Non-denatured Protein Research Articles

GPMelt: A hierarchical Gaussian process framework to explore the dark meltome of thermal proteome profiling experiments.

Thermal proteome profiling (TPP) is a proteome wide technology that enables unbiased detection of protein drug interactions as well as changes in post-translational state of proteins between different biological conditions. Statistical analysis of temperature range TPP (TPP-TR) datasets relies on comparing protein melting curves, describing the amount of non-denatured proteins as a function of temperature, between different conditions (e.g. presence or absence of a drug). However, state-of-the-art models are restricted to sigmoidal melting behaviours while unconventional melting curves, representing up to 50% of TPP-TR datasets, have recently been shown to carry important biological information. We present a novel statistical framework, based on hierarchical Gaussian process models and named GPMelt, to make TPP-TR datasets analysis unbiased with respect to the melting profiles of proteins. GPMelt scales to multiple conditions, and extension of the model to deeper hierarchies (i.e. with additional sub-levels) allows to deal with complex TPP-TR protocols. Collectively, our statistical framework extends the analysis of TPP-TR datasets for both protein and peptide level melting curves, offering access to thousands of previously excluded melting curves and thus substantially increasing the coverage and the ability of TPP to uncover new biology.

The Antioxidant and Antidepressant Properties of Dietary Proteins Derived from Egg and Bean Extracts and Their Acute Toxicity: A Journey from Nutrition to Pharmacognosy

This research reveals the previously unexplored pharmacognostic potential of antidepressants found in nutrients derived from both legume and animal sources. Through preclinical investigations involving mouse models, the study focused into antidepressant and antioxidant activities of non-denatured and denatured protein extracts from beans and eggs. Non-denatured protein extracts from beans and eggs, at saturation levels of 40% and 80%, were examined as macronutrients, while denatured protein extracts at equivalent saturation levels were considered micronutrients. The study employed the DPPH and hydrogen peroxide tests to assess antioxidant activity, and the forced swimming test and sucrose preference test to evaluate acute and chronic mild antidepressant effects, respectively. The acute toxicity study revealed that macronutrients from eggs at 40% and 80% saturation displayed non-toxic effects (LD50 >5 g/kg), while those from beans, specifically at saturation of 80%, exhibited a relatively low level of toxicity (LD50 = 2.5 g/kg). Evaluation of antioxidant activity using the DPPH test yielded inconclusive results due to the influence of ethanol precipitation. In contrast, the H2O2 test demonstrated significant antioxidant potential in both macronutrients and micronutrients extracted from beans and eggs at all saturation levels. In investigating antidepressant properties, both macronutrients and micronutrients of bean and egg protein extracts at 40% and 80% saturation exhibited notable antidepressant effects, particularly the micronutrients at saturation of 80%. This antidepressant effect was characterized by a reduction in immobility time and an increase in sucrose preference.

Characterization and comparison of structure, thermal and functional characteristics of various commercial pea proteins

Pea protein (PP) presents an attractive raw material for its excellent nutritional value and functional properties. Nevertheless, data of nutritional composition, structure and functional properties of various commercial PPs remain scarce. The purpose of this work is to comprehensively analyze and compare the composition, structural, thermal and functional characteristics of eight commercially available PPs from multiple batches and sources. Specifically, the contents of total, soluble and non-denatured protein of eight PP samples were in the range of 76%–85%, 57%–61% and 0.8%–7.0%, respectively. Additionally, the total contents of amino acids and hydrophilic amino acids of PP-Ⅳ sample were significantly higher than that of PP-Ⅰ, PP-Ⅴ, PP-Ⅵ and PP-Ⅶ (P < 0.05). Fourier transform infrared spectroscopy showed that PP-Ⅴ exhibited higher β-sheet content and better gelation properties than other PPs. Differential scanning calorimeter analysis suggested that denaturing temperature of PP-Ⅳ was 85.81 °C, which was higher than that of other PPs. Furthermore, the solubility and emulsion stability of PP-Ⅲ and PP-Ⅳ were significantly higher than other six PPs (P < 0.05). PP-Ⅳ also had higher significant foaming capacity and foaming stability than other seven samples (P < 0.05). Moreover, principal component analysis indicated that the total score of PP-Ⅳ was the highest among the eight PPs. This study is helpful to comprehensively evaluate the quality characteristics of PPs and provide theoretical reference for the industrial production of PP products with multifunctional characteristics.

Design and Development of a New Method for the Production of Nanotoxoids from Clostridium Perfringens Beta Toxin.

In recent years, a nanoparticle-based strategy has shown that non-denatured protein toxins can be used to enhance the appropriate immune response. Once the toxin reacts between the nanoparticles and the protein (toxin), it loses its toxicity because it does not attach to its ligand at the cell surface. The results of the nanoparticle and toxin complex show that the nanoparticles facilitate the internal release of the toxin. Clostridium perfringens beta toxin is produced by Clostridium perfringens type B and C, and diarrhea is the most important disease caused in newborn lambs. When beta toxin forms a complex with nanoparticles, the reaction between the toxin and the nanoparticle leads to the formation of a new form of nanoparticle in which the toxin loses its lethality due to its involvement; therefore, it becomes a toxoid. The nanoparticles used in this research are of poly lactic-co-glycolic acid (PLGA) type, one of the most developed biodegradable polymers. This study aimed to isolate and purify Clostridium perfringens beta toxin and produce its complex with PLGA nanoparticles to form a non-toxic structure. In this study, Clostridium perfringens beta toxin type B was isolated using ammonium sulfate precipitation and gel filtration chromatography. Toxin assay was performed in vivo (lethal dose [LD50]) and in vitro by sodium dodecyl sulphate-polyacrylamide gel electrophoresis at each stage, and the quantity of purified toxin was calculated to be 10 mg/ml. Afterward, the beta toxin antigen was used as the basis for the preparation of nanotoxoid candidates with nanoparticle formulation. Moreover, the PLGA polymer and water-oil-water methods were used to fabricate nanoparticles. Under optimal conditions, nanoparticles without antigen with an average size of 100 nm and zeta potential of -23.28 mV, as well as nanoparticles containing antigen with an average size of 120 nm and zeta potential of -18.2 mV, were prepared. When nanoparticles are injected into mice with the beta toxin, the toxin becomes a toxoid with no toxicity effects, and it cannot bind to its receptors and reveal its effects. In this study, the mice showed mild symptoms in one case, and none of them died. The beta and PLGA toxin model could also be applied as a candidate to study the release and immunization of the target animal. In order to achieve antigen regulation using natural polymers, it is recommended to conduct a comparative study between nanoparticles based on natural polymers.

A screening method for binding synthetic metallo-complexes to haem proteins

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV–Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.

Analysis of Target Molecules towards Anti-cancer Therapeutic Antibodies

Target molecules of existing anti-cancer therapeutic monoclonal antibodies (mAbs) are divided into 1) receptor-type tyrosine kinases, such as human epidermal growth factor receptor (HER) family, 2) differentiation antigens, such as CD20 (Rituxan target), 3) angiogenesis-related molecules, and 4) immune checkpoint molecules (PD-1, etc.). We have recently reported a novel therapy targeting lymphangiogenesis, but not angiogenesis, using an anti-LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) mAb. At present, many transporters are not considered to be target molecules for the cancer therapy; however, our study strongly suggested that the inhibition of cancer metabolism by mAbs against amino acid transporters will play a significant role in future cancer therapies. Most anti-cancer therapeutic mAbs bind cell-surface molecules on viable cancer cells: therefore, it is necessary to produce mAbs recognizing epitopes on the extracellular domains of native and non-denatured proteins. We concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti-cancer therapeutic mAbs. We introduce our efforts to develop seeds for therapeutic mAbs using whole cancer cells and transfectants as the immunogen. As many target candidates in the future are multi-pass membrane proteins, such as 12-pass amino acid transporter proteins belonging to the solute carrier (SLC) family, and their possible immunogenic extracellular regions are small, the production of specific mAbs is highly difficult. In this review, we summarize the successful preparation and characterization of mAbs recognizing the extracellular domain of oncoproteins, including transporters.

Vitamin D3 in High-Quality Cow Milk: An Italian Case Study.

The quality-labeling category of high-quality (HQ) milk defined by the Italian legislation must comply with specific requirements concerning rigorous breeder management, hygienic controls, fat and protein content, bacterial load, somatic cells, lactic acid content, and non-denatured soluble serum proteins. However, there is no specification for the vitamin D content of HQ milk. Moreover, the data on the vitamin D content of this milk category are very scarce. In the present study, the content of vitamin D3 was evaluated in HQ raw and pasteurized cow milk obtained from Italian cowsheds and supermarkets. The vitamin D3 content varied from not detected (less than 1 µg L−1) to 17.0 ± 2.0 µg L−1 milk and was not related to the milk fat content. These results represent a case study including a significant although not exhaustive part of the contemporary Italian market of HQ milk. It was shown for the first time that HQ raw milk does not necessarily contain more vitamin D3, even though non-expert consumers likely to buy milk labeled as HQ could expect it. The vitamin D3 content in HQ pasteurized whole milk should be reported on the label of the milk package as a best practice of consumer information policy.

Production and characterization of monoclonal antibodies against GII.6 norovirus virus-like particles

In this study we generated and characterized a series of monoclonal antibodies (mAbs) against GII.6 norovirus (NoV) virus like particles (VLPs). Mice were immunized with purified GII.6 NoV VLPs and peptide-bovine serum albumin (BSA) conjugates with the peptide sequence (31 aa) derived from the trypsin cleavage region. An indirect enzyme-linked immunosorbent assay was used to identify positive cell clones during cloning and subcloning, and an in vitro VLP-histo-blood group antigens (HBGAs) binding blockade assay was used to identify mAbs with blocking ability. A total of seven mAbs comprising five (1F7, 1F11, 2B6, 2C4, and 2E10) reactive with major capsid proteins (VP1) and two (1E5 and 2B2) reactive with both VP1 proteins and the peptide were identified. mAb 1F7, 1F11, and 2B6 were identified as blocking antibodies. Sandwich ELISA indicated that all these mAbs recognized soluble GII.6 NoV VLPs. Cross-reactivities with GI.7, GII.3, and GII.4 NoV VLPs were observed in indirect and sandwich ELISA. Western blot analysis indicated that all non-blocking mAbs recognized denatured GII.6 VP1 proteins and blocking mAbs only recognized non-denatured proteins. The in vitro VLP-HBGA binding blockade assay indicated that the three blocking antibodies exhibited blocking effects against GII.6 NoV VLPs, but not GI.7, GII.3, and GII.4 NoV VLPs. Epitope mapping and HBGA blocking assay indicated that mAbs targeting the predicted surface-exposed loop region did not have blocking effects, suggesting a possible important role of this region in regulating NoV-HBGA interactions. This is the first report regarding the characterization of mAbs with blocking ability against GII.6 NoV VLPs. These mAbs might be useful in facilitating our understanding of this group of viruses.

A next generation setup for pre-fractionation of non-denatured proteins reveals diverse albumin proteoforms each carrying several post-translational modifications

Proteomic biomarker search requires the greatest analytical reproducibility and detailed information on altered proteoforms. Our protein pre-fractionation applies orthogonal native chromatography and conserves important features of protein variants such as native molecular weight, charge and major glycans. Moreover, we maximized reproducibility of sample pre-fractionation and preparation before mass spectrometry by parallelization and automation. In blood plasma and cerebrospinal fluid (CSF), most proteins, including candidate biomarkers, distribute into a multitude of chromatographic clusters. Plasma albumin, for example, divides into 15-17 clusters. As an example of our technique, we analyzed these albumin clusters from healthy volunteers and from dogs and identified cluster-typical modification patterns. Renal disease further modifies these patterns. In human CSF, we found only a subset of proteoforms with fewer modifications than in plasma. We infer from this example that our method can be used to identify and characterize distinct proteoforms and, optionally, enrich them, thereby yielding the characteristics of proteoform-selective biomarkers.

The cysteine-rich whey protein supplement, Immunocal®, preserves brain glutathione and improves cognitive, motor, and histopathological indices of traumatic brain injury in a mouse model of controlled cortical impact

Traumatic brain injury (TBI) is a major public health problem estimated to affect nearly 1.7 million people in the United States annually. Due to the often debilitating effects of TBI, novel preventative agents are highly desirable for at risk populations. Here, we tested a whey protein supplement, Immunocal®, for its potential to enhance resilience to TBI. Immunocal® is a non-denatured whey protein preparation which has been shown to act as a cysteine delivery system to increase levels of the essential antioxidant glutathione (GSH). Twice daily oral supplementation of CD1 mice with Immunocal® for 28 days prior to receiving a moderate TBI prevented an ~ 25% reduction in brain GSH/GSSG observed in untreated TBI mice. Immunocal® had no significant effect on the primary mechanical injury induced by TBI, as assessed by MRI, changes in Tau phosphorylation, and righting reflex time or apnea. However, pre-injury supplementation with Immunocal® resulted in statistically significant improvements in motor function (beam walk and rotarod) and cognitive function (Barnes maze). We also observed a significant preservation of corpus callosum width (axonal myelination), a significant decrease in degenerating neurons, a reduction in Iba1 (microglial marker), decreased lipid peroxidation, and preservation of brain-derived neurotrophic factor (BDNF) in the brains of Immunocal®-pretreated mice compared to untreated TBI mice. Taken together, these data indicate that pre-injury supplementation with Immunocal® significantly enhances the resilience to TBI induced by a moderate closed head injury in mice. We conclude that Immunocal® may hold significant promise as a preventative agent for TBI, particularly in certain high risk populations such as athletes and military personnel.

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Extending the Compatibility of the SP3 Paramagnetic Bead Processing Approach for Proteomics

The diversity in protein and peptide biochemistry necessitates robust protocols and reagents for efficiently handling and enriching these molecules prior to analysis with mass spectrometry (MS) or other techniques. Further exploration of the paramagnetic bead-based approach, single-pot solid-phase-enhanced sample preparation (SP3), is carried out toward updating and extending previously described conditions and experimental workflows. The SP3 approach was tested in a wide range of experimental scenarios, including (1) binding solvents (acetonitrile, ethanol, isopropanol, acetone), (2) binding pH (acidic vs neutral), (3) solvent/lysate ratios (50-200%, v/v), (4) mixing and rinsing conditions (on-rack vs off-rack rinsing), (5) Enrichment of nondenatured proteins, and (6) capture of individual proteins from noncomplex mixtures. These results highlight the robust handling of proteins in a broad set of scenarios while also enabling the development of a modified SP3 workflow that offers extended compatibility. The modified SP3 approach is used in quantitative in-depth proteome analyses to compare it with commercial paramagnetic bead-based HILIC methods (MagReSyn) and across multiple binding conditions (e.g., pH and solvent during binding). Together, these data reveal the extensive quantitative coverage of the proteome possible with SP3 independent of the binding approach utilized. The results further establish the utility of SP3 for the unbiased handling of peptides and proteins for proteomic applications.

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10–30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.

Rheological properties and antioxidant activity of protein gels-like systems made from crayfish concentrate and hydrolysates

Crayfish protein obtained from a renewable, available, and low-cost raw material, may be regarded as an excellent alternative for the development of innovative food products on the basis of its nutritional value and bioactivity.Thermal gelation behaviour and antioxidant activity of gels made from non-denatured crayfish protein concentrate or their hydrolysates were studied at three different pH values (2.0, 6.5 and 8.0). Some physicochemical and rheological properties, as well as water holding capacity of the final gel-like systems were also determined.Both the pH and the degree of hydrolysis exerted a strong influence on the gelation behaviour and the properties of the final gels. The results obtained may be explained in terms of the protein interactions involved in gel formation. A remarkable antioxidant activity against ABTS and DPPH compounds was observed. The effects of pH and the hydrolysis degree on antioxidant activity were only moderate.

The effect of non-standard heat treatment of sheep's milk on physico-chemical properties, sensory characteristics, and the bacterial viability of classical and probiotic yogurt.

Classical and probiotic set yogurt were made using non-standard heat treatment of sheep's milk at 60°C/5min. Physico-chemical properties, sensory characteristics, and the viability of bacteria that originated from cultures in classical and probiotic yogurt were analysed during 21days of storage at 4°C. For the production of yogurt, a standard yogurt culture and a probiotic strain Lactobacillus rhamnosus GG were used. At the end of storage time of the classical and probiotic yogurt the totals of non-denatured whey proteins were 92.31 and 91.03%. The viability of yogurt culture bacteria and Lactobacillus rhamnosus GG were higher than 106cfu/g. The total sensory score (maximum - 20) was 18.49 for the classical and 18.53 for the probiotic. In nutritional and functional terms it is possible to produce classical and probiotic sheep's milk yogurt by using a non-standard temperature of heat treatment with a shelf life of 21days.

Improved Extraction and Sample Cleanup of Tri-glycoalkaloids α-Solanine and α-Chaconine in Non-denatured Potato Protein Isolates

Glycoalkaloids in potato-derived products result in bitter taste and potentially toxic effects at high intakes. Generally, extraction of glycoalkaloids prior to HPLC analysis is carried out by dilute acetic acid. For most potato-derived extracts including heat-coagulated potato proteins, this extraction method is sufficient to achieve satisfying tri-glycoalkaloid (TGA) recoveries. Soluble potato proteins obtained by non-denaturing processes show different requirements for glycoalkaloid extraction. TGA extraction was optimized for two commercially available native potato protein isolates and compared to heat-coagulated potato protein. The highest TGA levels were determined in the extract when extraction was carried out at 40 °C by at least 5 % acetic or propionic acid supplemented with 20 mM Na-1-heptanesulfonate (HSA). Addition of HSA results in substantially improved TGA extraction and induces precipitation of soluble protein which enhanced sample cleanup. On the contrary, extraction of TGA from coagulated potato protein in the presence of HSA showed a reduced TGA extraction efficiency. This improved TGA extraction procedure for soluble non-denatured potato protein isolates results in reliable quantification of bitter tasting and toxic glycoalkaloid levels. This contributes to a non-bitter and safe use of the nutritional and functional benefits of this plant protein in food applications.

Controlling the Ratio between Native-Like, Non-Native-Like, and Aggregated β-Lactoglobulin after Heat Treatment.

The amount of heat-denatured whey protein is typically determined by pH 4.6 precipitation. Using this method, a significant amount of nondenatured protein was reported even after long heating times. Apparently, a fraction of the unfolded protein refolds into the "native" state rather than form aggregates. This fact is known and has been explained using kinetic models. How the conditions affect the refolding and aggregation is, however, not fully understood. Therefore, this study investigates the unfolding, refolding, and aggregation process of β-lactoglobulin using circular dichroism and size-exclusion chromatography to characterize different folding variants and to quantify their content. The proteins remaining in solution at pH 4.6 were confirmed to be native-like. The nonaggregated fraction contains proteins with a native-like and two types of non-native-like conformations. The nonaggregated fraction increased with decreasing temperature (60-90 °C) and concentration (1-50 g/L) and increasing electrostatic repulsion (pH 7-8; 0-50 mM). The native-like fraction in the nonaggregated fraction was independent of pH, ionic strength, and concentration but increased with decreasing temperature.

Oligomerization States of LRRC8A and LRRC8B, Essential Components of the Volume-Regulated Anion Channel VRAC

Cellular volume regulation plays an important role in many cellular processes. VRAC is crucial for regulating volume decrease by allowing a net efflux of chloride and organic anions. Its molecular nature remained unclear until 2014 when Qiu et al. (Cell 157, 447-458) and Voss et al. (Science 344, 634-638) identified leucine-rich repeat-containing 8 (LRRC8) membrane proteins as subunits of VRAC. LRRC8 proteins, which occur in five isoforms (LRRC8A-E), have been suggested to share a common ancestor and accordingly a hexameric quaternary structure with pannexins (Abascal & Zardoya, BioEssays 34, 551-560, 2012). Here, we expressed two human isoforms, hLRRC8A and hLRRC8B, in Xenopus laevis oocytes, purified the LRRC8 proteins by Ni-NTA chromatography in dodecyl maltoside, and assessed their oligomeric structure by blue native polyacrylamide gel electrophoresis (BN-PAGE). Detergent screening revealed that maltoside detergents with alkyl chain length from 10 to 12 were similarly well suited to purify hLRRC8A as a stable homomultimeric protein complex. The non-denatured homomeric hLRRC8A protein and the heteromeric hLRRC8A+B protein (with calculated non-glycosylated protomer masses of 95 and 92 kDa, respectively) approximately co-migrated in the BN-PAGE gel with the GFP-tagged tetrameric hTRPV1 channel (calculated mass close to 500 kDa). Slight denaturing treatments of the heteromeric hLRRC8A+B protein complex resulted in the appearance of lower order oligomers in the BN-PAGE gel, which we could tentatively identify as dimers, tetramers and hexamers by comparison with the migration patterns of concatenated hLRRC8A/A and A/B dimers and A/A/A trimers. We conclude that hLRRC8A and hLRR8A+B assemble as homo- and heterohexamers, respectively, in X. laevis oocytes. In contrast to the hLRRC8A isoform, hLRRC8B requires co-assembly with hLRRC8A to achieve a defined (hexameric) assembly state and plasma membrane localization.

Investigation and optimization of a novel enzymatic approach for the isolation of proteins from potato pulp

Potato proteins have been associated with several functional and beneficial properties. A novel enzymatic approach, based on the use of combination of polysaccharide degrading biocatalysts, was investigated for the isolation of non-denatured proteins from potato pulp. The removal of starch was found to be essential for the effectiveness of the enzymatic approach, as protein recovery increased from 47% to 63–75% with starch removal. Contrary to endo-arabinase (Arase), endo-1,4-β-galactanase (GLase) and polygalacturonase MI (PGase) improved the protein recovery. A 5-level, 5-variable central composite rotatable design was performed using as independent variables temperature (⁰C; 30.0, 33.8, 37.5, 41.3, 45.0), time (h; 1.50, 9.00, 16.5, 24.0, 31.5), potato pulp concentration (kg/m3; 80.0, 110, 140, 170, 200), PGase units (U; 1.5, 11.0, 20.5, 30.0, 39.5), and GLase units (U; 1.5, 11.0, 20.5, 30.0, 39.5); and the responses were protein recovery yield, recovered patatin concentration, and recovered protease inhibitors concentration. For yield and recovered patatin, the most significant interaction was that between temperature and units of PGase with an antagonistic relationship. The most significant interaction for recovered protease inhibitors was found to be that between pulp concentration and temperature, showing a positive correlation. Comparison of predicted and experimental values validated the established predicted models, which can be used to identify the conditions for the isolation of potato proteins with selected composition.

A Cystine-Rich Whey Supplement (Immunocal(®)) Delays Disease Onset and Prevents Spinal Cord Glutathione Depletion in the hSOD1(G93A) Mouse Model of Amyotrophic Lateral Sclerosis.

Depletion of the endogenous antioxidant, glutathione (GSH), underlies progression of the devastating neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Thus, strategies aimed at elevating GSH may yield new therapeutics for ALS. Here, we investigated the effects of a unique non-denatured whey protein supplement, Immunocal®, in the transgenic Gly position 93 to Ala (G93A) mutant hSOD1 (hSOD1G93A) mouse model of ALS. Immunocal® is rich in the GSH precursor, cystine, and is therefore capable of bolstering GSH content. Transgenic hSOD1G93A mice receiving Immunocal® displayed a significant delay in disease onset compared to untreated hSOD1G93A controls. Additionally, Immunocal® treatment significantly decreased the rate of decline in grip strength and prevented disease-associated reductions in whole blood and spinal cord tissue GSH levels in end-stage hSOD1G93A mice. However, Immunocal® did not extend survival, likely due to its inability to preserve the mitochondrial GSH pool in spinal cord. Combination treatment with Immunocal® and the anti-glutamatergic compound, riluzole, delayed disease onset and extended survival in hSOD1G93A mice. These findings demonstrate that sustaining tissue GSH with Immunocal® only modestly delays disease onset and slows the loss of skeletal muscle strength in hSOD1G93A mice. Moreover, the inability of Immunocal® to rescue mitochondrial GSH in spinal cord provides a possible mechanism for its lack of effect on survival and is a limiting factor in the potential utility of this supplement as a therapeutic for ALS.