Membrane Fraction Research Articles

High Carbohydrate Diet Increased Glucose Transporter Protein Levels in Jejunum but Did Not Lead to Enhanced Post-Exercise Skeletal Muscle Glycogen Recovery.

We examined the effect of dietary carbohydrate intake on post-exercise glycogen recovery. Male Institute of Cancer Research (ICR) mice were fed moderate-carbohydrate chow (MCHO, 50%cal from carbohydrate) or high-carbohydrate chow (HCHO, 70%cal from carbohydrate) for 10 days. They then ran on a treadmill at 25 m/min for 60 min and administered an oral glucose solution (1.5 mg/g body weight). Compared to the MCHO group, the HCHO group showed significantly higher sodium-D-glucose co-transporter 1 protein levels in the brush border membrane fraction (p = 0.003) and the glucose transporter 2 level in the mucosa of jejunum (p = 0.004). At 30 min after the post-exercise glucose administration, the skeletal muscle and liver glycogen levels were not significantly different between the two diet groups. The blood glucose concentration from the portal vein (which is the entry site of nutrients from the gastrointestinal tract) was not significantly different between the groups at 15 min after the post-exercise glucose administration. There was no difference in the total or phosphorylated states of proteins related to glucose uptake and glycogen synthesis in skeletal muscle. Although the high-carbohydrate diet significantly increased glucose transporters in the jejunum, this adaptation stimulated neither glycogen recovery nor glucose absorption after the ingestion of post-exercise glucose.

Inhibition of gastric H+,K+-ATPase by new dihydropyrazole derivative KYY-008

The gastric proton pump (H+,K+-ATPase) responsible for the H+ secretion of gastric acid is an essential therapeutic target for acid-related diseases. H+,K+-ATPase belongs to a P2-type ATPase family. Here, we examined the effects of a newly synthesized dihydropyrazole derivative KYY-008 on the H+,K+-ATPase. KYY-008 concentration-dependently inhibited the enzyme activity of the ATPase in the membrane fractions prepared from isolated hog gastric mucosa and from human kidney HEK293 cells in which gastric H+,K+-ATPase is exogenously expressed. The IC50 values in these samples were 3.4 μM and 3.7 μM, respectively. In addition, KYY-008 significantly inhibited the H+,K+-ATPase-derived H+ uptake into the tightly sealed vesicles prepared from the hog gastric mucosa. In contrast, KYY-008 has no effect on the activities of other P2-type ATPases such as Na+,K+-ATPase and Ca2+-ATPase. KYY-008 did not change the ionic currents of voltage-dependent Ca2+ channels, that were potential targets for some dihydropyrazole derivatives. Together, we discovered a new dihydropyrazole derivative which acts as a selective inhibitor of gastric H+,K+-ATPase.

α2A- and α2C-adrenoceptor expression and functionality in postmortem prefrontal cortex of schizophrenia subjects

Previous evidence suggests that α2-adrenoceptors (α2-AR) may be involved in the pathophysiology of schizophrenia. However, postmortem brain studies on α2-AR expression and functionality in schizophrenia are scarce. The aim of our work was to evaluate α2A-AR and α2C-AR expression in different subcellular fractions of prefrontal cortex postmortem tissue from antipsychotic-free (absence of antipsychotics in blood at the time of death) (n = 12) and antipsychotic-treated (n = 12) subjects with schizophrenia, and matched controls (n = 24). Functional coupling of α2-AR to Gα proteins induced by the agonist UK14304 was also tested. Additionally, Gα protein expression was also evaluated. In antipsychotic-free schizophrenia subjects, α2A-AR and α2C-AR protein expression was similar to controls in all the subcellular fractions. Conversely, in antipsychotic-treated schizophrenia subjects, increased α2A-AR expression was found in synaptosomal plasma membrane and postsynaptic density (PSD) fractions (+60% and +79% vs controls, respectively) with no significant changes in α2C-AR. [35S]GTPγS SPA experiments showed a significant lower stimulation of Gαi2 and Gαi3 proteins by UK14304 in antipsychotic-treated schizophrenia subjects, whereas stimulation in antipsychotic-free schizophrenia subjects remained unchanged. Gαo protein stimulation was significantly decreased in both antipsychotic-free and antipsychotic-treated schizophrenia subjects compared to controls. Expression of Gαi3 protein did not differ between groups, whereas Gαi2 levels were increased in PSD of schizophrenia subjects, both antipsychotic-free and antipsychotic-treated. Gαo protein expression was increased in PSD of antipsychotic-treated subjects and in the presynaptic fraction of antipsychotic-free schizophrenia subjects. The present results suggest that antipsychotic treatment is able to modify in opposite directions both the protein expression and the functionality of α2A-AR in the cortex of schizophrenia patients.

Changes in synaptic proteins of the complex PSD-95/NMDA receptor/nNOS and mitochondrial dysfunction after levocabastine treatment

Nitric oxide generation is related to the activity of certain proteins located at synaptic sites. Previous findings show that NOS activity, nNOS protein expression, respiratory parameters and mitochondrial complex activities are altered in rat cerebral cortex by administration of levocabastine, an antagonist of histamine H1 and neurotensin NTS2 receptors. ATP provision by mitochondria may play an important role in the functional interaction between synaptic proteins NMDA receptor and PSD-95 with NO synthesis. In this context, our purpose was to evaluate the effect of levocabastine administration on protein expression of PSD-95, GluN2B and iNOS, as well as on mitochondrial ATP production. Male Wistar rats received a single (i.p.) dose of levocabastine (50 μg/kg) or saline solution (controls) and were decapitated 18 h later. Mitochondrial and synaptosomal membrane fractions were isolated from cerebral cortex by differential and sucrose gradient centrifugation. Expression of synaptic proteins was evaluated by Western blot assays in synaptosomal membrane fractions. Oxygen consumption, mitochondrial membrane potential and ATP production rate were determined in fresh crude mitochondrial fractions. After levocabastine treatment, protein expression of PSD-95, GluN2B and β-actin decreased 97, 45 and 55%, respectively, whereas that of iNOS enhanced 3.5-fold versus controls. In crude mitochondrial fractions levocabastine administration reduced roughly 15% respiratory control rate as assayed with malate-glutamate or succinate as substrates, decreased mitochondrial membrane potential (21%), and ATP production rates (57%). Results suggested that levocabastine administration induces alterations in synaptic proteins of the protein complex PSD-95/NMDA receptor/nNOS and in neuron cytoskeleton. Mitochondrial bioenergetics impairment may play a role in the functional link between synaptic proteins and NO synthesis.

Biological activity of 2α,3β,23-trihydroxyolean-12-ene on glucose homeostasis

We studied the effect and the mechanisms of action of 2α,3β,23-trihydroxyolean-12-ene (THO), from Croton heterodoxus Baill. (Euphorbiaceae), in glucose uptake in hyperglycemic rats. The effect of in vivo pretreatment with THO in hyperglycemic rats was analyzed. The in vitro effects of THO were observed in adipocytes and in adipose tissue. THO reduced glycemia, in part by increasing serum insulin and augmenting the disposal of glucose as glycogen in hepatocytes but did not change the serum concentration of glucagon-like peptide-1. THO increased glucose uptake in adipocytes and in adipose tissue by a mechanism dependent on phosphatidylinositol 3-kinase vesicular traffic and on the process of vesicle fusion at the plasma membrane in regions containing cholesterol, indicating the involvement of glucose transporter-4 (GLUT4). This triterpene may act solely via the activation and translocation of GLUT4 (rather than via nuclear actions, such as upregulation of GLUT4 synthesis), since THO did not alter the amount of GLUT4 mRNA or the content of GLUT4. Consistent with these data, the stimulatory effect of this triterpene on the quantity of GLUT4 in the membrane fraction was dependent upon p38 phosphorylation. In this experimental model, orally administered 10 mg/kg THO did not modulate extracellular serum lactate dehydrogenase. In conclusion, THO decreases hyperglycemia by increasing serum insulin and hepatic glycogen content. The THO mechanism of action on adipose tissue for glucose uptake is suggested to be via GLUT4 translocation stimulation mediated by a p38-dependent mechanism. THO is a potential antihyperglycemic agent that acts in a target tissue for glucose homeostasis.

Conversion of Whey into Value-Added Products through Fermentation and Membrane Fractionation

The cheese whey (95% composed of water) is an effluent produced in the cheese industry, of which more than 1.5 million tons are generated in Spain, constituting a serious environmental problem. The process starts with a new fermentative/enzymatic technology that totally converts whey, mainly composed by lactose, proteins, and salts, into a fermented product with higher added value. This new product is mainly composed by lactic acid bacteria biomass, ammonium lactate, and a protein hydrolysate. To separate valuable fractions, this fermented product is processed by a two-stage membrane system, which is a very innovative process in this type of fermented product. The first stage consists of ultrafiltration to separate all suspended solids. As a result of this stage, a product mainly constituted by lactic acid bacteria that have both agronomic applications, mainly as a biocontrol and biofertilizer/bio-stimulant, and applications in animal feeding as a probiotic, is obtained. The second stage consists of reverse osmosis used to concentrate the ultrafiltered permeate obtained earlier, leading to a microbiologically stable product and reducing transport costs. The concentrate is mainly composed of ammonium lactate and a protein hydrolysate, constituted by peptides and free amino acids, which has application both in agriculture as a bio-stimulant and in animal feeding, and the permeate is water, reusable in other industrial processes. This work demonstrates the technical feasibility of this valorization process to achieve the objective of “Waste 0” from a problematic by-product, while obtaining products with commercial utility.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulatesantioxidant gene expression andphosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclizeATP to enhance the cAMP basal level that regulatesantioxidant geneexpression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression wasupregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KDwas found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

The prognostic significance of immunostaining of Wnt signaling pathway molecules, E-cadherin and β-catenin in Colorectal Carcinoma

Background and study aimsColorectal carcinoma (CRC) is associated with high morbidity and mortality. The E-cadherin–catenin complex is crucial in the development and progression of carcinomas. This study was conducted to evaluate the relation between E-cadherin and β-catenin immunostaining and CRC outcome. Patients and methodsTissue microarrays were constructed from CRC, nodal metastases, adenomas, and normal mucosa. E-cadherin and β-catenin immunostaining was performed, and results were analyzed. ResultsFor E-cadherin, the membranous fraction (MF) was higher in normal mucosa, adenoma, CRC, and nodal metastasis than the cytoplasmic fraction (CF), but no difference in nodal metastasis was observed. A low MF in CRC was associated with disease relapse. For β-catenin, high MF and CF in normal mucosa, adenoma, CRC, and nodal metastasis were observed, whereas the nuclear fraction (NF) was high only in CRC. In CRC, a high CF was associated with nodal metastasis and the incidence of relapse and predicted nodal metastasis. A high NF could predict distance metastasis. A high CF in CRC was associated with favorable disease-free survival and overall survival. ConclusionReduced E-cadherin and β-catenin immunostaining in CRC is related to prognostic factors. The Wnt/β-catenin pathway may play a crucial role in CRC progression and help identify the high risk of adverse outcomes and indicate close follow-up.

1147-P: Pseudo Insulin Resistance: Palmitate Inactivates Insulin Signaling but Stimulates Basal Glucose Uptake in 3T3-L1 Adipocytes

Increased levels of circulating free fatty acids (FFA) is the major risk factor for type 2 diabetes in obesity. While the long-term effects of increased FFA on adipose tissue likely involve latent inflammation, the short-term effects on insulin-sensitive adipocytes are less clear. We treated fully differentiated 3T3-L1 adipocytes with increased concentrations (0.3-1 mM) of a typical FFA, palmitate, for 2 days, and measured insulin stimulation of 2-deoxyglucose (2-DG) uptake. The insulin cascade and AMPK activities, Glut-1 and Glut-4 levels were measured by western blots (WB). Glut-4 translocation was measured either by WB after cell membrane fractionation, or by fluorescence analysis after lentiviral transduction of adipocytes with Glut-4 construct containing C-terminal mCherry and outer Myc epitope insertion in the Glut-4 extracellular loop. While fluorescence of mCherry reported the total Glut-4, that of anti-Myc antibody revealed Glut-4 exposed on the plasma membrane. Palmitate dose dependently increased basal 2-DG uptake, whereas insulin-stimulated uptake and Glut-4 translocation to the plasma membrane were significantly reduced only by high (1 mM) palmitate. High palmitate did not alter Glut-1 and Glut-4 expression, but reduced IRS phosphorylation (Tyr612) and completely ceased that of Akt (Thr308/Ser473) and AS160 (Ser318) in the absence or presence of insulin, as well as phosphorylation of ACC (Ser-79) by AMPK. These results suggest that the short-term effects of palmitate on adipocytes are associated with “pseudo” insulin resistance, i.e., an increased basal glucose uptake likely due to the “pull” mechanism for increased triglyceride turnover, and the loss of insulin stimulation possibly due to fill-up of fat depots. Disclosure N. Podkuychenko: None. S. Michurina: None. I. Stafeev: None. A. Y. Khapchaev: None. V. P. Shirinsky: None. A. V. Vorotnikov: None. Funding Russian Foundation for Basic Research (20-015-00471)

Dual regulation of fatty acid synthase (FASN) expression by O-GlcNAc transferase (OGT) and mTOR pathway in proliferating liver cancer cells.

Fatty acid synthase (FASN) participates in many fundamental biological processes, including energy storage and signal transduction, and is overexpressed in many cancer cells. We previously showed in a context of lipogenesis that FASN is protected from degradation by its interaction with O-GlcNAc transferase (OGT) in a nutrient-dependent manner. We and others also reported that OGT and O-GlcNAcylation up-regulate the PI3K/AKT/mTOR pathway that senses mitogenic signals and nutrient availability to drive cell cycle. Using biochemical and microscopy approaches, we show here that FASN co-localizes with OGT in the cytoplasm and, to a lesser extent, in the membrane fraction. This interaction occurs in a cell cycle-dependent manner, following the pattern of FASN expression. Moreover, we show that FASN expression depends on OGT upon serum stimulation. The level of FASN also correlates with the activation of the PI3K/AKT/mTOR pathway in hepatic cell lines, and in livers of obese mice and in a chronically activated insulin and mTOR signaling mouse model (PTEN-null mice). These results indicate that FASN is under a dual control of O-GlcNAcylation and mTOR pathways. In turn, blocking FASN with the small-molecule inhibitor C75 reduces both OGT and O-GlcNAcylation levels, and mTOR activation, highlighting a novel reciprocal regulation between these actors. In addition to the role of O-GlcNAcylation in tumorigenesis, our findings shed new light on how aberrant activity of FASN and mTOR signaling may promote the emergence of hepatic tumors.

Entamoeba histolytica: Proteomics Bioinformatics Reveal Predictive Functions and Protein-Protein Interactions of Differentially Abundant Membrane and Cytosolic Proteins.

Amoebiasis is caused by Entamoeba histolytica and ranked second for parasitic diseases causing death after malaria. E. histolytica membrane and cytosolic proteins play important roles in the pathogenesis. Our previous study had shown several cytosolic proteins were found in the membrane fraction. Therefore, this study aimed to quantify the differential abundance of membrane and cytosolic proteins in membrane versus cytosolic fractions and analyze their predicted functions and interaction. Previous LC-ESI-MS/MS data were analyzed by PERSEUS software for the differentially abundant proteins, then they were classified into their functional annotations and the protein networks were summarized using PantherDB and STRiNG, respectively. The results showed 24 (44.4%) out of the 54 proteins that increased in abundance were membrane proteins and 30 were cytosolic proteins. Meanwhile, 45 cytosolic proteins were found to decrease in abundance. Functional analysis showed differential abundance proteins involved in the molecular function, biological process, and cellular component with 18.88%, 33.04% and, 48.07%, respectively. The STRiNG server predicted that the decreased abundance proteins had more protein–protein network interactions compared to increased abundance proteins. Overall, this study has confirmed the presence of the differentially abundant membrane and cytosolic proteins and provided the predictive functions and interactions between them.

Porphyromonas gingivalis induced up-regulation of PD-L1 in colon carcinoma cells.

Programmed death-ligand-1 (PD-L1) is a ligand for programmed death receptor (PD-1) that plays a major role in cell-mediated immune response; it regulates T-cell activation and regulates survival and functions of activated T cells. Expression of PD-L1 can induce chronic inflammation and activate mechanisms of immune evasion. PD-L1 is expressed in most of human carcinomas. Porphyromonas gingivalis (P. gingivalis) is a major keystone pathogen in periodontitis that invade host cells and disposes a variety of virulence factors. The aim of the present study was to clarify the signaling pathway of P. gingivalis molecules that induce PD-L1 up-regulation in colon carcinoma cells. Additionally, it was investigated which components of P. gingivalis are responsible for PD-L1 induction. Colon cancer cells (CL-11) were stimulated with total membrane (TM) fractions, peptidoglycans (PDGs) and viable P. gingivalis bacteria. Seven signaling molecule inhibitors were used: receptor-interacting serine/threonine-protein kinase 2 (RIP2) tyrosine kinase inhibitor, nucleotide-binding oligomerization domain (NOD)-like receptor 1&2 inhibitor, NOD-like receptor, nuclear factor kappa B inhibitor, c-Jun N-terminal kinases inhibitor, mitogen-activated protein/extracellular signal-regulated kinase inhibitor, mitogen activated kinase (MAPK) inhibitor. PD-L1 protein expression was examined by western blot analysis and quantitative real time PCR. It was demonstrated that the TM fraction and PDG induced up-regulation of PD-L1 expression in colon cancer cells. In conclusion, the results of this study suggest that PDG of P. gingivalis plays a major role in PD-L1 up-regulation in colon cancer cells. In addition, the mechanism of PD-L1 up-regulation depends on NOD 1 and NOD 2 and involves activation of RIP2 and MAPK signaling pathways.

Aspartame induces cancer stem cell enrichment through p21, NICD and GLI1 in human PANC-1 pancreas adenocarcinoma cells

This study aimed to investigate the molecular effects of the common natural sugar glucose and artificial sweetener aspartame on cancer stem cell (CSC) population and cancer aggressiveness of PANC-1 human pancreas adenocarcinoma cells. According to our findings while aspartame exposure significantly increased the CSC population, high glucose had no effect on it. The epithelial-mesenchymal transition marker N-cadherin increased only in the aspartame group. The findings indicate that a high level of glucose exposure does not effect the invasion and migration of PANC-1 cells, while aspartame increases both of these aggressiveness criteria. The findings also suggest that a high concentration of glucose maintains CSC population through induction of nuclear Oct3/4 and differentiation to parental cells via increasing cytoplasmic c-myc. Aspartame exposure to PANC-1 cells activated AKT and deactivated GSK3β by increasing levels of ROS and cytoplasmic Ca+2, respectively, through T1R2/T1R3 stimulation. Then p-GSK3β(Ser9) boosted the CSC population by increasing pluripotency factors Oct3/4 and c-myc via NICD, GLI1 and p21. In the aspartame group, T1R1 silencing further increased the CSC population but decreased cell viability and suppressed the p21, NICD and GLI activation. The presence and amount of T1R subunits in the membrane fraction of PANC-1 cells are demonstrated for the first time in this study, as is the regulatory effect of T1R1's on CSC population. In conclusion, the present study demonstrated that long-term aspartame exposure increases CSC population and tumor cell aggressiveness through p21, NICD, GLI1. Moreover, while aspartame had no tumorigenic effect, it could potentially advance an existing tumor.

Bioelectronic Measurement of Target Engagement to a Membrane-Bound Transporter.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable. However, the mass dependence of SPR signals may preclude the detection of binding events when the ligand has a significantly smaller mass than the target protein. In FRET-based experiments, protein labeling through modification may inadvertently alter protein dynamics. Graphene Bio-Electronic Sensing Technology (GBEST) aims to overcome these challenges. Label-free characterization takes place in a microfluidic chamber wherein a fluid lipid membrane is reconstituted directly above the GBEST sensor surface. By leveraging the high conductivity, sensitivity, and electrical properties of monolayer graphene, minute changes in electrostatic charges arising from the binding and unbinding of a ligand to a native IMP target can be detected in real time and in a mass-independent manner. Using crude membrane fractions prepared from cells overexpressing monocarboxylate transporter 1 (MCT1), we demonstrate the ability to (1) form a fluid lipid bilayer enriched with MCT1 directly on top of the GBEST sensor and (2) obtain kinetic binding data for an anti-MCT1 antibody. Further development of this novel technology will enable characterization of target engagement by both low- and high-molecular-weight drug candidates to native IMP targets in a physiologically relevant membrane environment.

Hydroxyurea-induced membrane fluidity decreasing as a characterization of neuronal membrane aging in Alzheimer's disease.

Aging is one of the significant risk factors for Alzheimer’s disease (AD). Therefore, this study aimed to propose a new hypothesis “membrane aging” as a critical pathogenesis of AD. The concept of “membrane aging” was reviewed, and the possible mechanisms of membrane aging as the primary culprit of AD were clarified. To further prove this hypothesis, a hydroxyurea-induced “membrane aging” model was established in vitro and in vivo. First, neuronal aging was validated by immunocytochemistry with age-related markers, and membrane aging phenotypes were confirmed. The alterations of membrane fluidity within APP/PS1 mice were re-proved by intracerebroventricular injection of hydroxyurea. Decreased membrane fluidity was found in vitro and in vivo, accompanied by increased total cholesterol concentration in neurons but decreased cholesterol levels within membrane fractions. The Aβ level increased considerably after hydroxyurea treatment both in vitro and in vivo. DHA co-treatment ameliorated membrane aging phenotypes and Aβ aggregation. The study revealed the AMP-activated protein kinase/acetyl CoA carboxylase/carnitine palmitoyl transferase 1 pathway involved in membrane aging processes. These results strongly supported the idea that membrane aging was a pathogenesis of AD and might serve as a new therapeutic target for AD.

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum.

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

Direct Bioelectrocatalytic Oxidation of Glucose by Gluconobacter oxydans Membrane Fractions in PEDOT:PSS/TEG-Modified Biosensors.

Recent years have witnessed an ever-increasing interest in developing electrochemical biosensors based on direct electron transfer-type bioelectrocatalysis. This work investigates the bioelectrocatalytic oxidation of glucose by membrane fractions of Gluconobacter oxydans cells on screen-printed electrodes modified with thermally expanded graphite and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Electrooxidation of glucose was shown to occur without the presence of electron transport mediators. Chronoamperometric and cyclic voltametric characteristics showed an increase of anodic currents at electrode potentials of 0–500 mV relative to the reference electrode (Ag/AgCl). The direct electron transfer effect was observed for non-modified PEDOT:PSS as well as for PEDOT:PSS linked with crosslinkers and conductive fillers such as polyethylene glycol diglycidyl or dimethyl sulfoxide. Bioelectrodes with this composite can be successfully used in fast reagent-free glucose biosensors.

A Novel Role of Nuclear and Membrane Receptor on Isoflavone-Induced Neuritogenesis and Synaptogenesis

Thyroid hormone (TH) receptor (TR) and estrogen receptor (ER) play crucial roles in brain development. TR and ER are involved in dendrite growth, spines, and synapse formation in neurons. Soybean isoflavones, such as genistein, daidzein, and daidzein metabolite, S-equol are known to exert their action through TR, ER, and GPER1, a G-protein-coupled ER. However, the mechanisms of isoflavones action on brain development, especially during neuritogenesis and synaptogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone augmented TH- or estradiol (E2)-mediated dendrite arborization of Purkinje cells. Such augmentation was suppressed by G15, a selective GPER1 antagonist, and ICI 182.780, an antagonist for ERs in both cultures. The knockdown of nuclear TRs or ERs also significantly reduced the dendrite arborization of Purkinje cells. It also increased the mRNA levels of TH-responsive genes, including Mbp, Bdnf, Rc3, Ntf3, Camk2b, Hr, and also Syn1, Syp, and Psd95 that are involved in synaptic plasticity. Isoflavones also increased the protein levels of synapsin-1, synaptophysin, and PSD95 in dendrite and membrane fraction of the cerebellar culture. To study further the molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite growth of Neuro-2A. The knockdown of TRs, ERs, and GPR30 by RNAi reduced isoflavones-induced neurite growth. Moreover, the co-culture study of Neuro-2A and astrocytes also showed an increase in isoflavones-induces neurite growth. In addition, isoflavones increased the localization of synapsin-1 or synaptophysin and F-actin in filopodia tips during Neuro-2A differentiation. The knockdown of nuclear ERs or GPR30 significantly reduced the number of filopodia and synapsin-1 or synaptophysin expression levels in neurite and membrane fractions. However, there are no significant effects of filopodia formation after co-culture with astrocytes. These results indicate that nuclear ERs and TRs play an essential role in isoflavones-induces neuritogenesis. Non-genomics signaling through membrane receptor and F-actin are necessary for the isoflavones-induces synaptogenesis. Astrocytes-neurons communication also increased isoflavones-induced neuritogenesis, but not synaptogenesis.

Structure and Function of Cannabinoid Receptor Interacting Protein 1a (CRIP1a)

Cannabinoid receptor interacting protein 1a (CRIP1a) is an 18 kDa protein that regulates the CB1 cannabinoid receptor by modifying G-protein-mediated signaling and competing for β-arrestin interactions with the receptor (Booth et al., Molecules. 2019; 24: 3672). Physiological functions associated with CRIP1a include involvement in certain cancers, embryonic development, and functions of the retina (reviewed in Oliver et al., Biomolecules. 2020, 10: 1609). We determined the CRIP1a structure in an effort to elucidate CRIP1a functions. The 1.55 Å resolution crystal structure shows that CRIP1a exhibits a 10-stranded, antiparallel b-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top that serve to exclude solvent. The DALI server identified CRIP1a as homologous to a family of lipidated protein-carriers. Based upon this structural knowledge, we tested the hypothesis that CRIP1a could serve as a carrier of lipidated proteins from cytosol to membranes. Using a mouse neuroblastoma N18TG2 cultured cell model, homogenates were sedimented, and cytosolic and membrane fractions were subjected to SDS-page electrophoresis and western analysis. We determined that CRIP1a is found in both cytosolic and membrane fractions. Immunoprecipitations using a CRIP1a antibody in the absence of detergents showed an apparent molecular weight shift for CRIP1a upon western blot analysis compared with purified, recombinant CRIP1a or CRIP1a in detergent extracts. The molecular weight shift in CRIP1a suggests the formation of a complex or interaction with other cellular proteins consistent with a role for CRIP1a in trafficking of cargo proteins.