteristics and molecular insights of Zein protein through structural modeling and conformational dynamics: a synergistic approach between machine learning and molecular dynamics simulations

This research article presents a comprehensive investigation into the three-dimensional structure, physicochemical characteristics and conformational stability of the Zein protein. Machine learning (ML) based homology modeling approach, was employed to predict the 3D structure of Zein protein. Convolutional neural networks (CNNs) were utilized for refining the model, capturing complex spatial features and improving decoy refinement. The predicted 3D structure of Zein protein showed a high-confidence score, i.e. C-score of 0.96. Physiochemical characteristic was also analyzed to investigate its protonation and deprotonation behavior across a range of pH values. A comprehensive analysis of the titration curve and electrostatic charges was performed to uncover valuable molecular insights into the zein protein’s charge distribution, electrostatic interactions and potential conformational changes. Molecular dynamics (MD) simulations were performed to analyze the zein structural behavior under different pH values (2.0, 4.5, 6.8, 10.0 and 12.5), ionic strengths (0 mM, 25 mM, 50 mM, 75 mM, 100 mM) and temperatures (300K, 350K, 375K). Our results demonstrated the influence of these factors on zein protein’s stability and conformational dynamics. At extreme pH values of 2.0 and 12.5, the Zein protein exhibited increased structural deviations and potential unfolding, while intermediate pH values closer to the protein’s isoelectric point (pI) demonstrated more compact and stable conformations. Analysis of root mean square deviation, radius of gyration, solvent accessible surface area and Ramachandran plot provided clear understandings of the protein’s compactness and surface exposure, confirming the impact of pH, ionic strength and temperature on the protein’s conformation.

Physicochemical and electrical properties of DPPC bilayer membranes in the presence of oleanolic or asiatic acid

This study aimed to investigate the effect of selected compounds from the group of triterpene sapogenins on model phosphatidylcholine membranes. Two types of biological membrane model systems were used in the work, i.e., liposomes (microelectrophoresis method) and spherical bilayers (interfacial tension method). Each model was modified with the tested sapogenin compounds, and the change in their physicochemical and electrical parameters was analyzed. Parameters characterizing the equilibrium in the membrane of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-oleanolic acid (OA) and DPPC-asiatic acid (AA) were determined). Based on the Young-Laplace equation, the interfacial tensions of spherical lipid bilayers were measured. The formation of 1:1 complexes was assumed in the DPPC-OA and DPPC-AA membrane systems, and the parameters characterizing the interactions in the formed complexes were calculated. Microelectrophoresis was used to study the surface charge density of lipid membranes. These values were obtained from electrophoretic mobility data using Smoluchowsky’s equation. The influence of pH on the electrolyte solution and the composition of the membranes was investigated. The results indicate that modifying DPPC membranes with selected triterpene sapogenins, both OA and AA, causes changes in the surface charge density and shifts of the isoelectric point. Data presented in this work, obtained through mathematical derivation and confirmed experimentally, are of great importance for interpreting phenomena occurring in lipid membranes. A quantitative description of equilibria between phosphatidylcholine and sapogenins lets us understand the processes on the membrane surface. The equilibria are particularly significant from the standpoint of cell functioning. Phosphatidylcholine-sapogenin interactions modulate a range of physicochemical properties of membranes, and they are important in the course of the multiple processes involving membranes in the living cell (e.g., transport mechanism).

Exploring Enzyme Encapsulation Efficiency in MOFs Using Eco-Friendly Approaches.

The encapsulation of protein enzymes in metal-organic frameworks (MOFs) has been recognized as an effective enzyme immobilization approach. In this study, we demonstrated the influence of enzyme amount and the isoelectric points (pI) of different enzymes on the enzyme loading capacity in both mechanochemical (ball-milling) and water-based approaches. We found that increasing enzyme amounts enhances MOF enzyme loading without compromising activity, while the MOF shell protects encapsulated enzymes from proteinase K degradation through its size-sheltering mechanism. However, an excess of enzymes can hinder the formation of ZIF-90. Moreover, enzymes with low pI values (e. g., catalase, pI 5.4) facilitate encapsulation in MOFs, whereas enzymes with high pI values (e. g., lysozyme, pI 11.35) are more challenging to encapsulate. The simulation results revealed that increasing the enzyme amounts and pI values raises the activation energy necessary for MOF formation. This study highlights the crucial role of enzyme properties in the encapsulation process within MOFs, providing valuable insights for fabricating enzyme-MOF biocomposites for diverse applications, such as protein drug delivery.

Identification of Olea europaea CBF/DREB1 Family Genes in Abnormal Temperature Stress Response

Olea europaea, native to the Mediterranean region, has been widely cultivated for the nutritional content of its fruits and leaves. To adapt to climates beyond the Mediterranean, research on the climatic adaptation of O. europaea is urgently needed. Therefore, in this study, the CBF/DREB1 family genes in olives, which are related to cold tolerance, were genome-wide characterized. In total, four OeCBFs were screened; their open reading frame (ORF) were 552~684 bp in length, the encoded proteins were 20,786.60~25,235.16 Da, with 183~227 amino acids, and the theoretical isoelectric point (pI) were 4.91~9.13. The transcription of four OeCBFs was significantly different in olive tissues, especially OeCBF2 and OeCBF4, which were substantially more highly-expressed in aboveground tissues (terminal buds, young leaves, old leaves, and stems) than in underground tissues (thick and fine roots). Exposed to cold stress, OeCBF2 and OeCBF4 were up-regulated more obviously in olive leaves and stems, while OeCBF3 was induced to higher levels in the roots, implying the adaptation potential of the OeCBFs to low temperature stress. A comparison of the expression of the four OeCBFs in two varieties, Picholine (more tolerant to the other one) and Arbequina, found that the expression of OeCBFs was higher in most tissues of Picholine than that of Arbequina. When exposed to cold stress, the expression of the OeCBFs was also higher in the leaves and roots of Picholine than in Arbequina, further confirming the correlation between OeCBFs and cold tolerance of olive trees. This study not only deepens our understanding of the CBF gene family in olives, but also provides significant genetic information for olive breeding and improvement.

Pecan (Carya illinoinensis (Wangenh.) K. Koch) nuts as an emerging source of protein: extraction, physicochemical and functional properties.

The increasing demand for sustainable alternatives to traditional protein sources, driven by population growth, underscores the importance of protein in a healthy diet. Pecan (Carya illinoinensis (Wangenh.) K. Koch) nuts are currently underutilized as plant-based proteins but hold great potential in the food industry. However, there is insufficient information available on pecan protein, particularly its protein fractions. This study aimed to explore the physicochemical and functional properties of protein isolate and the main protein fraction glutelin extracted from pecan nuts. The results revealed that glutelin (820.67 ± 69.42 g kg-1) had a higher crude protein content compared to the protein isolate (618.43 ± 27.35 g kg-1), while both proteins exhibited amino acid profiles sufficient for adult requirements. The isoelectric points of protein isolate and glutelin were determined to be pH 4.0 and pH 5.0, respectively. The denaturation temperature of the protein isolate (90.23 °C) was higher than that of glutelin (87.43 °C), indicating a more organized and stable conformation. This is further supported by the fact that the protein isolate had a more stable main secondary structure than glutelin. Both proteins demonstrated improved solubility, emulsifying, and foaming properties at pH levels deviating from their isoelectric points in U-shaped curves. Compared to the protein isolate, glutelin displayed superior water and oil absorption capacity along with enhanced gelling ability. The protein isolate and glutelin from pecan nuts exhibited improved stability and competitive functional properties, respectively. The appropriate utilization of these two proteins will support their potential as natural ingredients in various food systems. © 2024 Society of Chemical Industry.

Interfacial structure modification and enhanced emulsification stability of microalgae protein through interaction with anionic polysaccharides

Microalgae protein (MP) have emerged as a focal point of research within food processing due to its nutritional value and foaming properties. However, its isoelectric point around pH 4 leads to it susceptible to collision, binding, and precipitation. Additionally, MP has poor emulsification properties and only shows stability under strongly alkaline conditions. This study investigated the effects of food-grade anionic polysaccharides (guar gum (GG), gum arabic (GA), low acyl gellan gum (LG), and pectin (PT)) on the molecular structure and emulsification properties of MP. Results indicated that these anionic polysaccharides enhanced the UV absorption of MP near 620 nm, especially at 14 % content, while fluorescence intensity decreased due to amino acid residues masking without structural changes. The addition of polysaccharides resulted in bimodal or multimodal particle size distributions, with LG and PT showing larger particle sizes. At pH 4, negatively charged polysaccharides formed stable complexes with near-neutral MP, improving solution stability via electrostatic repulsion and diminishing turbidity. The droplet distribution analysis indicated that higher anionic polysaccharide ratios (1:4, 1:2, and 1:1) correlated with smaller droplet sizes and increased emulsion stability. Zeta-potential measurements revealed negative charges for emulsions, with LG-MP and PT-MP complexes displaying higher absolute values (15.0 to 20.7 mV) compared to GG-MP and GA-MP complexes, indicating superior stability. Storage stability analysis showed that LG-MP and PT-MP complexes stabilized emulsions had minimal delamination over two weeks. Rheological assessments showed that increasing GG and GA contents from 14 % to 50 % had negligible effects on apparent viscosity, while LG-MP (1:1) complexes stabilized emulsion displayed higher viscosity compared to PT-MP emulsions. Frequency sweep results showed that GG-MP, GA-MP, and LG-MP emulsions had greater elastic moduli (G') than viscous moduli (G"), indicating elastic behavior, whereas PT-MP emulsions transitioned from liquid-like to solid-like behavior as frequency increased. This study illustrates the advantages of high LG and PT content in preventing particle aggregation and enhancing emulsion stability, providing a theoretical and practical foundation for MP applications in food processing.

Structural and functional properties of proteins isolated from four species of microalgae and their application in air-water interface stabilization

Herein, we extracted proteins from four microalgae: Chlamydomonas reinhardtii (CHR), Euglena gracilis (EUG), Spirulina platensis (SPP), and Spirulina maxima (SPM). Subsequently, their physicochemical and functional properties, as well as air-water interface adsorption behavior were investigated. Results demonstrated that the solubility and emulsifying properties of these proteins were significantly affected by pH, with minimum values observed near their isoelectric point (3.5) and maximum values at pH 11.0. Interestingly, the emulsifying properties of the four proteins were superior to soy protein isolate in the pH range of 3.0–7.0, indicating that these microalgae proteins may be suitable for applying as emulsifiers in foods. EUG exhibited the highest water/oil binding capacity, emulsifying activity, and emulsifying stability, which were attributed to its high surface hydrophobicity and β-sheet content, as well as small particle size. Dynamic adsorption behavior analysis illustrated that EUG showed faster diffusion and rearrangement rates, as well as a higher final equilibrated surface pressure value, contributing to its highest foaming capacity and foaming stability. This study greatly enhances the understanding of microalgae proteins and is expected to establish a theoretical foundation for developing novel protein resources that can be utilized in the foamed and emulsion-based food fabrication, including whipped cream and functional beverages.

A novel antimicrobial peptide CpAMP identified from Chinese horseshoe crab, Tachypleus tridentatus.

Antibacterial peptide (AMP) is a crucial component of the innate immune system in most organism, play an important role in host defense processes. Many of these peptides have broad antimicrobial activity against Gram-negative, -positive bacteria and fungi. In the present study, a novel AMP named CpAMP was identified using transcriptome analysis in Chinese horseshoe crab. The preprotein of CpAMP consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. And the putative mature peptide CpAMP was 4.95 kDa with a theoretical isoelectric point (pI) of 8.73. The mature CpAMP showed α-helix and irregularly curled structure in the cys-stabilized region. CpAMP exhibited a broad spectrum of antimicrobial activity against Gram-negative, -positive bacteria and antibiotic-resistant strains. In addition, CpAMP can significantly increase the survival rate of zebrafish infected with bacteria. Due to its broad-spectrum antibacterial activity and the sensitivity of drug-resistant strains, CpAMP could be used as a new type of drug for inhibition of pathogenic microorganisms or as an immune enhancer in animals.

Functional characterization of peroxiredoxin 5 from yellowtail clownfish (Amphiprion clarkii): immunological expression assessment, antioxidant activities, heavy metal detoxification, and nitrosative stress mitigation

Peroxiredoxin 5 (Prdx5) is the last recognized member of Prdx family. It is a unique, atypical, 2-Cys antioxidant enzyme, protecting cells from death caused by reactive oxygen species (ROS). In this study, the Prdx5 ortholog of Amphiprion clarkii (AcPrdx5) was identified and characterized to explore its specific structural features and functional properties. The open reading frame of AcPrdx5 is 573 bp long and encodes 190 amino acids containing a mitochondrial targeting sequence, thioredoxin domain, and two conserved cysteine residues responsible for antioxidant function. The predicted molecular weight and theoretical isoelectric point of AcPrdx5 are 20.3 kDa and 9.01, respectively. AcPrdx5 sequences were found to be highly conserved across the other orthologs from various organisms and it distinctively clustered within the fish Prdx5 subclade of the phylogenetic tree. The expression of AcPrdx5 was ubiquitously detected among twelve tested tissues, with the highest level in the brain. Furthermore, the mRNA levels of AcPrdx5 in the blood and head-kidney tissues were significantly (p < 0.05) upregulated following polyinosinic-polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi immune challenge. A concentration-dependent antioxidant potential of recombinant AcPrdx5 was observed in insulin disulfide bond reduction, heavy metal detoxification, free radical and hydrogen peroxide (H2O2) scavenging assays. Additionally, AcPrdx5 overexpression in fathead minnow (FHM) cells upregulated the antioxidant-associated gene (Rrm1, MAPK, SOD2, and PRDX1) expression after H2O2 treatment, and promoted cell viability upon arsenic (As) exposure. In macrophages, AcPrdx5 overexpression effectively suppressed substantial nitric oxide production under lipopolysaccharide treatment. Collectively, our results suggest that AcPrdx5 may play roles in both antioxidant defense system and innate immune response against pathogenic invasions in A. clarkii.

Morphological Identification and Antimicrobial Activity Test to Water Quality Using Bandotan (Ageratum conyzoides) Leaves

Water cleaning technology using coagulation has already been carried out use a plants in the coagulation process has never been done before, especially using plants that have potential to be a source of natural coagulants is Bandotan (Ageratum conyzoides). Plants that have the potential to be a source of natural coagulants are plants that contain a lot of protein. Each type of protein has an isoelectric point at a different pH like isoelectric point, the protein will have a neutral charge. . The calculation results of the number of colonies in samples were B1, B2, and B3 with dilutions of 266. 105, 9. 107 and 104. 109 CFU/mL. The results of Gram staining identification showed that the B1, B2 and B3 isolates had the shape are basil, basil and coccus (round) with Gram positive. The bacteria thrive in different habitats, so bacteria from the sample have been very cloudy to pH of 6.0 and 8.0 because neutralophiles properties. From the B3 isolate sample have high clear zone for Gram positive (S. aureus) of 13 mm more than Gram negative ( E. coli) of 11 mm. So that, the bandotan is able to inhibit the growth of pathogenic bacteria in the water

FastProtein-an automated software for in silico proteomic analysis.

Although various tools provide proteomic information, each tool has limitations related to execution platforms, libraries, versions, and data output format. Integrating data generated from different software is a laborious process that can prolong analysis time. Here, we present FastProtein, a protein analysis pipeline that is user-friendly, easily installable, and outputs important information about subcellular location, transmembrane domains, signal peptide, molecular weight, isoelectric point, hydropathy, aromaticity, gene ontology, endoplasmic reticulum retention domains, and N-glycosylation domains. It also helps determine the presence of glycosylphosphatidylinositol and obtain functional information from InterProScan, PANTHER, Pfam, and alignment-based annotation searches. FastProtein provides the scientific community with an easy-to-use computational tool for proteomic data analysis. It is applicable to both small datasets and proteome-wide studies. It can be used through the command line interface mode or a web interface installed on a local server. FastProtein significantly enhances proteomics analysis workflows by producing multiple results in a single-step process, thereby streamlining and accelerating the overall analysis. The software is open-source and freely available. Installation and execution instructions, as well as the source code and test files generated for tool validation, are available at https://github.com/bioinformatics-ufsc/FastProtein.

Regulation on Aggregation Behavior and In Vitro Digestibility of Phytic Acid-Whey Protein Isolate Complexes: Effects of Heating, pH and Phytic Acid Levels.

The impact of heat treatment, pH and phytic acid (PA) concentration on the aggregation behavior and digestibility of whey protein isolate (WPI) was investigated. The experimental results indicated that below the isoelectric point of WPI, heat treatment and elevated PA levels significantly increased turbidity and particle size, leading to the aggregation of WPI molecules. No new chemical bonds were formed and the thermodynamic parameters ΔH < 0, ΔS > 0 and ΔG < 0 suggested that the interaction between PA and WPI was primarily a spontaneous electrostatic interaction driven by enthalpy. After the small intestine stage, increasing phytic acid levels resulted in a significant decrease in hydrolysis degree from 16.2 ± 1.5% (PA0) to 10.9 ± 1.4% (0.5% PA). Conversely, above isoelectric point of WPI, there was no significant correlation between the presence of PA and the aggregation behavior or digestion characteristics of WPI. These results were attributed to steric hindrance caused by PA-WPI condensates, which prevented protease binding to hydrolysis sites on WPI. In summary, the effect of PA on protein aggregation behavior and digestive characteristics was not simply dependent on its presence but largely on the aggregation degree of PA-WPI induced by heat treatment, pH and PA concentration. The findings obtained here suggested that phytic acid may be utilized as an agent to modulate the digestion characteristics of proteins according to production requirements. Additionally, the agglomerates formed by heating phytic acid and protein below the isoelectric point could also be utilized for nutrient delivery.

Molecular depiction and functional delineation of E3 ubiquitin ligase MARCH5 in yellowtail clownfish (Amphiprion clarkii)

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi, AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H2O2-treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii.

Antibacterial and cytotoxic properties of esterified α- lactalbumin and β-lactoglobulin

α -Lactalbumin ( α -La) or β -lactoglobulin ( β -Lg) were esterified with methanol. Native proteins and their methylated esters (Est. α -La and Est. β -Lg) were tested for their antibacterial and cytotoxic properties. Esterification extents for Est. α -La and Est. β -Lg were 92% and 88%, with isoelectric points at pH 8 and 10, respectively. The Est. α -La and Est. β -Lg were more inhibiting the growth of pathogenic bacteria compared to the corresponding native proteins. The impact of esterified α -La and β -Lg on MCF-7, A549, and Caco-2 cell lines was assessed and revealed that the concentration inhibiting 50% cellular growth (IC50) for Est. α -La recorded 107.93 µg/mL against MCF-7, 616.44 µg/mL against A-549, and 388.23 µg/mL against Caco2. In parallel, Est. β -Lg achieved IC50 at 101.35, 420.84, and 234.16 µg/mL, against MCF-7, A-549, and Caco2, respectively. Esterified proteins may serve as therapeutic antimicrobial agents against pathogenic bacteria and cytotoxic agents against specific cancer cells.