Heat-induced Protein Research Articles

Improving the specific activity and stability of alkaline pectinase PEL3 through SpyTag/SpyCatcher cyclization.

PEL3, an alkaline pectinase, exhibited the highest activity among documented alkaline pectate lyases reported in our early study. Unfortunately, undesired thermal stability hampering its industrial application. The purpose of this study is to enhance the performance of wild-type PEL3 (W-PEL3) based on SpyTag/SpyCatcher-mediated cyclization. The cyclized PEL3 (C-PEL3) was observed to fold correctly and generate a spatial conformation in a head-to-tail manner in E. coli. C-PEL3 exhibited comparable optimum pH and temperature to those of W-PEL3. Moreover, the catalytic activity of C-PEL3 increased by 23% compared to W-PEL3, and the kcat/Km of C-PEL3 was 1.5-fold greater than that of the W-PEL3. Importantly, C-PEL3 showed improved stability compared to W-PEL3. Firstly, C-PEL3 displayed a 65% increase in residual activity after treatment at 55°C for 30min. Secondly, C-PEL3 was prone to resist heat-induced protein aggregation. Thirdly, C-PEL3 exhibited metal ion stability. Circular dichroism analysis revealed that C-PEL3 was more capable of maintaining its secondary structures than W-PEL3 upon heat treatment. C-PEL3, the initial example of a circular pectinase through SpyTag/SpyCatcher cyclization, exhibits superior performance and represents a highly encouraging contender for industrial utilization.

Multi-dimensional analysis of heat-induced soybean protein hydrolysate gels subjected to ultrasound-assisted pH pretreatment

This study aimed to evaluate the gelation characteristics of soybean protein hydrolysate (SPH) extracted by enzyme-assisted aqueous extraction. Specifically, the changes in gelation behaviors for heat-induced (95 °C, 20 min) SPH dispersions treated with pH (pH 3, 5, 9; pH 7 as control) and ultrasound (U; 240 W, 30 min) were investigated. The results showed that typical gel behavior with high elastic nature in the viscoelasticity and network structures were observed during the heating process, where the disulfide bond played a dominant role in the gel network formation of all the samples. Notably, the heat-induced aggregation in the SPH gels was mainly formed by the association of the basic B polypeptide in 11S and β subunit in 7S. The most superior SPH gel was formed at pH 7 when assisted by ultrasonication during the heating process. This as-synthesized gel showed a uniform filamentous structure and exhibited the more excellent textural, rheological and thermal properties than those of the samples formed under acidic and alkaline conditions. These results are of great value in revealing the gelation mechanism of SPH.

The effect of sweet tea polysaccharide on the physicochemical and structural properties of whey protein isolate gels

In this study, we investigated the effect of sweet tea polysaccharide (STP) on the physicochemical and structural properties of heat-induced whey protein isolate (WPI) gels, and explored the potential mechanism. The results indicated that STP promoted the unfolding and cross-linking of WPI to form a stable three-dimensional network structure, and significantly improved the strength, water-holding capacity and viscoelasticity of WPI gels. However, the addition of STP was limited to 2 %, too much STP would loosen the gel network and affect the gel properties. The results of FTIR and fluorescence spectroscopy suggested that STP affected the secondary and tertiary structures of WPI, promoted the movement of aromatic amino acids to the protein surface and the conversion of α-helix to β-sheet. In addition, STP reduced the surface hydrophobicity of the gel, increased the free sulfhydryl content, and enhanced the hydrogen bonding, disulfide bonding, and hydrophobic interactions between protein molecules. These findings can provide a reference for the application of STP as a gel modifier in the food industry.

Effects of different ph on properties of heat-induced Auricularia auricula-judae polysaccharide-whey protein isolate composite gels

The purpose of this study was to investigate the effects of different pH conditions on the properties of heat-induced composite gels of Auricularia auricula-judae polysaccharide (AP) and whey protein isolate (WPI). The color, hydration properties, texture, thermal stability, rheological properties, sulfhydryl content, surface hydrophobicity, and microstructure of the gels were evaluated. Due to the Maillard reaction, the color of the gel changed from white to brown with increasing pH. In contrast to the trend observed for color, the gels formed at pH 8 had better water holding capacity, springiness, hardness, thermal stability compared to pH 4, pH 6, and pH 10. Furthermore, rheological measurements showed that the gels after different pH treatments maintained their morphology by non-covalent interactions (hydrophobic interactions and electrostatic interactions), the gels exhibited shear thinning behavior. The free sulfhydryl content and surface hydrophobicity of the gels further confirmed the contribution of hydrophobic interactions in the gel network. The observation of the microstructure showed that the gel changed from a granular gel with rough surface to a gel with uniform and smooth surface with the increase of pH. This study provides some basic data references for improving the properties of AP-WPI composite gels and developing new gel-type products.

In vitro investigations of biological activities of Thymus willdenowii and Thymus atlanticus polyphenol-rich extracts

Thyme species produce a wide variety of phenolic compounds including tannins, phenolic acids, and flavonoids. Thymus atlanticus (T. atlanticus) and Thymus willdenowii (T. willdenowii) are important thyme species in the southeast of Morocco, with numerous biological properties. The polyphenolic extracts of these two thyme species were obtained using ethanol through Soxhlet apparatus. Antioxidant (DPPH, FRAP, and TAC methods), antihemolytic (2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) induced hemolysis test), hypolipidemic (3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity inhibition test), and anti-inflammatory (protein denaturation inhibition) effects of extracts were carried out using in vitro methods. The results showed that the polyphenolic extracts of these two species revealed important amounts of phenolic compounds. The contents of flavonoids were significant in the two species, while the contents of tannins and anthocyanin were very low. T. atlanticus showed an important antioxidant activity and a considerable antihemolytic effect in AAPH-induced hemolysis test (IC50 = 0.29 mg/mL), while T. willdenowii showed an important anti-inflammatory activity in heat-induced protein denaturation test (IC50 = 1.61 mg/mL). Moreover, both extracts at a dose of 20 µg/mL showed an important in vitro hypolipidemic activity by inhibiting HMG-CoA reductase activity (T. willdenowii: 51.16 %; T. atlanticus: 62.83 %). In conclusion, T. willdenowii and T. atlanticus extracts have considerable antioxidant, antihemolytic, hypolipidemic, and anti-inflammatory effects. The richness of these species in polyphenols gives them a large specter of biological properties, making them a valuable source of natural bioactive compounds that could prevent or treat various diseases.

CLPB3 is required for the removal of chloroplast protein aggregates and for thermotolerance in Chlamydomonas.

In the cytosol of plant cells, heat-induced protein aggregates are resolved by ClpB/Hsp100 family member HSP101, which is essential for thermotolerance. For chloroplast family member CLPB3 this is less clear with controversial reports on its role in conferring thermotolerance. To shed light onto this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TIG1 and the small heat shock proteins HSP22E/F in vivo and for conferring thermotolerance under heat stress. Although CLPB3 accumulates to similarly high levels as stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the ribosomal subunit PRPL1 and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that reduction in chloroplast protein synthesis capacity and increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely to the thylakoid membrane occupied area. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.

Evaluation of In-Vitro Anti-Inflammatory Activity of Leaves of Pongamia pinnata

Inflammation is a reaction of a living vascularised tissue to an injury. Conventional or synthetic drugs used in the treatment of inflammatory diseases are inadequate, it sometimes have serious side effects. So, number of herbal medicines is recommended for the treatment of inflammation that has no side effects. Pongamia pinnata, Pierre (Family- Fabaceae) commonly known as karanja. The seed and seed oil of the plant have been reported to be effective in various inflammatory and infectious diseases such as leucoderma, leprosy, lumbago, muscular and articular rheumatism, cutaneous affection including herpes and scabies etc. in folk medicine and Ayurveda, a traditional system of healing . Anti-inflammatory activities of this plant for various parts have been reported in various models. This study examined the anti-inflammatory potential of a methanolic leaves extract of Pongamia pinnata and reference drug Declofenac sodium, invitro using heat-induced protein denaturation technique. The concentrations of the leave extract used were 50-250μg/ml. While diclofenac sodium were 50, 100, 150, 200 and 250μg/ml. Both the Diclofenac sodium and extract showed the IC50 values 18.36 and 82.24µg/ml respectively. It is concluded that the methanolic leaves extract of Pongamia pinnata possess marked in vitro anti-inflammatory activity which is mediated possibly by flavonoids, tannins and alkaloids. This is a novel finding. Further the results scientifically justify the use of leaves of Pongamia pinnata in India traditional medicine to treat inflammation. Keywords: Inflammation, Pongamia pinnata, Fabaceae, heat-induced protein denaturation technique

Profiling the Hsp70 Chaperone Network in Heat-Induced Proteotoxic Stress Models of Human Neurons.

Heat stroke is among the most hazardous hyperthermia-related illnesses and an emerging threat to humans from climate change. Acute brain injury and long-lasting brain damage are the hallmarks of this condition. Hyperthermic neurological manifestations are remarkable for their damage correlation with stress amplitude and long-term persistence. Hyperthermia-induced protein unfolding, and nonspecific aggregation accumulation have neurotoxic effects and contribute to the pathogenesis of brain damage in heat stroke. Therefore, we generated heat-induced, dose-responsive extreme and mild proteotoxic stress models in medulloblastoma [Daoy] and neuroblastoma [SH-SY5Y] and differentiated SH-SY5Y neuronal cells. We show that heat-induced protein aggregation is associated with reduced cell proliferation and viability. Higher protein aggregation in differentiated neurons than in neuroblastoma precursors suggests a differential neuronal vulnerability to heat. We characterized the neuronal heat shock response through RT-PCR array analysis of eighty-four genes involved in protein folding and protein quality control (PQC). We identify seventeen significantly expressed genes, five of which are Hsp70 chaperones, and four of their known complementing function proteins. Protein expression analysis determined the individual differential contribution of the five Hsp70 chaperones to the proteotoxic stress response and the significance of only two members under mild conditions. The co-expression analysis reveals significantly high co-expression between the Hsp70 chaperones and their interacting partners. The findings of this study lend support to the hypothesis that hyperthermia-induced proteotoxicity may underlie the brain injury of heat stroke. Additionally, this study presents a comprehensive map of the Hsp70 network in these models with potential clinical and translational implications.

Attenuation of COX-2 enzyme by modulating H2O2-mediated NF-κB signaling pathway by monoamine oxidase inhibitor (MAOI): a further study on the reprofiling of MAOI in acute inflammation.

This study aims to investigate the anti-inflammatory mechanism of monoamine oxidase inhibitor (MAOI) in carrageenan (CARR) induced inflammation models to reprofile their use. We also aimed to explore the role of monoamine oxidase (MAO)-mediated H2O2-NF-κB-COX-2 pathway in acute inflammation. In vitro anti-inflammatory activity and hydrogen peroxide (H2O2) scavenging activity were performed according to the established procedure. Inflammation was induced using CARR in BALB/c mice at the foot paw and peritoneal cavity. Hourly measurement of paw swelling was performed. The level of nitric oxide (NO), myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and nuclear factor κB (NF-κB) was determined using enzyme-linked immunosorbent assay (ELISA). Peritoneal fluid was collected to investigate total count, differential count of leukocytes, and capillary permeability. In vitro anti-inflammatory evaluations revealed the potential role of MAOI to inhibit heat-induced protein denaturation and human red cell membrane destabilization. H2O2 inhibition activity of MAOI also proved their powerful role as an H2O2 scavenger. Treatment with MAOI in CARR-induced mice significantly reduced paw edema, leukocyte extravasation, and total and differential leukocyte count. The result of ELISA showed MAOI effectively reduce the level of COX-2, PGE2 and NF-κB in inflamed tissue. In short, this study demonstrates that inhibition of H2O2 by MAOI alleviates CARR-induced paw edema possibly by inhibiting the H2O2-mediated NF-κB-COX-2 pathway. The present investigation identifies MAOI might reprofile for the treatment of acute inflammation also, the MAO enzyme may use as a novel therapeutic target to design and develop new class of anti-inflammatory agents.

Inhibition of heat-induced protein aggregation by zirconium phthalocyanines.

Specific proteins found in food sources tend to aggregate into fibrils under heat treatment; studying these aggregation processes and developing tools to control protein heat-induced aggregation is an active area of research. Phthalocyanine complexes are known to exhibit antiprionic and anti-fibrillogenic activity. Thus, the anti-fibrillogenic effect of a series of Zr phthalocyanines with different out-of-plane coordinated ligands, namely positively charged (PcZrLys2 ), negatively charged (PcZrCitr2 ), and group able to form disulfide bridges (PcZrS2 ), on the heat-induced aggregation of such proteins as BLG, insulin, and lysozyme was studied. The inhibition of reaction activity up to about 90% was observed in the presence of these compounds for all proteins. The effective concentration of the inhibitor was calculated for the compound with the highest activity (PcZrS2 ) to be 10.6 ± 3.6 and 7.3 ± 1.2μM/L, respectively. Fluorescence spectroscopy studies demonstrated similar binding constants of three phthalocyanines binding with BLG globule. This is consistent with the results of molecular dynamics simulation, which imply the interaction of the globule with the tetrapyrrole macrocycle of phthalocyanine, leading to the globule stabilization. At the same time, TEM shows that in the presence of phthalocyanine PcZrS2 , thinner and longer fibrils were formed compared to control in all three proteins (BLG, insulin, and lysozyme). Thus, we can conclude that phthalocyanine PcZrS2 affects the amyloid aggregation's general mechanism, which is typical for proteins of different structures. Therefore, the phthalocyanine PcZrS2 is proposed as an anti-amyloidogenic agent suppressing heat-induced aggregation of proteins of different structures, making it potentially suitable for application in the food industry.

Poly-Arginine R18 Peptide Inhibits Heat-Induced Lysozyme Protein Aggregation: Implications for a Possible Therapeutic Role in Parkinson’s Disease

Poly-Arginine R18 Peptide Inhibits Heat-Induced Lysozyme Protein Aggregation: Implications for a Possible Therapeutic Role in Parkinson’s Disease

In vitro antioxidant and anti-inflammatory properties of Artocarpus altilis (Parkinson) Fosberg (seedless breadfruit) fruit pulp protein hydrolysates

Protein hydrolysates from dietary sources possess many physiological and biological properties. Artocarpus altilis is an evergreen multipurpose plant with many benefits. Therefore, this study evaluates in vitro antioxidant and anti-inflammatory properties of A. altilis protein hydrolysates. Protein was isolated from A. altilis and hydrolysed with pepsin and trypsin separately using different enzyme: substrate ratios (1:8, 1:16, 1:32). Antioxidant properties investigated included Fe2+-chelating, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydrogen peroxide radical scavenging activities. Anti-inflammatory activities were determined using effects on hypotonic solution-induced cell lysis on red blood cell membrane stabilisation and heat-induced protein denaturation. The degree of hydrolysis of trypsin hydrolysate increased with increasing enzyme–substrate ratio, while pepsin hydrolysate decreased as the enzyme–substrate ratio increased. The dominant amino acids in A. altilis protein and hydrolysates were glutamate, aspartate and leucine. Protein hydrolysates obtained from pepsin and trypsin digestion had DPPH scavenging abilities of 43.0 ± 0.01% and 22.2 ± 0.01%, respectively. However, trypsin-hydrolysed protein had a high Fe2+-chelating ability, while pepsin-hydrolysed protein had high hydrogen peroxide scavenging ability. Trypsin-hydrolysed protein showed good membrane stability and inhibition of protein denaturation. The results indicated that A. altilis protein hydrolysates possess significant antioxidant and anti-inflammatory effects and can further lend support to food industries as functional foods.

Modifying interprotein interactions for controlling heat-induced protein gelation

Globular proteins undergo heat-induced denaturation and eventually gelation at significantly elevated temperatures. The present study provides pathways to control temperature-driven protein gelation, having potential applications in a wide range of fields, from medicine to cosmetics to the food industry. The sol-gel transitions have been directed via modifying interprotein (electrostatic and hydrophobic) interactions by complexation of protein with amphiphiles. The heat-induced gelation of bovine serum albumin protein (anionic) is prevented in the presence of anionic sodium dodecyl sulfate (SDS) surfactant without significantly altering its native conformation. The specific binding of SDS monomers on the oppositely charged sites of protein increases the electrostatic repulsion between protein molecules, thereby suppressing the protein gelation. On the other hand, incorporating nonionic decaoxyethylene $n\ensuremath{-}\mathrm{dodecylether}$ (${\mathrm{C}}_{12}{\mathrm{E}}_{10}$) surfactant along with ionic surfactant reverses this scenario, and the solution state of the bovine serum albumin-SDS system undergoes gelation. The preferential binding of SDS with ${\mathrm{C}}_{12}{\mathrm{E}}_{10}$ nonionic surfactant forms detached mixed micelles, resulting in the release of the SDS monomers from protein, hence reverting the SDS-induced prevention of protein gelation. The results are counterintuitive and explained based on the interplay of hydrophobic and electrostatic interactions among protein molecules, as probed by small-angle neutron scattering, dynamic light scattering, and rheology.

Antibacterial, Anti-Inflammatory, and Antidiabetic Studies of the Amines Isolated from the Philippine Marine Sponge Desmacella sp

Aims: The study aimed to isolate and elucidate the structure of pure compounds from the polar extract of the marine sponge Desmacella sp. and explore their therapeutic potential. Background: Only a few marine organisms and their associated microorganisms collected from various parts of the Philippines have been documented and investigated in terms of their potential as sources of bioactive secondary metabolites. The sponge Desmacella sp. was collected off the coast of Agusan del Norte, Mindanao, Philippines, for the purpose of isolating compounds with medicinal prospects. Objective: The objective of the study was to explore the antibacterial, anti-inflammatory, and antidiabetic potentials of the pure isolates from the polar extract of the marine sponge Desmacella sp. Methods: The polar extract of the marine sponge Desmacella sp. from Agusan del Norte, Philippines, was subjected to purification using gravity column and medium pressure liquid chromatographic methods with TLC profiling as a guide. Pure isolates were obtained and subjected to NMR spectroscopy and mass spectrometry for structure elucidation, and the structures were confirmed by comparing the data with the literature. The pure compounds were explored for their antibacterial potential against B. subtilis BIOTECH 1679 and P. aeruginosa BIOTECH 1335 using a microplatebased antibacterial assay with resazurin indicator for the cell growth and ciprofloxacin as a positive control. Their anti-inflammatory potentials were determined using membrane stabilization and protein denaturation assays and were compared with the anti-inflammatory drug diclofenac and their antidiabetic potential by measuring their ability of inhibiting starch-hydrolyzing enzyme α-amylase. Results: The polar extract of the marine sponge Desmacella sp. from Agusan del Norte, Philippines, afforded three known compounds, namely, aaptamine, isopentylamine, and tyramine. This is the first report on the isolation of aaptamine, isopentylamine, and tyramine from the marine sponge genus Desmacella. The compounds aaptamine and isopentylamine exhibited antibacterial activity against B. subtilis, while tyramine and isopentylamine exhibited antibacterial activity against P. aeruginosa. These compounds showed the ability to inhibit heat-induced protein denaturation and effectively inhibited the erythrocytes' heat-induced hemolysis, which described in vitro antiinflammatory activity. These compounds were also evaluated for in vitro antidiabetic properties and showed their ability to inhibit the alpha-amylase enzyme activity of starch. Conclusion: Three known amines, namely, aaptamine, isopentylamine, and tyramine were isolated from the polar extract of the marine sponge Desmacella sp. collected off Mindanao coasts. Aaptamine and isopentylamine exhibited antibacterial properties against B. subtilis, while isopentylamine and tyramine exhibited antibacterial activity against P. aeruginosa. The anti-inflammatory potentials of aaptamine, isopentylamine, and tyramine were exhibited clearly in the membrane stabilization and protein denaturation assays and were compared with the anti-inflammatory drug diclofenac. Aaptamine, isopentylamine, and tyramine exhibited hypoglycemic properties and were compared with the antidiabetic drug acarbose in the inhibition of starch-hydrolyzing enzyme α-amylase assay.

Identification of subfunctionalized aggregate-remodeling J-domain proteins in Arabidopsis thaliana.

J-domain proteins (JDPs) are critical components of the cellular protein quality control machinery, playing crucial roles in preventing the formation and, solubilization of cytotoxic protein aggregates. Bacteria, yeast, and plants additionally have large, multimeric heat shock protein 100 (Hsp100)-class disaggregases that resolubilize protein aggregates. JDPs interact with aggregated proteins and specify the aggregate-remodeling activities of Hsp70s and Hsp100s. However, the aggregate-remodeling properties of plant JDPs are not well understood. Here we identify eight orthologs of Sis1 (an evolutionarily conserved Class II JDP of budding yeast) in Arabidopsis thaliana with distinct aggregate-remodeling functionalities. Six of these JDPs associate with heat-induced protein aggregates in vivo and co-localize with Hsp101 at heat-induced protein aggregate centers. Consistent with a role in solubilizing cytotoxic protein aggregates, an atDjB3 mutant had defects in both solubilizing heat-induced aggregates and acquired thermotolerance as compared with wild-type seedlings. Next, we used yeast prions as protein aggregate models to show that the six JDPs have distinct aggregate-remodeling properties. Results presented in this study, as well as findings from phylogenetic analysis, demonstrate that plants harbor multiple, evolutionarily conserved JDPs with capacity to process a variety of protein aggregate conformers induced by heat and other stressors.

Thickening mechanism of recombined dairy cream stored at 4 °C: Changes in the composition and structure of milk protein under different sterilization intensities

This work elucidates the mechanism involved in the effect of varying sterilization intensities on RDC thickening via comparative analysis of the changes in the composition and structure of RDC interfacial protein after storage at 4 °C and at 25 °C. The results showed that pasteurized RDCs (75 °C for 16 s, 90 °C for 5 min) and high-temperature sterilized RDCs (105 °C for 3 min, 115 °C for 7 min and 121 °C for 7 min) did not thicken during storage at 25 °C, and had lower viscosities and higher Ca2+ concentrations than those stored at 4 °C. Whey protein (WP) aggregates were found to have been adsorbed at the interface of high-temperature treated RDCs stored at 4 °C, leading to the aggregation of fat globules and, consequently, reversible thickening. However, high-temperature sterilized RDCs underwent into irreversible thickening at 10 d, 7 d and 3 d. This phenomenon was attributed to the large amount of heat-induced whey protein and κ-casein complex that was absorbed on the oil-water interface, with Ca2+ bonded to form bridging flocculation, which altered the secondary structure of the interfacial protein to one with increased β-sheet content and decreased random coil content.

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Chromatin-associated YTHDC1 coordinates heat-induced reprogramming of gene expression

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.

Gelation profile of laccase-crosslinked Bambara groundnut (Vigna subterranea) protein isolate

Enzymatic crosslinking has gained attention in improving plant protein heat-induced gels, which are composed of weak network structures. The aim of the study was to investigate the effect of laccase crosslinking on the rheological and microstructural properties of heat-induced Bambara groundnut protein gels. The rheological properties of laccase-modified Bambara groundnut protein isolate (BPI11BPI- Bambara groundnut protein isolate.) gel formed in situ were investigated. Changes in viscoelastic properties were monitored during heating and cooling ramps and gel structure fingerprints were analyzed by frequency sweep. Laccase addition induced an initial protein structure breakdown (G″>G′) at an enzyme dose-dependent (1–3 U/g) before gel formation and stabilization. Gel point temperatures were significantly decreased from 85°C to 29°C (∼3 folds) with increasing laccase activity (0 to 3 U/g protein, respectively). For laccase crosslinked gels, G′ was substantially greater than G” (>1 log) with no dependency on angular frequency, which suggests the formation of relatively well-structured gels. The highest gel strength (tan δ of 0.09, G* of 555.51 kPa & An of 468.04 kPa) was recorded at a laccase activity of 2 U/g protein and the gels formed at this activity appeared homogeneous with compact lath sheet-like structure. The crosslinking effects of laccase were corroborated by the decrease in thiol and phenolic contents as well as the crosslinking of amino acids in model reactions. Overall, the use of laccase improved gel properties and significantly altered the gelation profile of BPI. Laccase-modified Bambara groundnut protein gels have potential to be used in food texture improvement and development of new food products. For instance, they can be used in plant-based milk products such as yoghurt and cheese.

Heat-induced gelation of protein from mealworm (Tenebrio molitor): influence of pH and zinc concentration

Mealworm protein has the general potential to substitute environmentally less favourable, animal derived raw-materials as a gelling biomaterial for zinc enriched wound healing ointments. However, various factors affecting heat-induced gelation of mealworm protein have not yet been studied in detail. Therefore, we investigated the influence of pH (pH 5.5 and 7.5) and zinc concentration (0 M, 0.1 M and 0.3 M) on rheological properties and gel structure of heat-induced mealworm protein gels. Additionally, we used gel solubility experiments and rheological investigations to study the gels with regard to the involved types of interactions. Generally, all examined samples were able to form heat-induced gels. From gel solubility experiments and temperature sweeps, hydrophobic interactions and hydrogen bonds were found to be dominant in all samples. From higher storage moduli we derive the contribution of additional electrostatic and/or covalent interactions in samples with ZnSO4, while shorter linear viscoelastic regimes and more pronounced intracycle strain stiffening were related to less homogenous and more particulate gel structures. From our results strategies for the customisation of gel properties and gel composition may be deduced, that may advance the potential for future utilisation of mealworm protein in wound dressings.