Whey proteins as multifunctional food materials: Recent advancements in hydrolysis, separation, and peptidomimetic approaches.

Whey protein derived bioactives, including α-lactalbumin, ß-lactoglobulin, bovine serum albumin, lactoferrin, transferrin, and proteose-peptones, have exhibited wide ranges of functional, biological and therapeutic properties varying from anticancer, antihypertensive, and antimicrobial effects. In addition, their functional properties involve gelling, emulsifying, and foaming abilities. For these reasons, this review article is framed to understand the relationship existed in between those compound levels and structures with their main functional, biological, and therapeutic properties exhibited either in vitro or in vivo. The impacts of hydrolysis mechanism and separation techniques in enhancing those properties are likewise discussed. Furthermore, special emphasize is given to multifunctional effects of whey derived bioactives and their future trends in ameliorating further food, pharmaceutical, and nutraceutical products. The underlying mechanism effects of those properties are still remained unclear in terms of activity levels, efficacy, and targeted effectiveness. For these reasons, some important models linking to functional properties, thermal properties and cell circumstances are established. Moreover, the coexistence of radical trapping groups, chelating groups, sulfhydryl groups, inhibitory groups, and peptide bonds seemed to be the key elements in triggering those functions and properties. Practical Application: Whey proteins are the byproducts of cheese processing and usually the exploitation of these food waste products has increasingly getting acceptance in many countries, especially European countries. Whey proteins share comparable nutritive values to milk products, particularly on their richness on important proteins that can serve immune protection, structural, and energetic roles. The nutritive profile of whey proteins shows diverse type of bioactive molecules like α-lactalbumin, ß-lactoglobulin, lactoferrin, transferrin, immunoglobulin, and proteose peptones with wide biological importance to the living system, such as in maintaining immunological, neuronal, and signaling roles. The diversification of proteins of whey products prompted scientists to exploit the real mechanisms behind of their biological and therapeutic effects, especially in declining the risk of cancer, tumor, and further complications like diabetes type 2 and hypertension risk effects. For these reasons, profiling these types of proteins using different proteomic and peptidomic approaches helps in determining their biological and therapeutic targets along with their release into gastrointestinal tract conditions and their bioavailabilities into portal circulation, tissue, and organs. The wide applicability of those protein fractions and their derivative bioactive products showed significant impacts in the field of emulsion and double emulsion stabilization by playing roles as emulsifying, surfactant, stabilizing, and foaming agents. Their amphoteric properties helped them to act as excellent encapsulating agents, particularly as vehicle for delivering important vitamins and bioactive compounds. The presence of ferric elements increased their transportation to several metal-ions in the same time increased their scavenging effects to metal-transition and peroxidation of lipids. Their richness with almost essential and nonessential amino acids makes them as selective microbial starters, in addition their richness in sulfhydryl amino acids allowed them to act a cross-linker in conjugating further biomolecules. For instance, conjugating gold-nanoparticles and fluorescent materials in targeting diseases like cancer and tumors in vivo is considered the cutting-edges strategies for these versatile molecules due to their active diffusion across-cell membrane and the presence of specific transporters to these therapeutic molecules.

Diet Plus Inositols, α-Lactalbumin and Gymnema sylvestre: The Successful Combo to Restore Body Weight and Metabolic Profile in Obese and Dysmetabolic Patients.

The primary control of dysmetabolic patients is extremely challenging worldwide, with inadequate dietary habits and sporadic physical activity among the key risk factors for metabolic syndrome onset. Nowadays, there is no exclusive treatment for this condition, and considering that preventive measures usually fail, new therapeutic approaches need to be proposed and investigated. This present pilot study compared the effects of diet alone and in association with a combination of myo-inositol and d-chiro-inositol in their 40:1 ratio, α-lactalbumin, and Gymnema sylvestre on different metabolic parameters in obese dysmetabolic patients. To this purpose, 37 patients with BMI between 30 and 40 and fasting blood glucose between 100 and 125 mg/dL were divided into two groups: (i) the control group followed a hypocaloric Mediterranean diet, (ii) while the study group was also supplemented with a daily dosage of two sachets, each one containing 1950 mg myo-inositol, 50 mg d-chiro-inositol, 50 mg α-lactalbumin, and 250 mg Gymnema Sylvestre. After a 6-month treatment, all parameters improved in both groups. Nevertheless, the treated group experienced a greater improvement, especially concerning the variation from the baseline of HOMA index, triglycerides, BMI, body weight, and waist circumference. These findings support the supplementation with myo-inositol and d-chiro-inositol in the 40:1 ratio, α-lactalbumin, and Gymnema sylvestre as a therapeutical strategy to potentiate the beneficial effects induced via dietary programs in dysmetabolic patients.

α-Lactalbumin Peptide Asp-Gln-Trp Ameliorates Hepatic Steatosis and Oxidative Stress in Free Fatty Acids-Treated HepG2 Cells and High-Fat Diet-Induced NAFLD Mice by Activating the PPARα Pathway.

SCOPE: Dietary intervention has emerged as a promising strategy for the management of nonalcoholic fatty liver disease (NAFLD). The aim of this study is to investigate the ameliorative effects of the α-lactalbumin peptide Asp-Gln-Trp (DQW) against NAFLD and the underlying mechanism. METHODS AND RESULTS: The models of lipid metabolism disorders are established both in HepG2 cells and in C57BL/6J mice. The results demonstrate that DQW activates peroxisome proliferator-activated receptor α (PPARα) and subsequently ameliorates lipid deposition and oxidative stress in vitro. Interestingly, GW6471 markedly attenuates the modulatory effects of DQW on the PPARα pathway in HepG2 cells. Moreover, results of in vivo experiments indicate that DQW alleviates body weight gain, dyslipidemia, hepatic steatosis, and oxidative stress in high-fat-diet (HFD)-induced NAFLD mice. At the molecular level, DQW activates PPARα, subsequently enhances fatty acid ß-oxidation, and reduces lipogenesis, thereby ameliorating hepatic steatosis. Meanwhile, DQW may ameliorate liver injury and oxidative stress via activating the PPARα/nuclear-factor erythroid 2 (Nrf2)/heme-oxygenase 1 (HO-1) pathway. CONCLUSION: Those results indicate that α-lactalbumin peptide DQW may be an effective dietary supplement for alleviating NAFLD by alleviating lipid deposition and oxidative stress.

Tandem mass tag-based quantitative proteomics analysis reveals the effects of the α-lactalbumin peptides GINY and DQW on lipid deposition and oxidative stress in HepG2 cells.

The objective of this study was to investigate the mechanism by which the α-lactalbumin peptides Gly-Ile-Asn-Tyr (GINY) and Asp-Gln-Trp (DQW) ameliorate free fatty acid-induced lipid deposition in HepG2 cells. The results show that GINY and DQW reduced triglyceride, total cholesterol, and free fatty acid levels significantly in free fatty acid-treated HepG2 cells. Based on proteomic analysis, GINY and DQW alleviated lipid deposition and oxidative stress mainly through the peroxisome proliferator-activated receptor (PPAR) pathway, fatty acid metabolism, oxidative phosphorylation, and response to oxidative stress. In vitro experiments confirmed that GINY and DQW upregulated the mRNA and protein expression of fatty acid ß-oxidation-related and oxidative stress-related genes, and downregulated the mRNA and protein expression of lipogenesis-related genes by activating peroxisome proliferator-activated receptor α (PPARα). Meanwhile, GINY and DQW reduced free fatty acid-induced lipid droplet accumulation and reactive oxygen species generation, and enhanced the mitochondrial membrane potential and ATP levels. Furthermore, GINY and DQW enhanced carnitine palmitoyl-transferase 1a (CPT-1a) and superoxide dismutase activities, and diminished acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) activities in a PPARα-dependent manner. Interestingly, GW6471 (a PPARα inhibitor) weakened the effects of GINY and DQW on the PPARα pathway. Hence, our findings suggest that GINY and DQW have the potential to alleviate nonalcoholic fatty liver disease by activating the PPARα pathway.

The intracellular fate and transport mechanism of shape, size and rigidity varied nanocarriers for understanding their oral delivery efficiency.

Nanocarriers have become an effective strategy to overcome epithelial absorption barriers. During the absorption process, the endocytosis mechanisms, cell internalization pathways, and transport efficiency of nanocarriers are greatly impacted by their physical properties. To understand the relationship between physical properties of nanocarriers and their abilities overcoming multiple absorption barriers, nanocarriers with variable physical properties were prepared via self-assembly of hydrolyzed α-lactalbumin peptide fragments. The impacts of size, shape, and rigidity of nanocarriers on epithelial cells endocytosis mechanisms, internalization pathways, transport efficiency, and bioavailability were studied systematically. The results showed that nanospheres were mainly internalized via clathrin-mediated endocytosis, which was then locked in lysosomes and degraded enzymatically in cytoplasm. While macropinocytosis was the primary pathway of nanotubes and transported to the endoplasmic reticulum and Golgi apparatus, resulting in a high drug concentration and sustained release in cytoplasm. Besides, nanotubes can overcome the multi-drug resistance by inhibiting the P-glycoprotein efflux. Furthermore, nanotubes can open intercellular tight-junctions instantaneously and reversibly, which promotes transport into blood circulation. The aqueous solubility of hydrophobic bioactive mangiferin (Mgf) was improved by nanocarriers. Most importantly, the bioavailability of Mgf was the highest for cross-linked short nanotube (CSNT) which outperformed free Mgf and other formulations by in vivo pharmacokinetic studies. Finally, Mgf-loaded CSNT showed an excellent therapeutic efficiency in vivo for the intervention of streptozotocin-induced diabetes. These results indicate that cross-linked α-lactalbumin nanotubes could be an effective nanocarrier delivery system for improving the epithelium cellular absorption and bioavailability of hydrophobic bioactive compounds.

α-Lactalbumin peptide Asp-Gln-Trp alleviates hepatic insulin resistance and modulates gut microbiota dysbiosis in high-fat diet-induced NAFLD mice.

The progression of nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and gut microbiota. Dietary interventions have emerged as effective palliative strategies for NAFLD. The present study investigated the potential mechanisms by which α-lactalbumin peptide Asp-Gln-Trp (DQW) ameliorated insulin resistance and gut microbiota dysbiosis in high-fat diet (HFD)-induced NAFLD mice. The results demonstrated that DQW treatment alleviated HFD-induced body weight gain, hepatic steatosis, insulin resistance, and dyslipidemia. DQW treatment also increased the ratio of Bacteroides to Firmicutes in the gut, reduced the relative abundance of pathogenic bacteria (such as Bacteroides, Blautia, and Alistipes) and enhanced the relative abundance of short-chain fatty acid (SCFA)-producing bacteria (such as Muribaculaceae, Lachnospiraceae_NK4A136_group, and Rikenellaceae_RC9_gut_group). DQW treatment promoted the production of SCFAs and subsequently improved intestinal barrier integrity and inflammation. Furthermore, the results of real-time quantitative PCR (qRT-PCR) and western blotting further proved that the effects of DQW on the attenuation of hepatic insulin resistance were mediated by the PPARα and IRS1/PI3K/AKT pathways. Taken together, these results indicated that DQW treatment could attenuate HFD-induced NAFLD and insulin resistance by modulating gut microbiota composition, enhancing the SCFA levels, and activating the PPARα and IRS1/PI3K/Akt pathways.

The kinetic mechanism of cations induced protein nanotubes self-assembly and their application as delivery system.

The amphiphilic proteins can be used as building blocks (BBs) forming various self-assemblies. Understanding their self-assembly mechanism is important for designing novel nanomaterials. Herein, the BBs dimers were first prepared from carboxyl-abundant enzymolyzed α-lactalbumin (α-lac) at 50 °C. Then the unidentate coordination of Ca2+ between the BBs caused a ß-sheet stacking to further self-assemble into nanotubes (NTs). Compared with the traditional "one-pot" method, a step-wise new method was applied to study hydrolysis, aggregation and self-assembly processes separately. The α-lac was hydrolyzed into 11 kDa amphiphilic peptides independent of temperature while a BBs dimer was formed at 50 °C by hydrophobic interaction. Ca2+ induced a conformational change of BBs and promoted these BBs gradually aggregate into 10 strands of filaments, which twisted into helical ribbons by electrostatic repulsion. Ca2+ further induced the twisted helical ribbons closed into NTs driven by the reduction of line tension energy. Besides, the carboxyl-Ca2+ coordination dominated NTs elongation in the longitudinal direction and filaments aggregation in the lateral direction with the same binding stoichiometry of 1:1 respectively. Finally, NTs successfully encapsulated curcumin and improved the viscosity of liquid food. α-Lac NTs show a high potential as a delivery system for food applications.

Lipid bilayer composition as a determinant of cancer cell sensitivity to tumoricidal protein-lipid complexes.

Complexes formed by the alpha1 N-terminal peptide of alpha-lactalbumin and oleic acid (alpha1-oleate) interact with lipid bilayers. Plasma membrane perturbations trigger tumor cell death but normal differentiated cells are more resistant, and their plasma membranes are less strongly affected. This study examined membrane lipid composition as a determinant of tumor cell reactivity. Bladder cancer tissue showed a higher abundance of unsaturated lipids enriched in phosphatidylcholine, PC (36:4) and PC (38:4), and sphingomyelin, SM (36:1) than healthy bladder tissue, where saturated lipids predominated and the lipid extracts from bladder cancer tissue inhibited the tumoricidal effect of the complex more effectively than healthy tissue extracts. Furthermore, unsaturated PC in solution inhibited tumor cell death, and the complex interacted with giant unilamellar vesicles formed by PC, confirming the affinity of alpha1-oleate for fluid membranes enriched in PC. Quartz Crystal Microbalance with dissipation monitoring (QCM-D) detected a preference of the complex for the liquid-disordered phase, suggesting that the insertion into PC-based membranes and the resulting membrane perturbations are influenced by membrane lipid saturation. The results suggest that the membrane lipid composition is functionally important and that specific unsaturated membrane lipids may serve as "recognition motifs" for broad-spectrum tumoricidal molecules such as alpha1-oleate.

SIRT1 Mediates Neuroprotective and Neurorescue Effects of Camel α-Lactalbumin and Oleic Acid Complex on Rotenone-Induced Parkinson's Disease.

Parkinson's disease (PD) is the second most common devastating neurodegenerative disorder. Presently used therapies for PD have severe side effects and are limited to only temporary improvement. Therefore, a new therapeutic approach to treat PD urgently needs to be developed. α-Lactalbumin, the most abundant milk protein in camel milk, has been attributed to various medicinal properties. This study intended to investigate the neuroprotective efficacy of the camel α-lactalbumin and oleic acid (CLOA) complex. One mechanism postulated to underlie neuroprotection by the CLOA complex is the induction of silent information regulatory protein (SIRT1). SIRT1 is known to be involved in several pathological and physiological processes, and it has been suggested that SIRT1 plays a protective role in PD. Oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis are involved in PD pathogenesis. Our results revealed that SIRT1 inhibits oxidative stress by maintaining HIF-1α in a deacetylated state. SIRT1 upregulates the expression of FOXO3a and HSF-1, thus inhibiting apoptosis and maintaining the homeostasis of cellular proteins. Increased SIRT1 expression reduces the levels of TNF-α, IL-6, and IL-8, which in turn inhibits neuroinflammation. In addition to SIRT1, the CLOA complex also enhances the expression of survivin and leptin and promotes the survival of neuroblastoma cells. Altogether, our results suggest that the CLOA complex might be a novel therapeutic molecule that could ameliorate neuronal cell damage in PD.

Purification and Handling of the Chaperonin GroEL.

GroEL is an important model molecular chaperone. Despite being extensively studied, several critical aspects of its functionality are still in dispute due partly to difficulties in obtaining protein samples of consistent purity. Here I describe an easy-to-carry-out purification protocol that can reliably produce highly purified and fully functional GroEL protein in large quantities. The method takes advantage of the remarkable stability of the GroEL tetradecamer in 45% acetone which efficiently extracts and removes tightly bound substrate proteins that cannot be separated from GroEL by the usual chromatographic methods. The efficiency of the purification method can be assessed by the amount of residual tryptophan fluorescence associated with the purified GroEL sample. The functionality of the thus obtained GroEL sample is demonstrated by measuring its ATPase turnover both in the presence and absence of the GroEL model substrate protein α-lactalbumin.

UHT treatment and storage of liquid infant formula affects protein digestion and release of bioactive peptides.

Ready-to-feed liquid infant formulas (IF) were subjected to direct (D) or indirect (ID) ultra-high-temperature (UHT) treatment and then stored at 40 °C under aseptic conditions for 60-120 days simulating global transportation which accelerates the Maillard reaction. Low pasteurized and unstored IF (LP) was included as a control for the UHT treatments. Simulated infant in vitro digestion was conducted. SDS-PAGE indicated that protein aggregate formation correlated with thermal treatment, being greatest after 60 days of storage. Limited protein digestion was observed after pepsin treatment for 2 h. Beta-lactoglobulin (ß-Lg), alpha-lactalbumin (α-La) and protein aggregates remained undigested after 2 h of pepsin digestion in LP and D, but less ß-Lg and α-La remained in ID. The digestion of ß-Lg and α-La was enhanced in D and ID stored for 60 days, but aggregates remained undigested. After pepsin and pancreatin digestion, large amounts of ß-Lg remained undigested in the LP, but digestion increased after UHT treatment (ID > D) and increased further after storage for 60 and 120 days, indicating that heat treatment and storage facilitate the digestion of unaggregated proteins. No aggregates remained after pancreatin digestion of LP, D, ID and D stored for 60 days, but were present in ID stored for 60 days. Aggregates were mainly disulphide-linked, but dityrosine linkages were detected in D and ID stored for 120 days. LC-MS/MS indicated limited proteolysis arising from endogenous milk proteases prior to in vitro digestion, being highest in D. Peptide numbers increased following pepsin and further during pancreatin digestion (ß-casein > ß-Lg > ß-La), and released ß-Lg peptides, typically 5-8 amino acids in length, contained several bioactivities, e.g., dipeptidyl-peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibition.

Nicking and fragmentation are responsible for α-lactalbumin amyloid fibril formation at acidic pH and elevated temperature.

Amyloid fibrils are ordered aggregates that may be formed from disordered, partially unfolded, and fragments of proteins and peptides. There are several diseases, which are due to the formation and deposition of insoluble ß-sheet protein aggregates in various tissue, collectively known as amyloidosis. Here, we have used bovine α-lactalbumin as a model protein to understand the mechanism of amyloid fibril formation at pH 1.6 and 65°C under non-reducing conditions. Amyloid fibril formation is confirmed by Thioflavin T fluorescence and atomic force microscopy (AFM). Our finding demonstrates that hydrolysis of peptide bonds occurs under these conditions, which results in nicking and fragmentation. The nicking and fragmentation have been confirmed on non-reducing and reducing gel. We have identified the fragments by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The fragmentation may initiate nucleation as it coincides with AFM images. Conformational changes associated with monomer resulting in fibrillation are shown by circular dichroism and Raman spectroscopy. The current study highlights the importance of nicking and fragmentation in amyloid fibril formation, which may help understand the role of acidic pH and proteolysis under in vivo conditions in the initiation of amyloid fibril formation.

Enhanced cytotoxicity and antioxidant capacity of kaempferol complexed with α-lactalbumin.

As a dietary polyphenol, kaempferol exhibits numerous biological activities such as antioxidant and anticancer properties. However, its application is limited because of its poor solubility and low permeability. This work aims to investigate the interaction of kaempferol with α-lactalbumin. Multiple-spectroscopic techniques were used to prove the interaction between kaempferol and α-lactalbumin. UV-vis absorption spectra suggested that the conformation of α-lactalbumin could be changed via binding with kaempferol. The fluorescence quenching test showed that kaempferol significantly quenched the intrinsic fluorescence of α-lactalbumin. Circular dichroism spectroscopy showed that the percent helicity of α-lactalbumin secondary structure increased when combined with kaempferol. In addition, the α-lactalbumin-kaempferol complex showed stronger inhibition ability on the growth of HeLa cells compared with kaempferol alone. The complex also showed higher antioxidant capacity than kaempferol alone. Molecular docking provided three predicted binding sites of α-lactalbumin for kaempferol, as well as five predicted binding poses of kaempferol. The weak intermolecular interactions were the main forces to stabilize the α-lactalbumin-kaempferol complex. Besides, the binding stability between α-lactalbumin and kaempferol was explored by molecular dynamics simulation. In conclusion, this work provides a basis for the potential application of α-lactalbumin as a delivery carrier for kaempferol owing to its nontoxic and biocompatible properties.

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments.

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.

Structurally symmetric near-infrared fluorophore IRDye78-protein complex enables multimodal cancer imaging.

Rationale: Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current approved intraoperative contrast agents exemplified by indocyanine green (ICG) have a few drawbacks including the inability of pre-surgical localization. Alternative near-infrared (NIR) dyes including IRDye800cw are being explored in advanced clinical trials but often encounter low chemical yields and complex purifications owing to the asymmetric synthesis. A single contrast agent with ease of synthesis that works in multiple cancer types and simultaneously allows presurgical imaging, intraoperative deep-tissue three-dimensional visualization, and high-speed microscopic visualization of tumor margins via spatiotemporally complementary modalities would be beneficial. Methods: Due to the lack of commercial availability and the absence of detailed synthesis and characterization, we proposed a facile and scalable synthesis pathway for the symmetric NIR water-soluble heptamethine sulfoindocyanine IRDye78. The synthesis can be accomplished in four steps from commercially-available building blocks. Its symmetric resonant structure avoided asymmetric synthesis problems while still preserving the benefits of analogous IRDye800cw with commensurable optical properties. Next, we introduced a low-molecular-weight protein alpha-lactalbumin (α-LA) as the carrier that effectively modulates the hepatic clearance of IRDye78 into the preferred renal excretion pathway. We further implemented 89Zr radiolabeling onto the protein scaffold for positron emission tomography (PET). The multimodal imaging capability of the fluorophore-protein complex was validated in breast cancer and glioblastoma. Results: The scalable synthesis resulted in high chemical yields, typically 95% yield in the final step of the chloro dye. Chemical structures of intermediates and the final fluorophore were confirmed. Asymmetric IRDye78 exhibited comparable optical features as symmetric IRDye800cw. Its well-balanced quantum yield affords concurrent dual fluorescence and optoacoustic contrast without self-quenching nor concentration-dependent absorption. The NHS ester functionality modulates efficient covalent coupling to reactive side-chain amines to the protein carrier, along with desferrioxamine (DFO) for stable radiolabeling of 89Zr. The fluorophore-protein complex advantageously shifted the biodistribution and can be effectively cleared through the urinary pathway. The agent accumulates in tumors and enables triple-modal visualization in mouse xenograft models of both breast and brain cancers. Conclusion: This study described in detail a generalized strategic modulation of clearance routes towards the favorable renal clearance, via the introduction of α-LA. IRDye78 as a feasible alternative of IRDye800cw currently in clinical phases was proposed with a facile synthesis and fully characterized for the first time. This fluorophore-protein complex with stable radiolabeling should have great potential for clinical translation where it could enable an elegant workflow from preoperative planning to intraoperative deep tissue and high-resolution image-guided resection.

Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity.

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.

α-Lactalbumin, Amazing Calcium-Binding Protein.

α-Lactalbumin (α-LA) is a small (Mr 14,200), acidic (pI 4-5), Ca2+-binding protein. α-LA is a regulatory component of lactose synthase enzyme system functioning in the lactating mammary gland. The protein possesses a single strong Ca2+-binding site, which can also bind Mg2+, Mn2+, Na+, K+, and some other metal cations. It contains several distinct Zn2+-binding sites. Physical properties of α-LA strongly depend on the occupation of its metal binding sites by metal ions. In the absence of bound metal ions, α-LA is in the molten globule-like state. The binding of metal ions, and especially of Ca2+, increases stability of α-LA against the action of heat, various denaturing agents and proteases, while the binding of Zn2+ to the Ca2+-loaded protein decreases its stability and causes its aggregation. At pH 2, the protein is in the classical molten globule state. α-LA can associate with membranes at neutral or slightly acidic pH at physiological temperatures. Depending on external conditions, α-LA can form amyloid fibrils, amorphous aggregates, nanoparticles, and nanotubes. Some of these aggregated states of α-LA can be used in practical applications such as drug delivery to tissues and organs. α-LA and some of its fragments possess bactericidal and antiviral activities. Complexes of partially unfolded α-LA with oleic acid are cytotoxic to various tumor and bacterial cells. α-LA in the cytotoxic complexes plays a role of a delivery carrier of cytotoxic fatty acid molecules into tumor and bacterial cells across the cell membrane. Perhaps in the future the complexes of α-LA with oleic acid will be used for development of new anti-cancer drugs.

Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (ß-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/µg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.

Investigation and comparison of the protective activities of three functional proteins-lactoferrin, α-lactalbumin, and ß-lactoglobulin-in cerebral ischemia reperfusion injury.

The objective of this study was to evaluate the protection conferred by lactoferrin, α-lactalbumin, and ß-lactoglobulin in cerebral ischemia reperfusion (I/R) injury. Rat pheochromocytoma (PC12) cells were used to construct an oxygen and glucose deprivation model in vitro, and ICR mice underwent carotid artery "ligation-relaxation" to construct a cerebral I/R injury model in vivo. The levels of toll-like receptor 4 (TLR4) and downstream factors including nuclear factor-κB, tumor necrosis factor-α, and IL-1ß were measured. Metabonomics detection and data mining were conducted to identify the specific metabolic sponsor of the 3 proteins. The results showed that lactoferrin, α-lactalbumin, and ß-lactoglobulin protected neurons from cerebral I/R injury by increasing the level of bopindolol and subsequently inhibiting the TLR4-related pathway to different degrees; ß-lactoglobulin had the strongest activity of the 3 proteins. In summary, this study is the first to investigate and compare the protective effects of lactoferrin, α-lactalbumin, and ß-lactoglobulin in a cerebral stroke model. The results implicate TLR4 as a novel target of the 3 bioactive proteins to prevent cerebral I/R injury.

Generation and characterization of dipeptidyl peptidase-IV inhibitory peptides from trypsin-hydrolyzed α-lactalbumin-rich whey proteins.

Dipeptidyl peptidase-IV (DPP-IV) is an enzyme that break down the antidiabetic hormone glucagon-like peptide-1. Therefore, inhibition of DPP-IV could be an effective strategy to treat Type 2 diabetes (T2D). The α-lactalbumin-rich whey protein concentrate was hydrolyzed by trypsin, and the hydrolysates were then fractionated at a semi-preparative scale using a Superdex Gel filtration Chromatography. The peptides were analyzed by using HPLC coupled with tandem mass spectrometry (RP-HPLC-MS/MS), and their Dipeptidyl peptidase-IV inhibitory activity was determined by the enzymatic assay. Among tested fragments, a potent fragment (LDQWLCEKL), with the half-maximal inhibitory concentration (IC50) of 131 µM was obtained. Further analysis shows that the LDQWLCEKL peptide corresponds to the amino acid sequence of f(115-123) in α-lactalbumin. Furthermore, LDQWLCEKL exhibited a typical non-competitive mode of inhibition. The results indicate that α-lactalbumin contains active peptides with DPP-IV inhibitory activity that may be used to prevent and treat T2D.