Soluble Protein Fraction Research Articles

A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli.

Variable lymphocyte receptors (VLRs) are the antigen receptors of jawless vertebrates such as lamprey. VLRs are of growing biotechnological interest for their ability to bind certain antigenic targets with higher affinity than traditional immunoglobulins. However, VLRs are disulfide-bonded proteins that are often challenging to produce requiring genetic modifications, fusion partners, non-scalable host cell lines or inclusion body formation and refolding. As a potential VLR expression platform option, the SHuffle Escherichia coli strain has been genetically altered to allow cytoplasmic disulfide bond formation by mutations to thioredoxin reductase (trxB) and glutathione reductase (gor) to create an oxidative cytoplasm. Furthermore, the SHuffle strain expresses disulfide bond isomerase DsbC in the cytoplasm to promote correct disulfide bond pairing. Here, we demonstrate that the SHuffle strain can produce high yield VLRs with titers ranging from 2 to 32 mg of VLR per liter of SHuffle culture. Three VLRs (P1C10, RBC36, VLRA.R2.1) were expressed in SHuffle E. coli and the products were compared directly to those generated using the Rosetta E. coli strain. All VLRs were validated for correct sequence, purity, and activity. For all VLRs, SHuffle E. coli produced 2-9 times more soluble VLRs than Rosetta E. coli. Furthermore, the soluble protein fraction was 2-6 times greater in SHuffle E. coli than Rosetta E. coli for all VLRs. Overall, these results suggest that the E. coli SHuffle strain is a convenient and effective expression system for producing large amounts of VLRs.

Dehydroamino acids and their crosslinks in Alzheimer's disease aggregates.

Alzheimer's disease (AD) is characterized by the accumulation of protein aggregates, which are thought to be influenced by posttranslational modifications (PTMs). Dehydroamino acids (DHAAs) are rarely observed PTMs that contain an electrophilic alkene capable of forming protein-protein crosslinks, which may lead to protein aggregation. We report here the discovery of DHAAs in the protein aggregates from AD, constituting an unknown and previously unsuspected source of extensive proteomic complexity. We used mass spectrometry-based proteomics to discover 404 sites of DHAA formation in 171 proteins from protein aggregate-enriched human brain samples, 6-fold more sites than observed in the soluble protein fractions. The DHAA modifications are observed both directly and in the form of conjugates after reacting with abundant cellular nucleophiles or crosslinking to nucleophilic amino acid residues. We report 11 such crosslinks, including three in the Tau protein, which are 10-fold more abundant in AD samples compared with age-matched controls. Many of the proteins found to contain DHAAs and their conjugates are involved in protein aggregation or pathways dysregulated in AD. DHAAs are prevalent modifications in the AD brain proteome and give rise to protein crosslinks that may contribute to protein aggregation.

Pharmacological enhancement of slow-wave activity at an early disease stage improves cognition and reduces amyloid pathology in a mouse model of Alzheimer's disease.

Improving sleep in murine Alzheimer's disease (AD) is associated with reduced brain amyloidosis. However, the window of opportunity for successful sleep-targeted interventions, regarding the reduction in pathological hallmarks and related cognitive performance, remains poorly characterized. Here, we enhanced slow-wave activity (SWA) during sleep via sodium oxybate (SO) oral administration for 2 weeks at early (6 months old) or moderately late (11 months old) disease stages in Tg2576 mice and evaluated resulting neuropathology and behavioral performance. We observed that the cognitive performance of 6-month-old Tg2576 mice significantly improved upon SO treatment, whereas no change was observed in 11-month-old mice. Histochemical assessment of amyloid plaques demonstrated that SO-treated 11-month-old Tg2576 mice had significantly less plaque burden than placebo-treated ones, whereas ELISA of insoluble protein fractions from brains of 6-month-old Tg2576 mice indicated lower Aβ-42/Aβ-40 ratio in SO-treated group vs. placebo-treated controls. Altogether, our results suggest that SWA-dependent reduction in brain amyloidosis leads to alleviated behavioral impairment in Tg2576 mice only if administered early in the disease course, potentially highlighting the key importance of early sleep-based interventions in clinical cohorts.

Recombinant Production of Bovine αS1-Casein in Genome-Reduced Bacillus subtilis Strain IIG-Bs-20-5-1.

Cow's milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced Bacillus subtilis was applied for the recombinant production of bovine αS1-casein protein. A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine αS1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant αS1-casein production. After increasing the molecular abundance of the recombinant αS1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine αS1-casein. Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant αS1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mgcasein/gCDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native αS1-casein showed pH-mediated differences in aggregation behavior. The study demonstrates the potential of B. subtilis for the recombinant production of bovine αS1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.

Multiple forms of carbonic anhydrase in Tetragonia tetragonioides leaves and the impact of heavy metal ions on enzyme activity.

Tetragonia tetragonioides (Aizoaceae; Caryophyllales), an annual herbaceous plant, a halophyte native to the marine coasts of New Zealand is now cultivated worldwide as a minor salt-tolerant crop. The aim of this study was to evaluate the activity of carbonic anhydrase (CA, EC 4.2.1.1) and identify the CA isoforms in a fraction of total soluble proteins from T. tetragonioides leaves and to determine CA sensitivity to specific sulfonamide inhibitors and heavy metal (HM) ions. In higher plants, CA is one of the most abundant leaf enzymes catalyzing CO2 and HCO3- interconversions that maintain the balance between dissolved forms of inorganic carbon. Catalytically active CA isoforms in the soluble protein fraction isolated from T. tetragonioides leaves were identified by a protonography method which involves the detection of CA activity in the gel following SDS-PAGE and subsequent removal of SDS (De Luca et al., 2015). This approach enabled the detection of active oligomeric forms of CA in the gel, allowing for the separate assessment of their activity. The protonogram revealed the presence of five sources of CA activity, which correspond to isoforms with approximate molecular masses of 26, 35, 41, 52, and 166kDa. The total CA activity of T. tetragonioides leaf proteins was found to be inhibited by specific CA inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), within the same concentration range as CAs from other plants. The half-maximal inhibition of hydratase activity was estimated to be 16μM AZ and 2μM EZ. The in vitro impact of selected heavy metal ions (Cu2⁺, Cd2⁺, Zn2⁺, Pb2⁺, Hg2⁺, and Ag⁺) on the hydratase activity of the soluble T. tetragonioides protein fraction was examined. High sensitivity of CA activity to inhibition by silver (I50-0.5μM) and mercury ions (I50 ∼ 2μM) was shown. Copper and cadmium ions inhibited CA activity with I50 ∼ 40 and ∼12μM, respectively. Zinc ions (I50 ∼ 200μM) were weaker inhibitors, and lead ions in the concentration range of 50-500μM insignificantly stimulated the hydratase activity of soluble proteins of T. tetragonioides. The high sensitivity of T. tetragonioides CA to Ag⁺ and Hg2⁺ ions, which are known to act as sulfhydryl poisons, can be attributed to the presence of SH-containing cysteines within the active site of the plant CAs.

Characterizing the extractable proteins from tomato leaves - A proteomics study.

The ambition to utilize agricultural by-products has spotlighted tomato leaves as a promising source for plant-based proteins. High-yielding protein extractability is key for its industrial use, but previous studies reported decreased protein extractability at later stages of plant development. This study investigated the underlying factors in protein extractability through a comprehensive proteomics analysis across four plant developmental stages (vegetative, flowering, fruit-forming, mature-fruit). The findings linked reduced yields to a shift in leaf function, from anabolic to catabolic processes and (a)biotic stress responses. This functional shift is accompanied by decreased protein synthesis and increased protein degradation, leading to an overall decrease of the soluble protein fraction. Furthermore, incomplete extraction of soluble proteins from leaves of later developmental stages, suggested the presence of inhibitory molecules hindering the extraction process. These findings indicate that breeding strategies towards increased amounts of soluble proteins and reduced concentration of inhibitory molecules could enhance protein extraction yields.

Development of faba protein-tannic acid conjugate via free radical grafting: Evaluation of interaction mechanisms and antioxidative properties.

A soluble fraction of faba bean protein was conjugated with tannic acid via the free-radical grafting method using a mixture of ascorbic acid and hydrogen peroxide. Surface plasmon resonance showed a strong bonding between them, while the free amino and thiol group measurements indicated tannic acid's bonding with the amino groups and cysteine residues on the proteins. Structural analysis using intrinsic fluorescence and surface hydrophobicity demonstrated tannic acid's interaction with the aromatic and hydrophobic amino acids of the protein. The conjugate showed about 77% DPPH, 89% ABTS, and 83% hydroxyl radical scavenging activities and superior ferric-reducing ability compared to the protein alone and the mixture of protein and tannic acid. Electron paramagnetic resonance (EPR) spectroscopy revealed 97.8% radical scavenging ability of the conjugate, comparable to the pure tannic acid. The exceptional antioxidative properties of conjugate can be utilized to delay lipid oxidation in protein-stabilized oil-in-water emulsions.

Physicochemical and techno-functional characterization of soluble proteins extracted by ultrasound from the cricket Acheta domesticus

The growing interest in using insects for human consumption is due to their numerous benefits. Insects offer efficient protein generation, rapid growth rates, and high nutritional value. The objective of this work was to evaluate the physicochemical and techno-functional properties of the different soluble protein fractions of the cricket Acheta domesticus using various methods: grinding (CF), defatting (DCF), alkalinization (SPA), and ultrasound-assisted extraction (SPS). CF, DCF, SPA, and SPS were used as extenders in food models and compared with a control group prepared with meat and a commercial soy protein (SPI). Defatting increased the protein content (52 %) in CF, improving digestibility, while the SPS extraction method improved solid recovery (40.46 %), protein recovery (41.94 %), total protein content (53.85 %), and digestibility (53.7 %) compared to SPA. Proteins exhibited pH-dependent solubility, with higher solubility at pH 12–13 and an isoelectric point of 4.5. In techno-functional properties, SPS had the highest water/oil retention capacity (2.8 g/g, 3.49 g/g), foam formation (386.66 %), and emulsifying stability (32.96 m2/g). CF showed no foam formation, although defatting (DCF) improved foam formation (8.33 %) and emulsifying stability (6.23 m2/g). Heat coagulation was higher for CF and DCF (30.58 % and 30.33 % respectively). All meat models with SPA and SPS showed high elasticity and cohesiveness but low hardness, gumminess, and chewiness. The model prepared with 15 % SPS reduced cooking losses (0.91 %) and total expressible fluid separation (1 %), improved water retention capacity (83.02 %), and increased total soluble protein content (5.32 mg/mL). The ultrasound-assisted extraction method proved to be an efficient way to obtain soluble proteins from Acheta domesticus with techno-functional properties suitable for use as a food additive or meat extender.

Selection of a δ-latroinsectotoxin fragment from Latrodectus tredecimguttatus venom for efficient bacterial expression

Toxins from the venom of predatory and parasitoid arthropods, such as scorpions, spiders, and parasitoid wasps, are used as promising tools in plant protection against insect pests. In many cases, polyclonal antibodies can be created to study a single molecule in the laboratory, which allows for specific methods of detection and purification. In the present study, heterologous expression of δ-latroinsectotoxin from the venom of the black widow spider Latrodectus tredecimguttatus, as well as its N- and C- terminal fragments, was carried out in Escherichia coli cells at various temperatures and aeration conditions. Regardless of the expression conditions, the accumulation of the recombinant protein in large quantities in the bacterial cells as inclusion bodies was observed only for the C-terminal fragment of the toxin. The high efficiency of expression of this fragment allowed the use of the insoluble protein fraction of the bacterial homogenate for the immunization of mice and the production of antibodies to δ-latroinsectotoxin. The antigen-binding activity of the immunoglobulins was confirmed by immunoblotting of the synthesized toxin fragment in bacteria using a vector with removed leader sequences. The antibodies obtained can be used to detect the black widow spider toxin during its expression in various systems, such as in the creation of genetically modified entomopathogenic fungi or viruses.

Influence of stage lactation on quality and protein compositions of Kazakh mare milk and koumiss

Limited studies have examined the effects of geography, climate, and lactation on mare's milk in Kazakhstan. The study aimed to assess the protein components and quality of mare's milk and koumiss from 24 mares in southern Kazakhstan. Milk samples were collected monthly between July and December 2023. The soluble protein fraction was analysed via SDS-PAGE. Casein fractions were examined using SDS-PAGE polyacrylamide electrophoresis. The results indicated the presence of α-, β-, and κ-caseins, along with whey proteins such as α-lactalbumin and β-lactoglobulin in the milk and fermented products. The milk contained 9.02% total solids, 1.62% protein and 1.22% casein on average. The mare milk fat content was 0.71% in Almaty, and in the Zhambyl region, it was significantly higher - 1.24%. The fermented koumiss products had a fat content of 1.22% in Almaty, while in Zhambyl, it was significantly higher at 1.94%. Similarly, the casein content in the Zhambyl region was 1.38%, compared to 0.81% in Almaty. The results indicate that different zones significantly affect mare's milk's fat and protein composition.

Serum-Derived Extracellular Vesicles for the Treatment of Severe Ocular Surface Disease

PurposeAutologous serum is widely used for the treatment of severe ocular surface disease with mixed efficacy. Extracellular vesicles (EVs) are small membrane bound structures present in all body fluids, including serum. This study compared the proteomic, metabolomic, and inflammatory cytokine composition of serum-derived EVs (SDEVs) to that of the soluble free protein fraction and the subsequent capacity of SDEVs to induce corneal epithelial cell migration and inflammation. MethodsSDEVs were isolated from human serum using size exclusion chromatography. SDEVs were analyzed using nanoparticle tracking analysis, transmission electron microscopy, and western blotting. The effects of SDEVs on corneal epithelial cell migration were tested using a standard scratch assay. Inflammatory cytokines in SDEVs and the free protein fraction were quantified using a microarray. A mutli-omics approach was further used to define SDEV cargo. The ability of SDEVs to modulate inflammation in corneal epithelial cells was quantified using ELISAs. ResultsWestern blot and TEM confirmed the presence of SDEVs. Proinflammatory cytokines, along with complement proteins and TGF-β, were decreased in SDEVs compared to serum. Metabolites present in SDEVs were mostly involved in amino acid biosynthesis, the TCA cycle and oxidative phosphorylation. SDEVs exhibited pro-migratory effects similar to serum however, SDEVs did not induce secretion of IL-6 or IL-8. ConclusionsSDEVs exhibit reduced levels of pro-inflammatory cytokines while retaining the beneficial wound healing properties of serum. Unlike serum, SDEVs do not induce inflammation. SDEVs may represent an alternative option for patients with severe ocular surface disease where traditional autologous serum has failed.

Unlocking the secrets of celiac disease: the crucial role of HLA genes in diagnosis, symptoms, and prognosis

Celiac disease (CD) is a systemic autoimmune disorder triggered by gluten ingestion in genetically predisposed individuals, primarily manifesting as gastrointestinal symptoms. The primary environmental factor is gluten, (the soluble alcohol fraction of proteins found in certain cereals such as wheat, rye, and barley ; but genetic predisposition is crucial, localized on chromosome 6 within the major histocompatibility complex known as Human Leukocytes Antigens (HLA) in humans : over 90% of celiac patients express HLA DQ2, and the remaining patients express almost all HLA DQ8. We discuss the implications of these HLA genes in predisposition, diagnosis, symptomatology, and prognosis of CD.

Abstract We054: Mitochondria-containing Extracellular Vesicles Mediate Heart Failure Sterile Inflammation

Introduction: Sterile inflammation has long been postulated to exacerbate heart failure (HF); however, a lack of viable biological targets and thorough understanding of the underlying mechanism posed formidable challenges in the development of novel therapeutic interventions. Hypothesis: Given proinflammatory mitochondrial DNA (mtDNA) are known to be elevated in HF plasma; and that cells release mitochondrial components in extracellular vesicles, we hypothesize that proinflammatory circulating Mitochondria-containing Extracellular Vesicle (MitoEV) is a mediator of HF sterile inflammation. Methods/Results: Using plasma samples of 12 Stage-D HFrEF and 12 healthy participants, plasma microparticles (MP) are co-stained with mitochondrial- and cytoplasmic-specific markers then quantified by volume-metric flow analysis. We found that there are significantly more mitochondrial-cytoplasmic double-positive MP (designated MitoEV, accounts for >70% of total MP) in HF relative to control. Next, using size-exclusion chromatography, we found MitoEV fractions are significantly more potent than soluble protein fractions to induce cytokine production in macrophage, implicating MitoEV as the source of HF plasma immunogenicity. Surface marker analysis by flow suggests that CD14+ monocytes is a major source of HF plasma MitoEV. Using THP-1 monocytes, we found that TLR9 activation by mtDNA results in injured mitochondria to be released as MitoEV (rather than removed by mitophagy), a pathway negatively regulated by beclin-1. Finally, boosting NAD by nicotinamide riboside preserves mitochondrial dysfunction and significantly reduces MitoEV release in primary monocytes. Conclusion: Our findings implicate circulating MitoEV released by monocytes upon mtDNA activation as a pivotal mediator of sterile inflammation in HF, demonstrating a promising avenue for potential therapeutic targeting. The study also provides a potential cellular mechanism to the anti-inflammatory effect of boosting NAD. Model: In HF circulation, mtDNA activates TLR9 of monocytes, injuring mitochondria, which are released as MitoEV (containing mtDNA) to amplify and propagate inflammation in an autocrine manner. Becn1, a key mediator of mitophagy, negatively regulates MitoEV release. Activated monocytes also release cytokines to further injure the heart, resulting in a vicious cycle. Boosting NAD protects against mtDNA-induced mitochondrial dysfunction, attenuates the MitoEV release, and ameliorates inflammation.

Tau protein profiling in tauopathies: a human brain study

Abnormal accumulation of misfolded and hyperphosphorylated tau protein in brain is the defining feature of several neurodegenerative diseases called tauopathies, including Alzheimer’s disease (AD). In AD, this pathological change is reflected by highly specific cerebrospinal fluid (CSF) tau biomarkers, including both phosphorylated and non-phosphorylated variants. Interestingly, despite tau pathology being at the core of all tauopathies, CSF tau biomarkers remain unchanged in certain tauopathies, e.g., progressive supranuclear palsy (PSP), Pick’s disease (PiD), and corticobasal neurodegeneration (CBD). To better understand commonalities and differences between tauopathies, we report a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry capable of detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, including those carrying multiple phosphorylations. We investigated the tau proteoforms in soluble and insoluble fractions of brain tissue from subjects with autopsy-confirmed tauopathies, including sporadic AD (n = 10), PSP (n = 11), PiD (n = 10), and CBD (n = 10), and controls (n = 10). Our results demonstrate that non-phosphorylated tau profiles differ across tauopathies, generally showing high abundance of microtubule-binding region (MTBR)-containing peptides in insoluble protein fractions compared with controls; the AD group showed 12–72 times higher levels of MTBR-containing aggregates. Quantification of tau isoforms showed the 3R being more abundant in PiD and the 4R isoform being more abundant in CBD and PSP in the insoluble fraction. Twenty-three different phosphorylated peptides were quantified. Most phosphorylated peptides were measurable in all investigated tauopathies. All phosphorylated peptides were significantly increased in AD insoluble fraction. However, doubly and triply phosphorylated peptides were significantly increased in AD even in the soluble fraction. Results were replicated using a validation cohort comprising AD (n = 10), CBD (n = 10), and controls (n = 10). Our study demonstrates that abnormal levels of phosphorylation and aggregation do indeed occur in non-AD tauopathies, however, both appear pronouncedly increased in AD, becoming a distinctive characteristic of AD pathology.

The Influence of Milling Granulation on The Proportion of Fermentable and Non-Fermentable Ingredients in Mash Obtained from Mixed Endosperm Wheat

The use of purpose-selected wheat in brewing is limited because it is either not suitable for the application of usual agrotechnical measures or, more often, is not adapted to specific climatic conditions. As unmalted wheat in brewing can cause process problems and problems with the quality composition of mash/wort and consequently beer, by using the appropriate type and variety, but also by adjusting the malting process, these problems can be completely avoided or significantly reduced at the start. Two varieties were selected for the research, which in previous research showed good brewing/malting properties and which have significantly different starting values for the share of total protein (TP) in the grain. The goal of this research was to determine the effect of grain milling granulation on the quality of mash obtained from a hard type of wheat, i.e., the changes in the fermentable and non-fermentable extract ratio during mashing. Varieties with higher and lower initial contents of total protein (TP) in the grain were tested. It was found that regardless of the initial difference in TP concentration, the concentrations of total soluble proteins (TSP) and high molecular protein fraction (HMW N) are similar for varieties with higher and lower initial TP and that the difference in initial TP concentrations is shown as a difference in the concentration of insoluble protein fractions, which are eliminated during fermentation. By increasing the milling granulation in both kinds of wheat, there is a significant increase in the medium molecular protein fraction (MMW N), which is more pronounced in wheat with a lower starting concentration of TP, even though the starting concentrations of TSP in fine (Ø 0.2 mm) and the superfine milling (Ø < 0.2 mm) were similar. A significant drop in fermentability at the superfine granulation of the grain was noted.

High hydrostatic pressure for decontamination of soluble insect proteins prevents protein denaturation better than blanching

Insect production currently involves thermal processes to ensure microbial safety and inactivate endogenous enzymes, but these processes negatively affect the protein's technological properties. Therefore, this study compared high hydrostatic pressure (HHP) at 200/400/600 MPa for 10 min to blanching at 90 °C for 10 min, on soluble protein fractions (pH 3) from house crickets (Acheta domesticus) and lesser mealworms (Alphitobius diaperinus). HHP significantly reduced the microbial load, especially at 400 and 600 MPa. For aerobic counts of mealworms, blanching (from ∼6 to ∼3 log CFU/mL) was more effective than HHP (∼4 log CFU/mL). HHP preserved the secondary protein structure better than blanching, but was less effective in protease inactivation, though only evident in mealworms. HHP did not affect foaming and emulsification properties but improved protein solubility after pH adjustment to 7 compared to untreated fractions by 14–22%. We thus demonstrate that HHP can be an effective alternative to conventional blanching treatments. Industrial relevanceThe emerging edible insect industry is currently struggling with several challenges. Conventional thermal processes that are often applied can negatively affect the techno-functional protein properties. We demonstrated that high hydrostatic pressure (HHP) can be an effective milder alternative to thermal processes. The evaluated blanching condition did not always negatively affect the functional protein properties, and blanching was more effective in protease inactivation compared to HHP for lesser mealworms. HHP can be of industrial relevance for inactivating house cricket fractions with high protein solubility intended for emulsion applications. Blanching could be better suited to provide proteolytically stable fractions.

Adult-onset deactivation of autophagy leads to loss of synapse homeostasis and cognitive impairment, with implications for alzheimer disease

ABSTRACT A growing number of studies link dysfunction of macroautophagy/autophagy to the pathogenesis of diseases such as Alzheimer disease (AD). Given the global importance of autophagy for homeostasis, how its dysfunction can lead to specific neurological changes is puzzling. To examine this further, we compared the global deactivation of autophagy in the adult mouse using the atg7iKO with the impact of AD-associated pathogenic changes in autophagic processing of synaptic proteins. Isolated forebrain synaptosomes, rather than total homogenates, from atg7iKO mice demonstrated accumulation of synaptic proteins, suggesting that the synapse might be a vulnerable site for protein homeostasis disruption. Moreover, the deactivation of autophagy resulted in impaired cognitive performance over time, whereas gross locomotor skills remained intact. Despite deactivation of autophagy for 6.5 weeks, changes in cognition were in the absence of cell death or synapse loss. In the symptomatic APP PSEN1 double-transgenic mouse model of AD, we found that the impairment in autophagosome maturation coupled with diminished presence of discrete synaptic proteins in autophagosomes isolated from these mice, leading to the accumulation of one of these proteins in the detergent insoluble protein fraction. This protein, SLC17A7/Vglut, also accumulated in atg7iKO mouse synaptosomes. Taken together, we conclude that synaptic autophagy plays a role in maintaining protein homeostasis, and that while decreasing autophagy interrupts normal cognitive function, the preservation of locomotion suggests that not all circuits are affected similarly. Our data suggest that the disruption of autophagic activity in AD may have relevance for the cognitive impairment in this adult-onset neurodegenerative disease. Abbreviations: 2dRAWM: 2-day radial arm water maze; AD: Alzheimer disease; Aβ: amyloid-beta; AIF1/Iba1: allograft inflammatory factor 1; APP: amyloid beta precursor protein; ATG7: autophagy related 7; AV: autophagic vacuole; CCV: cargo capture value; Ctrl: control; DLG4/PSD-95: discs large MAGUK scaffold protein 4; GFAP: glial fibrillary acidic protein; GRIN2B/NMDAR2b: glutamate ionotropic receptor NMDA type subunit 2B; LTD: long-term depression; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; m/o: months-old; PNS: post-nuclear supernatant; PSEN1/PS1: presenilin 1; SHB: sucrose homogenization buffer; SLC32A1/Vgat: solute carrier family 32 member 1; SLC17A7/Vglut1: solute carrier family 17 member 7; SNAP25: synaptosome associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYP: synaptophysin ; SYT1: synaptotagmin 1; Tam: tamoxifen; VAMP2: vesicle associated membrane protein 2; VCL: vinculin; wks: weeks.

Exploring textural, solubility and rheological characteristics of high‐moisture extruded meat analogues: effects of wheat gluten and rice protein incorporation in pea protein isolate and feed moisture levels

SummaryThis research explores the development of high‐moisture meat analogues (HMMA) by blending wheat gluten (WG) and rice protein (RP) with pea protein isolate (PPI) in a twin‐screw extruder under high‐moisture extrusion conditions. The study investigates the effects of incorporating RP and WG with PPI on textural, rheological and colour properties, as well as the underlying protein–protein interactions through protein solubility. From the compositional analysis of these secondary ingredients, there was no significant difference between them. However, results from the extrusion indicated that WG‐PPI showed good fibration and higher instrumental anisotropy than RP‐PPI blend extrudates. RP‐PPI blends exhibit inferior textural and rheological properties, attributed to RP's limited crosslinking ability within the protein matrix. This has resulted in a poor structure and lower textural values at higher incorporation of RP (50%) in the pea protein matrix. This was due to the insoluble protein fraction of the RP resulting in a non‐melting in the extrusion barrel. These findings underscore the importance of understanding protein interactions in developing optimised HMMA formulations, paving the way for further exploration into developing modified RP with improved physiochemical properties which can be integrated into meat analogues.

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.

Reconsideration of the laminin receptor 67LR in colorectal cancer cells.

The 67 kDa laminin receptor (67LR) was identified as the first laminin receptor shown to be involved in the carcinogenesis of various cancers, including colorectal cancer. While the exact composition of this 67 kDa receptor remains unknown, it has been reported to be formed by the 37 kDa ribosomal protein SA (RPSA) covalently attached to another unidentified protein. The goal of this study was to clarify the molecular structure of 67LR to enhance our understanding of its role in malignancies. Using cell fractionation of colorectal cancer cells, the 67 kDa immunoreactive protein corresponding to 67LR was found in the soluble protein fraction, while some of the 37 kDa RPSA exhibited plasma membrane-like properties. Proteomic analysis of the 67 kDa fraction revealed the absence of RPSA but identified the β-galactosidase-related 67 kDa elastin-binding protein (67EBP), another laminin binding receptor which presents amino acid sequence similarities that can explain the immune cross reactivity with RPSA. The downregulation of β-galactosidase through short hairpin RNA (shRNA) led to a reduction in both 67LR and 67EBP immunoreactive proteins, confirming the misidentification of 67LR and 67EBP in colorectal cancer cells. Based on these findings, we propose to redefine the 67LR as the RPSA-containing laminin receptor (RCLR) to avoid confusion with the 67EBP.