Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins.

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αß-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4ß engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

Potential DPP IV Inhibitory Peptides from Dry-Cured Pork Loins after Hydrolysis: An In Vitro and In Silico Study.

Peptidyl peptidase IV (DPP-IV) is a pharmacotherapeutic target in type 2 diabetes, and inhibitors of this enzyme are an important class of drugs for the treatment of type 2 diabetes. In the present study, peptides (<7 kDa) isolated from dry-cured pork loins after pepsin and pancreatin hydrolysis were identified by mass spectrometry and tested as potential inhibitors of DPP-IV by the in silico method. Two peptides, namely WTIAVPGPPHS from myomesin (water-soluble fraction, A = 0.9091) and FKRPPL from troponin (salt-soluble fraction, A = 0.8333), were selected as the most promising inhibitors of DPP-IV. Both peptides were subjected to ADMET analysis. Fragments of these peptides showed promising drug-likeness properties as well as favorable absorption, distribution, metabolism, excretion, and toxicity functions, suggesting that they are novel leads in the development of DPP-IV inhibitors from food.

Synergistic recovery and enhancement of gelling properties of oxidatively damaged myofibrillar protein by l-lysine and transglutaminase.

The influence of combined Lysine (Lys) and transglutaminase (TG) on the conformation and gelling properties of oxidatively damaged myofibrillar protein (MP) was investigated. The addition of Lys (5 mM) significantly increased the α-helix content (by 47.8%) and decreased the particle size of oxidatively damaged MP, and improved the cooking yield (by 16.8%) and the breaking strength of MP gels (by 65.5%). The treatment with TG (E:S = 1:500) led to a slightly reduced α-helix content but improved breaking strength (by 41.8%) and cooking loss (by 13.3%) of the gels. Their combination (Lys + TG) showed the greatest and synergistic overall improvement, with the set gel displaying a fine, smooth and compact network structure. Notably, the gelling ability of oxidatively damaged MP upon Lys + TG treatment was significantly stronger than that of non-oxidized MP far exceeding its recovery. Therefore, significantly enhanced gelling properties of oxidatively damaged MP can be attained through the combination Lys and TG.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Thermal denaturation of proteins in the muscle fibre and connective tissue from bovine muscles composed of type I (masseter) or type II (cutaneous trunci) fibres: DSC and FTIR microspectroscopy study.

The changes in secondary structure of proteins with heating were characterised and compared for bovine masseter (fibre type I) and cutaneous trunci (fibre type II) muscles by Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR) microspectroscopy. Heating led to a decrease in α- helices, and an increase in aggregated strands, random coils and aromatic side chains in the muscle fibres of both muscles. In the intramuscular connective tissue (IMCT) of both muscles, a decrease in α- helix, turn and unordered structures was complemented with an increase in aggregated strands. At temperatures < 60 °C, the greater thermal denaturation of proteins in cutaneous trunci than in masseter (FTIR), supported by a myosin associated peak at 55.8 °C for cutaneous trunci and no peak for masseter (DSC), indicates that myosin in type II fibres is more sensitive to thermal denaturation than myosin in type I fibres and this should be considered in thermal meat processing.

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

Optimization of Extraction of Bioactive Peptides from Monkfish (Lophius litulon) and Characterization of Their Role in H2O2-Induced Lesion.

BACKGROUND: Marine fish meat has been widely used for the extraction of bioactive peptides. This study was aimed to optimize the preparation of monkfish muscle peptides (LPs) using response surface methodology (RSM) and explore the antioxidant activities of <1 kDa LPs. METHODS: Peptides were prepared from the muscles of monkfish (Lophius litulon), and five proteases were tested to hydrolyze muscle proteins. The hydrolysate that was treated using neutrase showed the highest degree of hydrolysis (DH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities. RESULTS: The optimized conditions were as follows: water/material ratio of 5.4:1, a time span of 5 h, pH of 7.0, enzyme concentration of 2000 U/g, and temperature of 45 °C; the maximum DPPH scavenging activity and DH were 92.861% and 19.302%, respectively. LPs exhibited appreciable antioxidant activities, including DPPH radical, hydroxyl radical, 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radical, and superoxide anion scavenging activities. LPs attenuated H2O2-related oxidative injury in RAW264.7 cells, reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels. CONCLUSION: We concluded that LPs could be an ideal source of bioactive peptides from monkfish and also have pharmaceutical potential.

High-yield skeletal muscle protein recovery from TRIzol after RNA and DNA extraction.

Extraction of DNA, RNA and protein from the same sample would allow for direct comparison of genomic, transcriptomic and proteomic information. Commercially available kits exhibit poor protein yield and the TRIzol® reagent produces a protein pellet that is extremely difficult to solubilize. In response to these limitations, this study presents an optimized method for the extraction of protein from the organic phase of TRIzol that allows for higher yield recovery of skeletal muscle protein compared with direct homogenization in a common protein lysis buffer. The presented method is inexpensive, simple and fast, requires no additional treatment of the protein pellet for dissolution, and is compatible with downstream western blot applications.

Kinetic parameters of human aspartate/asparagine-ß-hydroxylase suggest that it has a possible function in oxygen sensing.

Human aspartate/asparagine-ß-hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor-like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1-2, 3-4, 5-6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases.

Effects of high-speed shear homogenization on the emulsifying and structural properties of myofibrillar protein under low-fat conditions.

BACKGROUND: Emulsification is important for food quality and processing functionality. Most emulsification occurs under high-fat conditions that eventually cause health concerns. Protein emulsifiers also have drawbacks such as lower dispersity. This study considered the effects of different high-speed shear homogenization (HSH) speeds on the emulsifying and structural properties of myofibrillar proteins (MPs) under low-fat conditions. RESULTS: High-speed shear homogenization significantly increased the emulsifying activity and emulsifying stability of MPs at lower speeds (8000 to 14 500 rpm). The primary structure of MP was not altered significantly by HSH, whereas its secondary, tertiary, and quaternary structures were changed. Particle size decreased first and then increased significantly, and reached a minimum when the HSH speed was 14 500 rpm. The absolute zeta potential values increased significantly and the dendritic fibrous structure of sample was destroyed when the speed exceeded 14 500 rpm. High-speed shear homogenization (14 500 rpm) decreased the particle size and unfolded the protein, which improved the emulsifying properties of MPs. Excessive HSH speeds (20 500 rpm or higher) caused an aggregation of MP molecules, which was not conducive to improving their emulsifying properties. CONCLUSION: Optimal HSH speed was achieved at 14 500 rpm to modify MPs' emulsifying and structural properties under low-fatconditions. © 2019 Society of Chemical Industry.

Oxidative stability of isoelectric solubilization/precipitation-isolated PSE-like chicken protein.

The effects of a hydroxyl radical generating system (Fe3+/H2O2) at different H2O2 concentrations (0, 1 and 10 mM) on the chemical and structural properties of isoelectric solubilization/precipitation (ISP)-isolated PSE (pale, soft, exudative)-like chicken protein were investigated. Both acid and alkaline treatments effectively reduced the pro-oxidant contents in PSE-like meat systems, such as lipids, pigments and myoglobins. The ISP samples generated less carbonyl derivatives and Schiff base but yielded higher sulfhydryl and free amine loss in response to oxidation. Correspondingly, sulfur-contained amino acids in ISP samples were more easily converted, even at the 1-mM H2O2 concentration, than in the PSE-like meat paste. Moreover, the ISP-isolated proteins have possibly maintained their gelling properties after oxidation compared to the PSE-like meat paste. In regards to chemical and structural modification, the ISP treated protein showed a different susceptibility to oxidation in vitro.

Comparative studies on the yield and characteristics of myofibrillar proteins from catfish heads and frames extracted by two methods for making surimi-like protein gel products.

Fish protein isolates (FPI) were recovered from catfish heads and frames by alkaline extraction (AE) and salt extraction (SE) and made into surimi-like gels. Protein patterns and content, moisture, color, and texture of cooked protein gels were compared with commercial products. Sodium-dodecyl-sulfate poly acrylamide gel electrophoreses (SDS-PAGE) indicated that the integrity of major myofibrillar proteins was maintained during the extraction process, and the protein patterns were almost the same with that of the commercial surimi products. The yields of AE-FPI (heads: 36%; frames: 55%) were much higher (p < 0.05) than that of SE-FPI (heads: 9%; frames: 16%). Firmness of cooked protein gels made from heads was similar with that made from frames. Firmness of cooked protein gels made from FPI extracted by the SE method (heads: 0.45 kg/cm2; frames: 0.43 kg/cm2) was significantly lower (p < 0.05) than that made from FPI extracted by the AE method (heads: 1.96 kg/cm2; frames: 1.85 kg/cm2).

Potential of recovered proteins from invasive green crabs (Carcinus maenas) as a functional food ingredient.

BACKGROUND: Invasive green crabs contain high-quality proteins that have potential as functional ingredients in formulated foods. This study evaluated the functional properties and compositional characteristics of green crab proteins recovered by isoelectric solubilization/precipitation (ISP). RESULTS: Mechanically separated green crab mince (control) was solubilized at pH 2 (PP2) and pH 10 (PP10), then proteins were precipitated at pH 5.5 and subsequently dried. Yield of recovered protein powder was approximately 1.5 times higher for PP2 than for PP10. Compared with the control (230 g kg-1 ), ash content was reduced in PP2 (54 g kg-1 ) and PP10 (23 g kg-1 ) samples. PP2 contained predominantly large-molecular-weight proteins, while small-molecular-weight proteins were distributed in PP10. With regard to functional properties, at pH 7 and 8, solubility of PP10 was significantly higher than that of PP2. At pH 7.5, PP10 exhibited significantly higher emulsifying activity (1482 m2 g-1 ) than PP2 (858 m2 g-1 ) and the control (958 m2 g-1 ). PP2 showed statistically higher gelation activity and had higher L* value than PP10 and the control. CONCLUSION: The results indicate that recovered green crab proteins have functional properties potentially useful for formulated foods, and that these functional properties can be modified by the solubilization pH during the recovery process. © 2018 Society of Chemical Industry.

Effects of high-pressure homogenization on functional properties and structure of mussel (Mytilus edulis) myofibrillar proteins.

Mussel myofibrillar proteins (MMP) suspensions (10.6% ±â€¯0.5%, w/v) were treated by high-pressure homogenization (HPH) at 0 (control), 20, 40, 60, 80 or 100 MPa for 3 cycles. Particle size distribution, zeta potential, solubility, water and oil holding capacity, emulsifying, foaming properties, secondary structure, free sulfhydryl and surface hydrophobicity of the obtained suspensions were analyzed. The results showed that functional properties of MMP significantly (P < 0.05) improved after HPH treatment. Absolute zeta potential, emulsifying activity index, emulsion stability index, foaming ability and foaming stability increased by 23.64 mV, 14.99 m2/g, 4.3 min, 17.3% and 29.7% at 80 MPa, protein solubility and oil holding capacity increased by 7.4% and 1300% at 100 MPa. However, HPH treatment significantly (P < 0.05) decreased particle size and water holding capacity. HPH treatment altered secondary structure, tertiary and quaternary structure. Functionality improvements mainly resulted from changes in structure and decrease in particle size. The results showed that HPH has potential for improving functional properties of MMP, thus expand its application in food industry.

Purification and identification of anti-inflammatory peptides from spent hen muscle proteins hydrolysate.

Spent hens have low market value and incur significant costs for disposal. This study explored the use of spent hens as a source of bioactive peptides. Spent hen hydrolysates prepared by Protease M or Protex 50FP exhibited interleukin (IL)-6 inhibitory activity in endotoxin-activated macrophage-like U937 cells (p < .05). The potential peptides from Protex 50FP hydrolysate were further fractionated using a combination of ultrafiltration, solid-phase extraction and high-performance liquid chromatography; 17 novel peptides encoded in major muscle proteins were identified by mass spectrometry analysis, of which 7 were chemically synthesized and assayed for the IL-6 inhibitory activity. At a concentration of 100 µg/mL, peptide FLWGKSY induced a 79% reduction of IL-6 production in endotoxin-activated macrophage-like U937 cells, which is comparable to results reported from other food sources. Our results indicate that spent hens have potential to be a source of bioactive peptides for anti-inflammatory applications.

Interactions and phase behaviors in mixed solutions of κ-carrageenan and myofibrillar protein extracted from Alaska Pollock surimi.

In the present work, we provide insight into electrostatic interactions and phase behaviors in mixtures of myofibrillar protein (MP) and κ-carrageenan (KC) of various pHs (8.0-3.0) and biopolymer weight ratios [R, from 1:1 to 20:1; total concentration=0.05% (w/w)] through turbidimetric analysis, dynamic light scattering (DLS) and zeta potential analysis, and optical microscopy. At R=1: 1, critical pH values (i.e., pHc, pHΦ1, and pHΦ2), which indicate phase transitions interrelated to the formation of soluble or insoluble MP-KC complexes, were observed at pH7.6, 6.8, and 3.6, respectively. As the ratio increased, the pHmax shifted from 4.9 to 5.8. A similar trend was observed for the isoelectric point of MP-KC mixtures, as determined by zeta potential measurements. The maximum interaction indicated by the highest turbidity occurred at pHmax=5.3, at a ratio of 5:1, whereas pHc remained constant during acidification. The changes in electrostatic interactions and transformations of phase behaviors accompanying the complex formation and disassociation processes were further supported by particle size distribution analysis and optical microscopic observations of MP-KC mixtures (R=1:1) at different pH values. This work fills the previous lack of studies on phase behaviors of surimi protein and colloidal polysaccharide in liquid system, and lay the foundation to provide a new way to solve problems of interactions between protein and polysaccharide during the processing of surimi products.

Proteomic analysis of cardiac ventricles: baso-apical differences.

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.

Depletion of Myofibril-Associated Proteins Using Selective Protein Extraction as a Tool in Cardiac Proteomics.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.

Polyphosphate and myofibrillar protein extract promote transglutaminase-mediated enhancements of rheological and textural properties of PSE pork meat batters.

The objective of this study was to improve the rheological (storage modulus G'; phase angle δ) and textural (hardness; breaking force) properties of nitrite-cured sausage batters prepared from pale, soft, exudative (PSE) pork with microbial transglutaminase (TG) using isolated myofibrillar protein (MP) or MP extracted by sodium tripolyphosphate (TPP) as substrates. While TG alone significantly enhanced G', δ and hardness, its combination with TPP was more pronounced as these product quality parameters were increased to the level equal to that of the counterpart batter made from normal (red, firm, non-exudative, RFN) pork. The addition of MP had negligible such effect on TG functions. Cooking loss of TG-treated RFN and PSE meat with TPP was minimal but the batters were less bright (L*) than other treatments. Redness (a*) was variable between treatments. Therefore, texture-related properties of comminuted PSE meat products can be restored to the RFN level by TG cross-linking of protein that is extracted by TPP.

Two novel peptides with angiotensin I converting enzyme inhibitory and antioxidative activities from Scorpaena notata muscle protein hydrolysate.

Fish protein hydrolysate was prepared from muscle of small red scorpionfish (Scorpaena notata) by treatment with a protease from the fungus Penicillium digitatum. Protein hydrolysate was found to strongly inhibit the angiotensin I converting enzyme and exhibited high antioxidative activity through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. After ultrafiltration, peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high-performance liquid chromatography with a high purification yield of 2.5 mg of peptide per gram of initial protein. Two major peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS), corresponding to the following sequences: Leu-Val-Thr-Gly-Asp-Asp-Lys-Thr-Asn-Leu-Lys (1,204.665 Da) and Asp-Thr-Gly-Ser-Asp-Lys-Lys-Gln-Leu (992.511 Da). These peptides, mainly composed of hydrophilic amino acids, showed high antioxidative and angiotensin I converting enzyme inhibitory activities. These data suggest that the two novel peptides isolated from the muscle hydrolysate of small red scorpionfish can be a beneficial ingredient for functional foods or pharmaceuticals against hypertension and oxidative stress.