A small molecule compound 759 inhibits the wnt/beta-catenin signaling pathway via increasing the Axin protein stability.

Wnt/ß-catenin signaling plays important role in cancers. Compound 759 is one of the compounds previously screened to identify inhibitors of the Wnt/ß-catenin pathway in A549 cells [Lee et al. in Bioorg Med Chem Lett 20:5900-5904, 2010]. However, the mechanism by which Compound 759 induces the inhibition of the Wnt/ß-catenin pathway remains unknown. In our study, we employed various assays to comprehensively evaluate the effects of Compound 759 on lung cancer cells. Our results demonstrated that Compound 759 significantly suppressed cell proliferation and Wnt3a-induced Topflash activity and arrested the cell cycle at the G1 stage. Changes in Wnt/ß-catenin signaling-related protein expression, gene activity, and protein stability including Axin, and p21, were achieved through western blot and qRT-PCR analysis. Compound 759 treatment upregulated the mRNA level of p21 and increased Axin protein levels without altering the mRNA expression in A549 cells. Co-treatment of Wnt3a and varying doses of Compound 759 dose-dependently increased the amounts of Axin1 in the cytosol and inhibited ß-catenin translocation into the nucleus. Moreover, Compound 759 reduced tumor size and weight in the A549 cell-induced tumor growth in the in vivo tumor xenograft mouse model. Our findings indicate that Compound 759 exhibits potential anti-cancer activity by inhibiting the Wnt/ß-catenin signaling pathway through the increase of Axin1 protein stability.

BMP Stimulation Differentially Affects Phosphorylation and Protein Stability of ß-Catenin in Breast Cancer Cell Lines.

Increased expression and nuclear translocation of ß-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting ß-CATENIN are not as efficient as desired. Therefore, detailed understanding of ß-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing ß-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of ß-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce ß-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces ß-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on ß-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced ß-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding ß-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.

Influence of flash heating and aspergillopepsin I supplementation on must and wine attributes of aromatic varieties.

The protein instability with haze formation represents one of the main faults occurring in white and rosé wines. Among the various solutions industrially proposed, aspergillopepsin I (AP-I) supplementation coupled with must heating (60-75 °C) has been recently approved by OIV and the European Commission for ensuring protein stability of wines. This study investigates the impact of AP-I either applied independently or in combination with flash pasteurization on the chemical composition of grape must and wines derived from Sauvignon Blanc and Gewürztraminer. The efficacy on protein stability of a complete treatment combining heat (70 °C) and AP-I (HP) was confirmed through heat test and bentonite requirement, although no differences were observed between must heating and HP treatments. However, high-performance liquid chromatography analysis of unstable pathogenesis-related proteins revealed that AP-I supplementation reduced chitinases and thaumatin-like proteins compared to the non-enzymed samples, with and without must heating. Amino acid increase was reported only in HP musts, particularly in Sauvignon Blanc. The concentration of yeast-derived aroma compounds in Gewürztraminer wines was increased by must heating; compared to controls, flash pasteurization rose the overall acetate esters content of 85 % and HP of 43 %, mostly due to isoamyl acetate. However, heat treatments -with or without AP-I- reduced terpenes up to 68 %. Despite the different aroma profiles, no differences were observed for any descriptor for both varieties in wine tasting, and only a slight decrease trend was observed for the floral intensity and the typicality descriptors in heated wines.

Novel protein stabilization in white wine: A study on thermally treated zirconia-alumina composites.

A major concern for wineries is haze formation in white wines due to protein instability. Despite its prevalent use, the conventional bentonite method has shortcomings, including potential alteration of color and aroma, slow processing times, and notable wine wastage. Zirconium oxide (ZrO2) effectively removes proteins without affecting wine characteristics. However, producing cost-effective ZrO2 materials with efficient protein removal capabilities poses a significant challenge. This research aims to assess the viability of designing a porous material impregnated with zirconia to remove turbidity-causing proteins effectively. For this purpose, the support material alone (Al2O3) and the zirconia-impregnated support (ZrO2/Al2O3) were subjected to different calcination temperatures. It was observed that high-temperature treatments (750 °C) enhanced wine stability and protein adsorption capacity. The optimal adsorbent achieved a notable reduction in turbidity, decreasing the ΔNTU from 42 to 18, alongside a significant 44 % reduction in the total protein content, particularly affecting proteins in the molecular weight range of 10 to 70 kDa. This result is attributed to modifying the textural properties of ZrO2/Al2O3, characterized by the reduction of acidic sites, augmented pore diameters from 4.81 to 7.74 nm, and the emergence of zirconia clusters across the surface of the porous support. In summary, this study presents the first application of zirconia on the alumina support surface for protein stabilization in white wine. Combining ZrO2/Al2O3 and a high-temperature treatment emerges as a promising, cost-efficient, and environmentally sustainable strategy for protein removal in white wine.

Bioactive peptides in dry-cured ham: A comprehensive review of preparation methods, metabolic stability, safety, health benefits, and regulatory frameworks.

Dry-cured hams contain abundant bioactive peptides with significant potential for the development of functional foods. However, the limited bioavailability of food-derived bioactive peptides has hindered their utilization in health food development. Moreover, there is insufficient regulatory information regarding bioactive peptides and related products globally. This review summarizes diverse bioactive peptides derived from dry-cured ham and by-products originating from various countries and regions. The bioactivity, preparation techniques, bioavailability, and metabolic stability of these bioactive peptides are described, as well as the legal and regulatory frameworks in various countries. The primary objectives of this review are to dig deeper into the functionality of dry-cured ham and provide theoretical support for the commercialization of bioactive peptides from food sources, especially the dry-cured ham.

Electrostatics introduce a trade-off between mesophilic stability and adaptation in halophilic proteins.

Extremophile organisms have adapted to extreme physicochemical conditions. Halophilic organisms, in particular, survive at very high salt concentrations. To achieve this, they have engineered the surface of their proteins to increase the number of short, polar and acidic amino acids, while decreasing large, hydrophobic and basic residues. While these adaptations initially decrease protein stability in the absence of salt, they grant halophilic proteins remarkable stability in environments with extremely high salt concentrations, where non-adapted proteins unfold and aggregate. The molecular mechanisms by which halophilic proteins achieve this, however, are not yet clear. Here, we test the hypothesis that the halophilic amino acid composition destabilizes the surface of the protein, but in exchange improves the stability in the presence of salts. To do that, we have measured the folding thermodynamics of various protein variants with different degrees of halophilicity in the absence and presence of different salts, and at different pH values to tune the ionization state of the acidic amino acids. Our results show that halophilic amino acids decrease the stability of halophilic proteins under mesophilic conditions, but in exchange improve salt-induced stabilization and solubility. We also find that, in contrast to traditional assumptions, contributions arising from hydrophobic effect and preferential ion exclusion are more relevant for haloadaptation than electrostatics. Overall, our findings suggest a trade-off between folding thermodynamics and halophilic adaptation to optimize proteins for hypersaline environments.

Engineering G protein-coupled receptors for stabilization.

G protein-coupled receptors (GPCRs) are one of the most important families of targets for drug discovery. One of the limiting steps in the study of GPCRs has been their stability, with significant and time-consuming protein engineering often used to stabilize GPCRs for structural characterization and drug screening. Unfortunately, computational methods developed using globular soluble proteins have translated poorly to the rational engineering of GPCRs. To fill this gap, we propose GPCR-tm, a novel and personalized structurally driven web-based machine learning tool to study the impacts of mutations on GPCR stability. We show that GPCR-tm performs as well as or better than alternative methods, and that it can accurately rank the stability changes of a wide range of mutations occurring in various types of class A GPCRs. GPCR-tm achieved Pearson's correlation coefficients of 0.74 and 0.46 on 10-fold cross-validation and blind test sets, respectively. We observed that the (structural) graph-based signatures were the most important set of features for predicting destabilizing mutations, which points out that these signatures properly describe the changes in the environment where the mutations occur. More specifically, GPCR-tm was able to accurately rank mutations based on their effect on protein stability, guiding their rational stabilization. GPCR-tm is available through a user-friendly web server at https://biosig.lab.uq.edu.au/gpcr_tm/.

Decoding the mechanism governing the structural stability of wheat germ agglutinin and its isolated domains: A combined calorimetric, NMR, and MD simulation study.

Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

Studying protein stability in crowded environments by NMR.

Most proteins perform their functions in crowded and complex cellular environments where weak interactions are ubiquitous between biomolecules. These complex environments can modulate the protein folding energy landscape and hence affect protein stability. NMR is a nondestructive and effective method to quantify the kinetics and equilibrium thermodynamic stability of proteins at an atomic level within crowded environments and living cells. Here, we review NMR methods that can be used to measure protein stability, as well as findings of studies on protein stability in crowded environments mimicked by polymer and protein crowders and in living cells. The important effects of chemical interactions on protein stability are highlighted and compared to spatial excluded volume effects.

Identification of sorbitol esterification of glutamic acid by LC-MS/MS in a monoclonal antibody stability assessment.

The stability of monoclonal antibodies (mAbs) is vital for their therapeutic success. Sorbitol, a common mAb stabilizer used to prevent aggregation, was evaluated for any potential adverse effects on the chemical stability of mAb X. An LC-MS/MS based analysis focusing on the post-translational modifications (PTMs) of mAb X was conducted on samples that had undergone accelerated aging at 40°C. Along with PTMs that are known to affect mAbs' structure function and stability (such as deamidation and oxidation), a novel mAb PTM was discovered, the esterification of glutamic acid by sorbitol. Incubation of mAb X with a 1:1 ratio of unlabeled sorbitol and isotopically labeled sorbitol (13C6) further corroborated that the modification was the consequence of the esterification of glutamic acid by sorbitol. Levels of esterification varied across glutamic acid residues and correlated with incubation time and sorbitol concentration. After 4 weeks of accelerated stability with isotopically labeled sorbitol, it was found that 16% of the total mAb possesses an esterified glutamic acid. No esterification was observed at aspartic acid sites despite the free carboxylic acid side chain. This study unveils a unique modification of mAbs, emphasizing its potential significance for formulation and drug development.

A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin.

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Stereospecific NANOG PEST Stabilization by Pin1.

NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.

Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability.

Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═Oi···Hi+4-N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both i/i + 4 hydrogen bonds and i/i + 1 n → π* interactions. This hypothesis was tested in peptides X-AKAAAAKAAAAKAAGY-NH2, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and iso-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water-amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide-water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity.

HdeA and HdeB are dimeric ATP-independent acid-stress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB's intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.

Differential stability of Gcn4p controls its cell-specific activity in differentiated yeast colonies.

Gcn4p belongs to conserved AP-1 transcription factors involved in many cellular processes, including cell proliferation, stress response, and nutrient availability in yeast and mammals. AP-1 activities are regulated at different levels, such as translational activation or protein degradation, which increases the variability of regulation under different conditions. Gcn4p activity in unstructured yeast liquid cultures increases upon amino acid deficiency and is rapidly eliminated upon amino acid excess. Gcn2p kinase is the major described regulator of Gcn4p that enables GCN4 mRNA translation via the uORFs mechanism. Here, we show that Gcn4p is specifically active in U cells in the upper regions and inactive in L cells in the lower regions of differentiated colonies. Using in situ microscopy in combination with analysis of mutants and strains with GFP at different positions in the translational regulatory region of Gcn4p, we show that cell-specific Gcn4p activity is independent of Gcn2p or other translational or transcriptional regulation. Genetically, biochemically, and microscopically, we identified cell-specific proteasomal degradation as a key mechanism that diversifies Gcn4p function between U and L cells. The identified regulation leading to active Gcn4p in U cells with amino acids and efficient degradation in starved L cells differs from known regulations of Gcn4p in yeast but shows similarities to the activity of AP-1 ATF4 in mammals during insulin signaling. These findings may open new avenues for understanding the parallel activities of Gcn4p/ATF4 and reveal a novel biological role for cell type-specific regulation of proteasome-dependent degradation.IMPORTANCEIn nature, microbes usually live in spatially structured communities and differentiate into precisely localized, functionally specialized cells. The coordinated interplay of cells and their response to environmental changes, such as starvation, followed by metabolic adaptation, is critical for the survival of the entire community. Transcription factor Gcn4p is responsible for yeast adaptation under amino acid starvation in liquid cultures, and its activity is regulated mainly at the level of translation involving Gcn2p kinase. Whether Gcn4p functions in structured communities was unknown. We show that translational regulation of Gcn4p plays no role in the development of colony subpopulations; the main regulation occurs at the level of stabilization of the Gcn4p molecule in the cells of one subpopulation and its proteasomal degradation in the other. This regulation ensures specific spatiotemporal activity of Gcn4p in the colony. Our work highlights differences in regulatory networks in unorganized populations and organized structures of yeast, which in many respects resemble multicellular organisms.

GEN1 as a risk factor for human congenital anomalies of the kidney and urinary tract.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are prevalent birth defects. Although pathogenic CAKUT genes are known, they are insufficient to reveal the causes for all patients. Our previous studies indicated GEN1 as a pathogenic gene of CAKUT in mice, and this study further investigated the correlation between GEN1 and human CAKUT. METHODS: In this study, DNA from 910 individuals with CAKUT was collected; 26 GEN1 rare variants were identified, and two GEN1 (missense) variants in a non-CAKUT group were found. Mainly due to the stability results of the predicted mutant on the website, in vitro, 10 variants (eight CAKUT, two non-CAKUT) were selected to verify mutant protein stability. In addition, mainly based on the division of the mutation site located in the functional region of the GEN1 protein, 8 variants (six CAKUT, two non-CAKUT) were selected to verify enzymatic hydrolysis, and the splice variant GEN1 (c.1071 + 3(IVS10) A > G) was selected to verify shear ability. Based on the results of in vitro experiments and higher frequency, three sites with the most significant functional change were selected to build mouse models. RESULTS: Protein stability changed in six variants in the CAKUT group. Based on electrophoretic mobility shift assay of eight variants (six CAKUT, two non-CAKUT), the enzymatic hydrolysis and DNA-binding abilities of mutant proteins were impaired in the CAKUT group. The most serious functional damage was observed in the Gen1 variant that produced a truncated protein. A mini-gene splicing assay showed that the variant GEN1 (c.1071 + 3(IVS10) A > G) in the CAKUT group significantly affected splicing function. An abnormal exon10 was detected in the mini-gene splicing assay. Point-mutant mouse strains were constructed (Gen1: c.1068 + 3 A > G, p.R400X, and p.T105R) based on the variant frequency in the CAKUT group and functional impairment in vitro study and CAKUT phenotypes were replicated in each. CONCLUSION: Overall, our findings indicated GEN1 as a risk factor for human CAKUT.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Predicted deleterious variants in ABCA1, LPL, LPA and KIF6 are associated with statin response and adverse events in patients with familial hypercholesterolemia and disturb protein structure and stability.

OBJECTIVES: This study explored the association of deleterious variants in pharmacodynamics (PD) genes with statin response and adverse effects in patients with familial hypercholesterolemia (FH) and analyzed their potential effects on protein structure and stability. METHODS: Clinical and laboratory data were obtained from 144 adult FH patients treated with statins. A panel of 32 PD genes was analyzed by exon-targeted gene sequencing. Deleterious variants were identified using prediction algorithms and their structural effects were analyzed by molecular modeling studies. RESULTS: A total of 102 variants were predicted as deleterious (83 missense, 8 stop-gain, 4 frameshift, 1 indel, 6 splicing). The variants ABCA1 rs769705621 (indel), LPA rs41267807 (p.Tyr2023Cys) and KIF6 rs20455 (p.Trp719Arg) were associated with reduced low-density lipoprotein cholesterol (LDLc) response to statins, and the LPL rs1801177 (p.Asp36Asn) with increased LDLc response (P < 0.05). LPA rs3124784 (p.Arg2016Cys) was predicted to increase statin response (P = 0.022), and ABCA1 rs769705621 to increase the risk of statin-related adverse events (SRAE) (P = 0.027). LPA p.Arg2016Cys and LPL p.Asn36Asp maintained interactions with solvent, LPA p.Tyr2023Cys reduced intramolecular interaction with Gln1987, and KIF6 p.Trp719Arg did not affect intramolecular interactions. DDMut analysis showed that LPA p.Arg2016Cys and p.Tyr2023Cys and LPL p.Asp36Asn caused energetically favorable changes, and KIF6 p.Trp719Arg resulted in unfavorable energetic changes, affecting protein stability. CONCLUSION: Deleterious variants in ABCA1, LPA, LPL and KIF6 are associated with variability in LDLc response to statins, and ABCA1 rs769705621 is associated with SRAE risk in FH patients. Molecular modeling studies suggest that LPA p.Tyr2023Cys and KIF6 p.Trp719Arg disturb protein conformational structure and stability.