Computational design and experimental confirmation of a disulfide-stapled YAP helixα1-trap derived from TEAD4 helical hairpin to selectively capture YAP α1-helix with potent antitumor activity.

Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.

Incorporation of cross-linked/acetylated tapioca starches on the gelling properties, rheological behaviour, and microstructure of low-salt myofibrillar protein gels: Perspective on phase transition.

This study investigated the gelling properties, rheological behaviour, and microstructure of heat-induced, low-salt myofibrillar protein (MP) gels containing different levels (2%, 4%, 6%, and 8%, w/w) of cross-linked (CTS) or acetylated (ATS) tapioca starch. The results indicated that either CTS or ATS significantly enhanced the gel strength and water-holding capacity of low-salt MP gels (P < 0.05), an outcome verified by the rheological behaviour test results under different modes. Furthermore, iodine-staining images indicated that the MP-dominated continuous phase gradually transited to a starch-dominated phase with increasing CTS or ATS levels, and 4% was the critical point for this phase transition. In addition, hydrophobic interactions and disulphide bonds constituted the major intermolecular forces of low-salt MP gels, effectively promoting phase transition. In brief, modified tapioca starches possess considerable potential application value in low-salt meat products.

Effects of high-intensity ultrasound on physicochemical and gel properties of myofibrillar proteins from the bay scallop (Argopecten irradians).

Myofibrillar proteins (MPs) have a notable impact on the firmness and flexibility of gel-based products. Therefore, enhancing the gelation and emulsification properties of scallop MPs is of paramount significance for producing high-quality scallop surimi products. In this study, we investigated the effects of high-intensity ultrasound on the physicochemical and gelation properties of MPs from bay scallops (Argopecten irradians). The carbonyl content of MPs significantly increased with an increase in ultrasound power (150, 350, and 550 W), indicating ultrasound-induced MP oxidation. Meanwhile, high-intensity ultrasound treatment (550 W) enhanced the emulsifying capacity and the short-term stability of MPs (up to 72.05 m2/g and 153.05 min, respectively). As the ultrasound power increased, the disulfide bond content and surface hydrophobicity of MPs exhibited a notable increase, indicating conformational changes in MPs. Moreover, in the secondary structure of MPs, the α-helix content significantly decreased, whereas the ß-sheet content increased, thereby suggesting the ultrasound-induced stretching and flexibility of MP molecules. Sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and scanning electron microscopy analysis further elucidated that high-intensity ultrasound induced MP oxidation, leading to modification of amino acid side chains, intra- and intermolecular cross-linking, and MP aggregation. Consequently, high-intensity ultrasound treatment was found to augment the viscoelasticity, gel strength, and water-holding capacity of MP gels, because ultrasound treatment facilitated the formation of a stable network structure in protein gels. Thus, this study offers theoretical insights into the functional modification of bay scallop MPs and the processing of its surimi products.

Molecular structural modification of myofibrillar protein from oyster (Crassostrea gigas) with oligosaccharides for improving its gel properties.

Glycation is a promising approach to enhance protein gel characteristics in the food industry. The impact of oyster myofibrillar protein (MP) being glycosylated with six oligosaccharides (dextran [Dex]-1 kDa, 5 kDa, 6 kDa, and 10 kDa, xylan [Xyla], and xyloglucan [Xyg]) on structural properties, aggregation behavior and gel properties was investigated in this study. The findings demonstrated that oligosaccharides significantly increased the glycation degree of MP by forming a stable tertiary conformation, increasing the contents of the disulfide bond and hydrogen bonds. Additionally, particle sizes decreased and solubility increased after glycation, improving the gel's strength, water-holding capacity, thermal stability, elastic modulus, and ordered network layout. It was determined that MP-Dex 5 had the best gel properties. The gel strength and water holding capacity of MP-Dex 5 increased by 70.59% and 32.27%, respectively. Molecular dynamics simulations results showed van der Waals energy and electrostatic interactions favor myosin binding to Dex or Xyla units. This study will provide insights into the relationship between molecular structure, aggregation behavior and gel property of oyster MP-oligosaccharide couples, and expand the application of oyster MP in food gels.

Effects of multi-frequency ultrasound-assisted immersion freezing processing on myofibrillar protein structure and lipid oxidation of large yellow croaker (Larimichthys crocea) during long-time frozen storage.

In this study, large yellow croaker (Larimichthys crocea) was frozen using multi-frequency ultrasound-assisted freezing (MUIF) with different powers (160 W, 175 W, and 190 W, respectively) and stored at -18 °C for ten months. The effect of different ultrasound powers on the myofibrillar protein (MP) structures and lipid oxidation of large yellow croaker was investigated. The results showed that MUIF significantly slowed down the oxidation of MP by inhibiting carbonyl formation and maintaining high sulfhydryl contents. These treatments also held a high activity of Ca2+-ATPase in the MP. MUIF maintained a higher ratio of α-helix to ß-sheet during frozen storage, thereby protecting the secondary structure of the tissue and stabilizing the tertiary structure. In addition, MUIF inhibited the production of thiobarbituric acid reactive substances value and the loss of unsaturated fatty acid content, indicating that MUIF could better inhibit lipid oxidation of large yellow croaker during long-time frozen storage.

Potential mechanisms and effects of ultrasound treatment combined with pre- and post-addition of κ-carrageenan on the gelling properties and rheological behavior of myofibrillar proteins under low-salt condition.

This study investigated the effect of ultrasound (US) combined with pre- and post-addition of κ-carrageenan (KC) on the gelling properties, structural characteristics and rheological behavior of myofibrillar proteins (MP) under low-salt conditions. The results showed that US combined with either pre- or post-addition of KC rendered higher gel strength and water holding capacity (WHC) of MP gels than those treated with US alone and added with KC alone (P < 0.05). US combined with pre-addition of KC facilitated the binding between MP and KC, which enhanced the gel strength and WHC of the mixed MP gels and significantly improved the rheological behavior of MP. This was also confirmed by the highest surface hydrophobicity, disulfide bonds and ß-sheet content of the MP gels with US combined with pre-addition of KC. Moreover, microstructural results reflected a denser structure for the pre-addition of KC in combination with US. However, US combined with post-addition of KC resulted in limited MP unfolding and relatively weak hydrophobic interactions in the composite gels, which were less effective in improving the gel properties of the MP gels. This study provides potential strategies for enhancing the gelling properties of low-salt meat products via application of US and KC.

Thawed drip and its membrane-separated components: Role in retarding myofibrillar protein gel deterioration during freezing-thawing cycles.

Myofibrillar proteins are crucial for gel formation in processed meat products such as sausages and meat patties. Freeze-thaw cycles can alter protein properties, impacting gel stability and product quality. This study aims to investigate the potential of thawed drip and its membrane-separated components as potential antifreeze agents to retard denaturation, oxidation and gel deterioration of myofibrillar proteins during freezing-thawing cycles of pork patties. The thawed drip and its membrane-separated components of > 10 kDa and < 10 kDa, along with deionized water, were added to minced pork at 10 % mass fraction and subjected to increasing freeze-thaw cycles. Results showed that the addition of thawed drip and its membrane separation components inhibited denaturation and structural changes of myofibrillar proteins, evidenced by reduced surface hydrophobicity and carbonyl content, increased free sulfhydryl groups, protein solubility and α-helix, as compared to the deionized water group. Correspondingly, improved gel properties including water-holding capacity, textural parameters and denser network structure were observed with the addition of thawed drip and its membrane separation components. Denaturation and oxidation of myofibrillar proteins were positively correlated with gel deterioration during freezing-thawing cycles. We here propose a role of thawed drip and its membrane separation components as cryoprotectants against myofibrillar protein gel deterioration during freeze-thawing cycles.

Thermal aggregation behavior of silver carp myofibrillar protein at low salt content: Effect of oat ß-glucan combined with ultrasound-assisted heating.

The effects of oat ß-glucan (OG) combined with ultrasound-assisted treatment on thermal aggregation behavior of silver carp myofibrillar protein (MP) under low salt concentration were investigated. The particle size and turbidity of MP were increased to higher levels by OG participation or ultrasound treatment during the two-stage heating. Both OG and ultrasonic treatment promoted the unfolding of MP structure, evidenced by the gradual decrease of α-helix content and fluorescence intensity, as well as the increase of ß-sheet content, surface hydrophobicity and sulfhydryl content. Compared to solely OG or ultrasonic treatment, the combination of OG and ultrasound further promoted the unfolding of MP and more sulfhydryl groups were exposed in the pre-heating stage, which was conducive to strengthen the chemical forces between MP molecules. Additionally, AFM analysis revealed that the apparent morphology of the OG combined with ultrasonic treated group exhibited a smoother surface and a more uniform distribution of aggregates.

Effect of two-stage low-temperature tempering process assisted by electrostatic field application on physicochemical and structural properties of myofibrillar protein in frozen longissimus dorsi of tan mutton.

The effects of refrigerator tempering, two-stage low-temperature tempering (TLT), and a combination of TLT with electrostatic field tempering (TLT-1500/2000/2500/3000) on the physicochemical and structural properties of the myofibrillar protein (MPs) in Longissimus dorsi of Tan mutton were investigated. The results from differential scanning calorimetry and dynamic rheology indicated that TLT-2000/2500 had the least impact on the thermal stability of MPs. While the carbonyl and dityrosine contents of MPs in TLT-2000/2500 were the lowest, the total sulfhydryl content and Ca2+-ATPase activity were the highest, suggesting that TLT-2000/2500 preserved the properties of MPs more effectively. The smaller and uniformly distributed particle size, highest zeta potential, and SDS-PAGE analysis confirmed that TLT-2000/2500 had minimal impact on the aggregation and degradation of MPs. Additionally, results from surface hydrophobicity, Fourier transform infrared spectroscopy, intrinsic fluorescence, and UV second-derivative absorption spectra suggested that TLT-2000/2500 was more conducive to stabilizing the primary, secondary, and tertiary structures of MPs.

Comparative analysis of the longissimus muscle proteome of European wild boar and domestic pig in response to thermal processing.

This study tries to fill the knowledge gap regarding differences in the expression of proteins in the meat of European wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica), considering the impact of thermally induced degradation. We assessed relative protein changes between cooked longissimus thoracis et lumborum (LTL) muscle proteomes by using mass spectrometry, chemometric, label-free proteomic, and bioinformatic tools. Among 30 differentially abundant proteins identified MyHC-2a, ATPs-α, CK-S, ADP/ATPt1, IDH2, and MyBP-C1 were upregulated (x > 1) whereas NEB, γ-ENO and EPSF were downregulated (x < 1) in wild boar. ShinyGO and KEGG database pathway analyses revealed that these proteins are mainly involved in processes related to muscle contraction and various pathways of glucose metabolism and energy production. Protein expression changes could have been caused by the different muscle activity of wild animals in response to prolonged movement associated with foraging for food in the natural environment.

Mechanisms underlying the changes in the structural, physicochemical, and emulsification properties of porcine myofibrillar proteins induced by prolonged pulsed electric field treatment.

This study aimed to explore how pulsed electric field (PEF) treatment affects the structural, physicochemical, and emulsification properties of porcine-derived myofibrillar proteins (MPs). Increasing PEF treatment induced partial polarization and protein unfolding, resulting in notable denaturation that affected both the secondary and tertiary structures. PEF treatment also improved the solubility and emulsification ability of MPs by reducing their pH and surface hydrophobicity. Confocal laser scanning microscopy confirmed the effective adsorption of MPs and PEF-treated MPs at the oil/water interface, resulting in well-fabricated Pickering emulsions. A weak particle network increased the apparent viscosity in short-term PEF-treated Pickering emulsions. Conversely, in emulsions with long-term PEF-treated MP, rheological variables decreased, and dispersion stability increased. These results endorse the potential application of PEF-treated porcine-derived MPs as efficient Pickering stabilizers, offering valuable insights into the creative use of PEF for enhancing high-quality meat products, meeting the increasing demand for clean-label choices.

Effect of sodium metabisulfite-mediated self-assembly on the quality of silver carp myofibrillar protein-EGCG composite gels.

Myofibrillar protein (MP) gels are susceptible to oxidation, which can be prevented by complexing with hydrophilic polyphenols, but may cause gel deterioration. Sodium metabisulfite (Na2S2O5) has been used to induce self-assembly of MP and analyze the impact of self-assembly on the quality of composite gels containing high amounts of (-)-epigallocatechin gallate (EGCG). Hydrophobic forces were confirmed as the main driver of self-assembly. Self-assembly reduced the size of the MP-EGCG complex to approximately 670 nm and increased the gel's hydrophobic force by approximately 3.6-fold. The maximum hardness of the Na2S2O5-treated MP-EGCG composite gel was 52.43 g/kg, which was approximately 49% greater than pure MP gel. After oxidative treatment, the Na2S2O5-treated MP-EGCG composite gel had considerably lower carbonyl and dityrosine levels (2.47-µmol/g protein and 450 a.u.) than the control (8.37-µmol/g protein and 964 a.u.). Therefore, Na2S2O5 shows potential as a cost-effective additive for alleviating MP limitations in the food industry.

Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

Influence of flaxseed-derived diglyceride-based high internal phase Pickering emulsions on the rheological and physicochemical properties of myofibrillar protein gels.

The study aimed to investigate the influence of flaxseed-derived diglyceride-based high internal phase Pickering emulsions (HIPPE) at different levels (0%, 10%, 20%, 30%, 40%, and 50%) on the rheological and physicochemical properties of myofibrillar protein (MPs) gels. The study indicated that with increasing HIPPE levels, there was a significant increase in whiteness while a decrease in water-holding capacity. The gels with 10% HIPPE levels had higher ionic bonds, while those with 40% and 50% HIPPE levels showed higher hydrogen bonds. By increasing HIPPE levels in the formation of MP gels, the T2 relaxation time was found to decrease. Additionally, in all MP gels, G' values were significantly higher than G" values over time. Adding lower contents of HIPPE levels resulted in a more compact microstructure. These findings indicate that flaxseed-derived diglyceride-based HIPPEs could be utilized as fat substitutes in meat products to enhance their nutritional quality.

Discovery of a temperature-dependent protease spoiling meat from Pseudomonas fragi: Target to myofibrillar and sarcoplasmic proteins rather than collagen.

Chilled meat frequently suffered microbial spoilage because bacteria can secrete various proteases that break down the proteins. In this study, Pseudomonas fragi NMC 206 exhibited a temperature-dependent secretion pattern, with the ability to release the specific protease only below 25 °C. It was identified as alkaline protease AprA by LC-MS/MS, with the molecular weight of 50.4 kDa, belonging to the Serralysin family metalloprotease. Its significant potential for meat spoilage in situ resulted in alterations in meat color and sensory evaluation, as well as elevated pH, total volatile basic nitrogen (TVB-N) and the formation of volatile organic compounds (VOCs). The hydrolysis of meat proteins in vitro showed that AprA possessed a considerable proteolysis activity and degradation preferences on meat proteins, especially its ability to degrade myofibrillar and sarcoplasmic proteins, rather than collagen. These observations demonstrated temperatures regulated the secretion of AprA, which was closely related to chilled chicken spoilage caused by bacteria. These will provide a new basis for the preservation of meat products at low temperatures.

The interplay of muscle and pea proteins in low-salt gels: An insight into in situ structure formation in hybrid meat alternatives.

The present study investigated thermal gelation of mixed sarcoplasmic (Sarc), myofibrillar (Myof), and pea proteins corresponding to partial meat replacements (0, 25, and 50%) by pea protein isolate (PPI) at reducing salt levels (0.6 â†’ 0.1 M NaCl) to understand in situ (simulated) structure-forming properties of hybrid meat analogues. The amount of soluble proteins in hybrids generally increased with salt concentrations and PPI substitution. While muscle proteins (mixed Sarc and Myof) had the strongest gelling capacity, hybrid proteins also exhibited moderate aggregation and gelling activity based on the sol→gel rheological transition and gel hardness testing. Sarc and pea 7S/11S globulins collectively compensated for the attenuated gelling capacity of mixed proteins due to diminishing Myof in the hybrids. Immobilized water within hybrid protein gels was tightly bonded (T2 from nuclear magnetic resonance), consistent with the dense and uniform microstructure observed. These findings offer a new knowledge base for developing reduced-salt hybrid meat analogues.

Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2.

Effect of ultrasound-assisted L-lysine treatment on pork meat quality and myofibrillar protein properties during postmortem aging.

This paper aimed to investigate the effects of ultrasound-assisted L-lysine treatment on meat quality and myofibrillar proteins (MPs) properties of pork longissimus dorsi during postmortem aging. The results revealed that the L-lysine (Lys) and/or ultrasound treatment significantly increased (p < 0.05) the water-holding capacity and tenderness of the pork during postmortem aging, while the ultrasound-assisted Lys treatment had the lowest cooking loss, pressurization loss, Warner-Bratzler shear force, and hardness. In addition, L-lysine and/or ultrasound treatment increased (p < 0.05) pH value, T21, and myofibrillar fragmentation index, while the ultrasound-assisted Lys treatment had the highest value. Meanwhile, the protein solubility was increased with Lys and/or ultrasound treatment during postmortem aging, and ultrasound-assisted Lys treatment had the highest solubility, reaching 88.19%, 92.98%, and 91.73% at 0, 1, and 3 days, respectively. The result of protein conformational characteristics showed that Lys and/or ultrasound treatment caused the unfolding of the α-helix structure, resulting in the exposure of more hydrophobic amino acids and buried sulfhydryl groups, ultimately enhancing MPs solubility. In summary, ultrasound-assisted Lys treatment altered the structure of MPs, resulting in the enhancement of the water-holding capacity and tenderness of the pork. PRACTICAL APPLICATION: This study showed that ultrasound-assisted L-lysine (Lys) treatment could enhance the water-holding capacity and tenderness of pork during postmortem aging. The results might provide a reference for the application of ultrasound-assisted Lys treatment on the improvement of pork meat quality. To facilitate practical applications in production, the development of medium and large-sized ultrasound equipment for conducting small-scale and pilot experiments is crucial for future research.

Salt reduction in myofibrillar protein gel via inhomogeneous distribution of sodium-containing encapsulated fish oil coacervate: Mucopenetration ability of sodium carboxymethyl cellulose.

The potential application of fish oil microcapsules as salt reduction strategies in low-salt myofibrillar protein (MP) gel was investigated by employing soy protein isolates/carboxymethyl cellulose sodium (SPI-CMC) coacervates enriched with 25 mM sodium chloride and exploring their rheological characteristics, taste perception, and microstructure. The results revealed that the SPI-CMC coacervate phase exhibited the highest sodium content under 25 mM sodium level, albeit with uneven distribution. Notably, the hydrophilic and adhesive properties of CMC to sodium facilitated the in vitro release of sodium during oral digestion, as evidenced by the excellent wettability and mucopenetration ability of CMC. Remarkably, the fish oil microcapsules incorporating SPI-CMC as the wall material, prepared at pH 3.5 with a core-to-wall ratio of 1:1, demonstrated the highest encapsulation efficiency, which was supported by the strong hydrogen bonding. Interestingly, the presence of SPI-CMC coacervates and fish oil microcapsules enhanced the interaction between MPs and strengthened the low-salt MP gel network. Coupled with electronic tongue analysis, the incorporation of fish oil microcapsules slightly exacerbated the non-uniformity of sodium distribution. This ultimately contributed to an enhanced perception of saltiness, richness, and aftertaste in low-salt protein gels. Overall, the incorporation of fish oil microcapsules emerged as an effective salt reduction strategy in low-salt MP gel.

Effects of Eleutherine bulbosa extract on the myofibrillar protein oxidation and moisture migration of yak meat under oxidation stress.

The influence of Eleutherine bulbosa (EB) extract at various levels (1, 4, 7, 10 or 13 g/kg) on the myofibrillar protein oxidation and moisture migration of yak meat in Fenton oxidation system was investigated. The results showed that inclusion of EB extract in yak meat efficiently inhibited carbonyl formation triggered by hydroxyl radicals. Supplementation of EB extract at 1-10 g/kg manifested more contents of the active sulfhydryl, ε-NH2 groups and α-helix structure, and higher solubility of myofibrillar proteins (MPs), but alleviated the turbidity of MPs. However, adding high level of EB extract (13 g/kg) induced the loss of free amine and α-helix content and resulted in more aggregation of MPs. The SDS-PAGE demonstrated that adding 1-7 g/kg EB extract had an obvious protective effect for myosin heavy chain and actin, whereas 10 or 13 g/kg EB extract led to weakened intensities of protein bands. DSC and LF-NMR analysis revealed that 7 g/kg EB extract had appreciable effects on thermal stabilities of MPs, and improved the hydration of yak meat induced by oxidation, while 13 g/kg EB extract accelerated MP structure destabilization and lowered water retention. Our results suggested that incorporation of low levels of EB extract (1-7 g/kg) effectively retarded the oxidative damage to MPs and EB extract could be a promising natural antioxidant in meat processing.