Plasma Membrane-enriched Fraction Research Articles

Involvement of the Membrane Nanodomain Protein, AtFlot1, in Vesicular Transport of Plasma Membrane H+-ATPase in Arabidopsis thaliana under Salt Stress.

The aim of this study was to elucidate whether the membrane nanodomain protein AtFlot1 is involved in vesicular transport pathways and regulation of the P-type H+-ATPase content in plasma membrane of A. thaliana under salt stress. Transmission electron microscopy revealed changes in the endosomal system of A. thaliana root cells due to knockout mutation SALK_205125C (Atflot1ko). Immunoblotting of the plasma membrane-enriched fractions isolated from plant organs with an antibody to the H+-ATPase demonstrated changes in the H+-ATPase content in plasma membrane in response to the Atflot1ko mutation and salt shock. Expression levels of the main H+-ATPase isoforms, PMA1 and PMA2, as well as endocytosis activity of root cells determined by endocytic probe FM4-64 uptake assay, were unchanged in the Atflot1ko mutant. We have shown that AtFlot1 participates in regulation of the H+-ATPase content in the plasma membrane. We hypothesized that AtFlot1 is involved in both exocytosis and endocytosis, and, thus, contributes to the maintenance of cell ion homeostasis under salt stress. The lack of a pronounced Atflot1ko phenotype under salt stress conditions may be due to the assumed ability of Atflot1ko to switch vesicular transport to alternative pathways. Functional redundancy of AtFlot proteins may play a role in the functioning of these alternative pathways.

Polycystin-1 Is a Crucial Regulator of BIN1 Expression and T-Tubule Remodeling Associated with the Development of Dilated Cardiomyopathy

Cardiomyopathy is commonly observed in patients with autosomal dominant polycystic kidney disease (ADPKD), even when they have normal renal function and arterial pressure. The role of cardiomyocyte polycystin-1 (PC1) in cardiovascular pathophysiology remains unknown. PC1 is a potential regulator of BIN1 that maintains T-tubule structure, and alterations in BIN1 expression induce cardiac pathologies. We used a cardiomyocyte-specific PC1-silenced (PC1-KO) mouse model to explore the relevance of cardiomyocyte PC1 in the development of heart failure (HF), considering reduced BIN1 expression induced T-tubule remodeling as a potential mechanism. PC1-KO mice exhibited an impairment of cardiac function, as measured by echocardiography, but no signs of HF until 7-9 months of age. Of the PC1-KO mice, 43% died suddenly at 7 months of age, and 100% died after 9 months with dilated cardiomyopathy. Total BIN1 mRNA, protein levels, and its localization in plasma membrane-enriched fractions decreased in PC1-KO mice. Moreover, the BIN1 + 13 isoform decreased while the BIN1 + 13 + 17 isoform was overexpressed in mice without signs of HF. However, BIN1 + 13 + 17 overexpression was not observed in mice with HF. T-tubule remodeling and BIN1 score measured in plasma samples were associated with decreased PC1-BIN1 expression and HF development. Our results show that decreased PC1 expression in cardiomyocytes induces dilated cardiomyopathy associated with diminished BIN1 expression and T-tubule remodeling. In conclusion, positive modulation of BIN1 expression by PC1 suggests a novel pathway that may be relevant to understanding the pathophysiological mechanisms leading to cardiomyopathy in ADPKD patients.

Comprehensive analysis of glycerolipid dynamics during tobacco pollen germination and pollen tube growth.

Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination.

The Identification and Validation of Plasma Membrane Proteins as Potential Therapeutic Targets for Histone H3 Mutant Paediatric High-Grade Gliomas

Abstract AIMS Paediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death in children. Histone H3.3 mutations are an important hallmark of pHGG, but for which their underlying oncogenic mechanisms are incompletely understood. This research aims to identify potential molecular effectors in these tumours that may be therapeutically effective. METHOD To assess potential molecular effectors, we employed proteomic profiling using LC-MS/MS and determined plasma membrane proteins of the surfactome that are differentially expressed on the surface of H3.3 mutant (G34R, G34V), compared to H3.3-wildtype human glioma cells in culture. RESULTS Profiling of plasma membrane-enriched fractions identified 34 membrane proteins exclusively detected from H3 G34V mutant cells compared to H3 wild-type cells, and 10 membrane proteins exclusively detected from H3 G34R mutant cells, with 39 membrane proteins shared between both H3 G34R/V genotypes. One of these shared membrane proteins, GLUT1 (glucose transporter) has very recently been reported to be over-expressed and linked to tumour phenotypes in mouse glioma cells manipulated to carry the most common DIPG-associated H3.3-K27M mutation [Miya et al. Neuro-Oncology Advances 2021]. Here, we explore the functional significance of GLUT1 differential expression in H3.3 mutant (G34R, G34V) human glioma cells.

Abstract P332: The Hypertension Induced By Clozapine Is Related With Increased Renal Sodium Transporters In Mice

Introduction: Hypertension is caused by multi-factors, and the pathogenesis is linked to, generally, the increases in the protein expressions and functions of renal sodium transport proteins along the nephron. One of the major side effects of clozapine (common antipsychotic drug) is hypertension. The mechanism is unknown. Hypothesis: the pathogenesis of the clozapine-induced hypertension is associated with increases of renal sodium transport proteins. Methods: we examined the blood pressures (BP) and protein expressions of major sodium transport proteins profiled along the renal tubules in the C57Bl6/J male mice (9 weeks old, n=4-5/group) treated with clozapine at three doses of 5,10 and 20 mg/ kg for 1 week via intraperitoneal injection. Result: blood pressures (tail-cuff, BP98A, softron, Japan) were higher in clozapine at 20mg/kg/day than vehicle (SBP: 130±1.64 vs 109±1.39, DBP: 87 ±4.12 vs 69±2.10 mmHg, n=5/group) and not altered at 5 & 10 mg/kg/day. There were no differences in BW, serum/urine creatinine and electrolytes except for a slight increase in serum sodium at dose of 20 mg/kg (152.8±0.71 vs 150.6±0.40, mmol/L).In renal homogenates, total protein abundance of NaPi2 (western blotting,199±26% of vehicle) NKCC2(184±20), NCC(213±14),and ENaC-α(217±47),were higher at 20 mg/kg than vehicle. No increases were found in protein expressions of NHE3, αNKA, ENaC-β and ENaC-γ. The expressions of renin, AT1R and ACE at 20mg/kg were similar between groups while ACE2 was decreased (42±10). No increases were found in protein expressions of pNKCC2 in 5&10mg/kg(100±50,45±15); NCC was increased at 5 mg/kg (175±9); α NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups.In plasma membrane enriched fraction (17k), NKCC2(204±43) and ENaC-α(381±28)were higher at 20 mg/kg than vehicle. In intracellular membrane enriched fraction (200k), NKCC2(521±127) was higher than vehicle. Phosphorylated NKCC2 was increased in renal homogenates(331±35),17k(427±87) 200k(521±127) but WNK4 was not changed. Conclusions: increases of renal sodium transport proteins, mainly pNKCC2 and NKCC2, contribute to the dose-related increase of BP in mice treated with clozapine and may play an important role in the pathogenesis of the clozapine-induced hypertension.

MODL-23. The identification and validation of plasma membrane proteins as potential therapeutic targets for histone H3 mutant paediatric high-grade gliomas.

Abstract Paediatric high grade gliomas (pHGG) are the leading cause of cancer-related death in children. Histone H3.3 mutations are an important hallmark of pHGG, but their underlying oncogenic mechanisms are incompletely understood. Towards identifying molecular effectors that may be therapeutically effective, we employed proteomic profiling and determined plasma membrane proteins of the surfactome that are differentially expressed on the surface of H3.3 mutant (G34R, G34V) compared to H3.3-wildtype human glioma cells in culture. Profiling of plasma membrane-enriched fractions identified 34 membrane proteins exclusively detected from H3 G34V mutant cells compared to H3 wild-type cells, and 10 membrane proteins exclusively detected from H3 G34R mutant cells, with 39 membrane proteins shared between both H3 G34R/V genotypes. One of these shared membrane proteins, GLUT1 (glucose transporter), has very recently been reported to be over-expressed and linked to tumour phenotypes in mouse glioma cells manipulated to carry the most common DIPG-associated H3.3-K27M mutation [Miya et al. Neuro-Oncology Advances 2021]. Here, we explore the functional significance of GLUT1 differential expression in H3.3 mutant (G34R, G34V) human glioma cells. Collective observations support a model where different H3.3 mutations converge on changes in GLUT1 expression and reinforce GLUT1 as a potential therapeutic target in paediatric high-grade gliomas.

The Hypertension Induced by Clozapine is Related with Increased Renal Sodium Transporters in Mice

IntroductionHypertension is caused by multi‐factors, and the pathogenesis is linked to, generally, the increases in the protein expressions and functions of renal sodium transport proteins along the nephron. One of the major side effects of clozapine (common antipsychotic drug) is hypertension. The mechanism is unknown.Hypothesisthe pathogenesis of the clozapine‐induced hypertension is associated with increases of renal sodium transport proteins.Methodswe examined the blood pressures (BP) and protein expressions of major sodium transport proteins profiled along the renal tubules in the C57Bl6/J male mice (9 weeks old, n=4‐5/group) treated with clozapine at three doses of 5,10 and 20 mg/ kg for 1 week via intraperitoneal injection.Resultblood pressures (tail‐cuff, BP98A, softron, Japan) were higher in clozapine at 20mg/kg/day than vehicle (SBP: 130±1.64 vs 109±1.39, DBP: 87 ±4.12 vs 69±2.10 mmHg, n=5/group) and not altered at 5 & 10 mg/kg/day. There were no differences in BW, serum/urine creatinine and electrolytes except for a slight increase in serum sodium at dose of 20 mg/kg (152.8±0.71 vs 150.6±0.40, mmol/L). In renal homogenates, total protein abundance of NaPi2 (western blotting,199±26% of vehicle) NKCC2(184±20), NCC(213±14),and ENaC‐α(217±47),were higher at 20 mg/kg than vehicle. No increases were found in protein expressions of NHE3, αNKA, ENaC‐β and ENaC‐γ. The expressions of renin, AT1R and ACE at 20mg/kg were similar between groups while ACE2 was decreased (42±10). No increases were found in protein expressions of pNKCC2 in 5&10mg/kg(100±50,45±15); NCC was increased at 5 mg/kg (175±9); α NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups.In plasma membrane enriched fraction (17k), NKCC2(204±43) and ENaC‐α(381±28)were higher at 20 mg/kg than vehicle. In intracellular membrane enriched fraction (200k), NKCC2(521±127) was higher than vehicle. Phosphorylated NKCC2 was increased in renal homogenates(331±35),17k(427±87) 200k(521±127) but WNK4 was not changed.Additional mice were treated with clozapine at 20mg/kg/day via osmotic mini‐pump for 1 week (mini‐pump) or administrated by intraperitoneal injection for 2 weeks (2wks). Increases of renal NKCC2 (mini‐pump:180±21, 2wks:153±18) and NCC (mini‐pump:171±22, 2 wks:160±23) were also found in those models.Conclusionsincreases of renal sodium transport proteins, mainly pNKCC2 and NKCC2, contribute to the dose‐related increase of BP in mice treated with clozapine and may play an important role in the pathogenesis of the clozapine‐induced hypertension.

Allotype-Specific Glycosylation and Cellular Localization of Human Leukocyte Antigen Class I Proteins.

Presentation of antigens by human leukocyte antigen (HLA) complexes at the cell surface is a key process in the immune response. The α-chain, containing the peptide-binding groove, is one of the most polymorphic proteins in the proteome. All HLA class I α-chains carry a conserved N-glycosylation site, but little is known about its nature and function. Here, we report an in-depth characterization of N-glycosylation features of HLA class I molecules. We observe that different HLA-A α-chains carry similar glycosylation, distinctly different from the HLA-B, HLA-C, and HLA-F α-chains. Although HLA-A displays the broadest variety of glycan characteristics, HLA-B α-chains carry mostly mature glycans, and HLA-C and HLA-F α-chains carry predominantly high-mannose glycans. We expected these glycosylation features to be directly linked to cellular localization of the HLA complexes. Indeed, analyzing HLA class I complexes from crude plasma and inner membrane-enriched fractions confirmed that most HLA-B complexes can be found at the plasma membrane, while most HLA-C and HLA-F molecules reside in the endoplasmic reticulum and Golgi membrane, and HLA-A molecules are more equally distributed over these cellular compartments. This allotype-specific cellular distribution of HLA molecules should be taken into account when analyzing peptide antigen presentation by immunopeptidomics.

Histone Deacetylase Activity and the Renin-Angiotensin-Aldosterone System: Key Elements in Cardiorenal Alterations Provoked by Chronic Malnutrition in Male Adult Rats.

Chronic malnutrition (M) affects >1 billion people worldwide. Epidemiological data point to long-term renal and cardiovascular outcomes (e.g. arterial hypertension, cardiorenal syndromes). The renin-angiotensin-aldosterone system (RAAS) has been implicated in the physiopathology of these disturbances, but M-induced alterations in RAAS-modulated renal Na+ handling and their cardiovascular repercussions are not known. Moreover, altered tissue-specific histone deacetylases (HDAC) results in arterial hypertension and the use of sodium Valproate (Val; a HDAC inhibitor) reduces blood pressure. However, there are no reports regarding the renal and cardiovascular effects of HDAC inhibition in M, or on the signaling pathways involved. The central aim of our study has been to investigate whether alterations in the HDAC/RAAS axis underpin alterations in active Na+ transport in the kidney and heart, and affects blood pressure. Male rats aged 28 days were given either a control (C) or a multideficient diet (Regional Basic Diet, RBD), which mimics alimentary habits from developing countries. Subgroups received Losartan (Los), a blocker of type 1 Angiotensin II receptors. When the rats reached 70 days, new subgroups received Val until they were 90 days of age. Homogenates and enriched plasma membrane fractions from renal cortex corticis and cardiomyocytes were obtained by differential centrifugation of the tissues. The activity of renal and cardiac deacetylases was assayed by measuring - after incubation with the membranes - the amount of deacetylated lysines in a substrate containing an acetylated lysine side chain. Protein kinases activities were measured following the incorporation of the γ-phosphoryl group of [γ-32P]ATP into Ser/Thr residues of histone type III-S. The activity of Na+-transporting ATPases (kidney and heart) was quantified by measuring the release of Pi from ATP that was sensitive to ouabain ((Na++K+)ATPase), or sensitive to furosemide (Na+-ATPase). Tail-cuff plethysmography was used to measure systolic blood pressure and heart rate. M provoked HDAC downregulation, which was reversed by Los and Val, either alone or in combination, with selective upregulation of protein kinases C and A (PKC, PKA) in renal cortex corticis, but not in left ventricle cardiomyocytes. The 2 kinases were strongly inhibited by Los and Val in both organs. Malnourished rats developed elevated systolic arterial pressure (SAP) and heart rate (HR) at 70 days of age; Los and Val restored the control SAP, but not HR. Functional and the above biochemical alterations were associated with the deregulation of renal and cardiac Na+-transporting ATPases. (Na++K+)ATPase activities were downregulated in M rats in both organs, and were further inhibited by the pharmacological treatments in the renal cortex corticis (C and M groups) and the left ventricle (only in C rats). No additional effect was found in cardiac (Na++K+)ATPase from M rats. Ouabain-resistant Na+-ATPase was upregulated in renal cortex corticis and downregulated in cardiomyocytes, returning to C values after administration of Los and Val. The HDAC/RAAS axis appears to be a key regulator of Na+-transporting ATPases in renal cortex corticis and cardiomyocytes via an appropriate balance of PKC and PKA activities. Modifications within the HDAC/RAAS axis provoked by chronic M - with repercussions in renal and cardiac Na+ transport - underpin alterations in bodily Na+ homeostasis that culminate with the onset of arterial hypertension and potential cardiorenal syndrome.

SMN protein promotes membrane compartmentalization of ribosomal protein S6 transcript in human fibroblasts

Alterations of RNA homeostasis can lead to severe pathological conditions. The Survival of Motor Neuron (SMN) protein, which is reduced in Spinal Muscular Atrophy, impacts critical aspects of the RNA life cycle, such as splicing, trafficking, and translation. Increasing evidence points to a potential role of SMN in ribosome biogenesis. Our previous study revealed that SMN promotes membrane-bound ribosomal proteins (RPs), sustaining activity-dependent local translation. Here, we suggest that plasma membrane domains could be a docking site not only for RPs but also for their encoding transcripts. We have shown that SMN knockdown perturbs subcellular localization as well as translation efficiency of RPS6 mRNA. We have also shown that plasma membrane-enriched fractions from human fibroblasts retain RPS6 transcripts in an SMN-dependent manner. Furthermore, we revealed that SMN traffics with RPS6 mRNA promoting its association with caveolin-1, a key component of membrane dynamics. Overall, these findings further support the SMN-mediated crosstalk between plasma membrane dynamics and translation machinery. Importantly, our study points to a potential role of SMN in the ribosome assembly pathway by selective RPs synthesis/localization in both space and time.

A ferric reductase of Trypanosoma cruzi (TcFR) is involved in iron metabolism in the parasite

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.

A Truncated Six Transmembrane Splice Variant MOR-1G Enhances Expression of the Full-Length Seven Transmembrane μ-Opioid Receptor through Heterodimerization.

The μ-opioid receptor gene undergoes extensive alternative splicing to generate an array of splice variants. One group of splice variants excludes the first transmembrane (TM) domain and contains six TM domains. These 6TM variants are essential for the action of a novel class of analgesic drugs, including 3-iodobenzoyl-6β-naltrexamide, which is potent against a spectrum of pain models without exhibiting the adverse side effects of traditional opiates. The 6TM variants are also involved in analgesic action through other drug classes, including δ-opioid and κ-opioids and α 2-adrenergic drugs. Of the five 6TM variants in mouse, mouse μ-opioid receptor (mMOR)-1G is abundant and conserved from rodent to human. In the present study, we demonstrate a new function of mMOR-1G in enhancing expression of the full-length 7TM μ-opioid receptor, mMOR-1. When coexpressed with mMOR-1 in a Tet-Off inducible CHO cell line, mMOR-1G has no effect on mMOR-1 mRNA expression but greatly increases mMOR-1 protein expression in a dose-dependent manner determined by opioid receptor binding and [35S] guanosine 5'-3-O-(thio)triphosphate binding. Subcellular fractionation analysis using OptiPrep density gradient centrifugation shows an increase of functional mMOR-1 receptor in plasma membrane-enriched fractions. Using a coimmunoprecipitation approach, we further demonstrate that mMOR-1G physically associates with mMOR-1 starting at the endoplasmic reticulum, suggesting a chaperone-like function. These data provide a molecular mechanism for how mMOR-1G regulates expression and function of the full-length 7TM μ-opioid receptor. SIGNIFICANCE STATEMENT: The current study establishes a novel function of mouse μ-opioid receptor (mMOR)-1G, a truncated splice variant with six transmembrane (TM) domains of the mouse μ-opioid receptor gene, in enhancing expression of the full-length 7TM mMOR-1 through a chaperone-like function.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Feeding cycle alters the biophysics and molecular expression of voltage‐gated Na+ currents in rat hippocampal CA1 neurones

The function of hippocampus as a hub for energy balance is a subject of broad and current interest. This study aims at providing more evidence on this regard by addressing the effects of feeding cycle on the voltage-gated sodium (Na+ ) currents of acutely isolated Wistar rat hippocampal CA1 neurones. Specifically, by applying patch clamp techniques (whole cell voltage clamp and single channel in inside-out patches) we assessed the influence of feeding and fasting conditions on the intrinsic biophysical properties of Na+ currents. Additionally, mass spectrometry and western blotting experiments were used to address the effect of feeding cycle over the Na+ channel population of the rat hippocampus. Na+ currents were recorded in neurones obtained from fed and fasted animals (here termed "fed neurones" and "fasted neurones", respectively). Whole cell Na+ currents of fed neurones, as compared to fasted neurones, showed increased mean maximum current density and a higher "window current" amplitude. We demonstrate that these results are supported by an increased single channel Na+ conductance in fed neurones and, also, by a greater Nav1.2 channel density in plasma membrane-enriched fractions of fed samples (but not in whole hippocampus preparations). These results imply fast variations on the biophysics and molecular expression of Na+ currents of rat hippocampal CA1 neurones, throughout the feeding cycle. Thus, one may expect a differentiated regulation of the intrinsic neuronal excitability, which may account for the role of the hippocampus as a processor of satiety information.

Cation-dependent mannose-6-phosphate receptor expression and distribution are influenced by estradiol in MCF-7 breast cancer cells.

Cancer cells secrete procathepsin D, and its secretion is enhanced by estradiol. Although alterations in the pro-enzyme intracellular transport have been reported, the mechanism by which it is secreted remains poorly understood. In this work, we have studied the influence of estradiol on the expression and distribution of the cation-dependent mannose-6-phosphate receptor (CD-MPR), which would be a key molecule to ensure the proper localization of the enzyme to lysosomes in breast cancer cells. Immunoblotting studies demonstrated that the expression of CD-MPR is higher in MCF-7 cells, as compared to other breast cancer and non-tumorigenic cells. This expression correlated with high levels of cathepsin D (CatD) in these cells. By immunofluorescence, this receptor mostly co-localized with a Golgi marker in all cell types, exhibiting an additional peripheral labelling in MCF-7 cells. In addition, CD-MPR showed great differences regarding to cation-independent mannose-6-phosphate receptor. On the other hand, the treatment with estradiol induced an increase in CD-MPR and CatD expression and a re-distribution of both proteins towards the cell periphery. These effects were blocked by the anti-estrogen tamoxifen. Moreover, a re-distribution of CD-MPR to plasma membrane-enriched fractions, analyzed by gradient centrifugation, was observed after estradiol treatment. We conclude that, in hormone-responsive breast cancer cells, CD-MPR and CatD are distributed together, and that their expression and distribution are influenced by estradiol. These findings strongly support the involvement of the CD-MPR in the pro-enzyme transport in MCF-7 cells, suggesting the participation of this receptor in the procathepsin D secretion previously reported in breast cancer cells.

Liver HFE protein content is posttranscriptionally decreased in iron-deficient mice and rats.

Although the relationship between hereditary hemochromatosis and mutations in the HFE gene was discovered more than 20 years ago, information on the in vivo regulation of HFE protein expression is still limited. The purpose of the study was to determine the response of liver HFE protein content to iron deficiency in mice and rats by immunoblotting. Attempts to visualize the HFE protein in whole liver homogenates were unsuccessful; however, HFE could be detected in liver microsomes or in plasma membrane-enriched fractions. Five-week-old male C57BL/6 mice fed an iron-deficient diet for 4 wk presented with a significant decrease in liver iron content and liver Hamp expression, as well as with a significant decrease in liver HFE protein content. Rats fed an iron-deficient diet for 4 wk also displayed significant decrease in liver Hamp expression and liver HFE protein content. These results suggest that the downregulation of HFE-dependent signaling may contribute to decreased Hamp gene expression in states of prolonged iron deficiency. It has recently been proposed that HFE protein could be a potential target of matriptase-2, a hepatocyte protease mutated in iron-refractory iron deficiency anemia. However, immunoblot analysis of HFE protein in the livers from Tmprss6-mutated mask mice did not show evidence of matriptase-2-dependent HFE protein cleavage. In addition, no indication of HFE protein cleavage was seen in iron-deficient rats, whereas the full-length matriptase-2 protein content in the same animals was significantly increased. These results suggest that HFE is probably not a major physiological target of matriptase-2. NEW & NOTEWORTHY Feeding of iron-deficient diet for 4 wk decreased liver HFE protein content in both mice and rats, suggesting that decreased HFE-dependent signaling may contribute to hepcidin downregulation in iron deficiency. There was no difference in HFE protein band appearance between matriptase-2-mutated mask mice and wild-type mice, indicating that HFE is probably not a major physiological substrate of matriptase-2-mediated protease activity in vivo.

Erythropoietin administration increases splenic erythroferrone protein content and liver TMPRSS6 protein content in rats

Erythroferrone (ERFE) and TMPRSS6 are important proteins in the regulation of iron metabolism. The objective of the study was to examine splenic ERFE and liver TMPRSS6 synthesis in rats treated with a combination of iron and erythropoietin (EPO).EPO was administered to female Wistar rats at 600U/day for four days, iron-pretreated rats received 150mg of iron before EPO treatment. Content of ERFE and TMPRSS6 proteins was determined by commercial antibodies.Iron pretreatment prevented the EPO-induced decrease in hepcidin expression. Content of phosphorylated SMAD 1,5,8 proteins was decreased in the liver by both EPO and iron plus EPO treatment.Fam132b expression in the spleen was increased both by EPO and iron plus EPO treatments; these treatments also significantly induced splenic Fam132a expression. ERFE protein content in the spleen was increased both by EPO and iron plus EPO to a similar extent.EPO administration increased TMPRSS6 content in the plasma membrane-enriched fraction of liver homogenate; in iron-pretreated rats, this increase was abolished.The results confirm that iron pretreatment prevents the EPO-induced decrease in liver Hamp expression. This effect probably occurs despite high circulating ERFE levels, since EPO-induced ERFE protein synthesis is not influenced by iron pretreatment.

Abstract LB-066: Proteomic-based senescent biomarker identification and characterization in non-small cell lung cancer

Abstract There is widespread interest in the relationship between aging and cancer, and the involvement of senescence in both processes. The accumulation of senescent cells imparts elevated risk of age-associated disease, including cancer; however, a continuing challenge in the field is the lack of senescence-specific biomarkers. Chemotherapy and radiotherapy are potent inducers of senescence, and the pro-inflammatory secretion of persistent senescent cells has been shown to propel tumor evolution and metastatic dissemination. Thus, there is a need to define how much senescence can be tolerated in tissue prior to physiologic dysfunction; however defining senescence ‘burden’ in aged tissue or cancer cannot be accurately determined with existing markers due to their redundancy. To address this shortcoming, we undertook a proteomic-based analysis to identify novel senescent biomarkers in K-Ras mutant non-small cell lung cancer (NSCLC). Importantly, quiescent cells were also included in the experimental design to enable the identification of proteins associated with general dormancy and not specific to senescence. Cell surface proteins from senescent, quiescent and asynchronous cancer cells were labeled with biotin, enriched and analyzed using tandem mass spectrometry. An average of 850 proteins were detected in each replicate, and 287 were unique to senescence. Gene ontology analysis of proteins detected in senescence indicated functions enriched for metabolism, DNA damage checkpoint integrity, membrane organization, wound healing and cytoskeletal remodeling; all consistent with a senescent phenotype. Data were filtered by strict criteria to derive a final list of 31 proteins unique to senescence. Initial validation of a subset of these for which specific commercial antibodies were available, was done by immunoblotting whole cell lysates and plasma membrane-enriched fractions. Some candidates had enriched expression in senescence; while others were increased in both senescence and quiescence and thus are more useful as dormancy markers. Additional validation by immunofluorescence was performed to deduce expression and localization of four senescent candidates. Novel patterns of localization were found whereby some candidates showed enrichment in structures resembling large vesicles and invadopodia-like extensions that connected with neighboring senescent cells. Lastly, immunohistochemical (IHC) analyses of formalin-fixed tissue obtained from chemotherapy-treated NSCLC patients revealed intense staining in enlarged cells with polymorphic nuclei, a characteristic of senescent cells. Thus, we have identified a unique cohort of senescence-specific biomarkers, some of which are amenable to IHC analyses. Ongoing studies will determine their utility as prognostic and predictive biomarkers of senescence tumor burden in NSCLC, with potential extrapolation to other malignancies and pathologies. Citation Format: Meagan Vogt, Chia-Ping H. Yang, Edward Nieves, Kenny Ye, Perry Cohen, Steven Keller, Hayley M. McDaid. Proteomic-based senescent biomarker identification and characterization in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-066.

Gel-based and gel-free search for plasma membrane proteins in chickpea (Cicer arietinum L.) augments the comprehensive data sets of membrane protein repertoire

Plasma membrane (PM) encompasses total cellular contents, serving as semi-porous barrier to cell exterior. This living barrier regulates all cellular exchanges in a spatio-temporal fashion. Most of the essential tasks of PMs including molecular transport, cell-cell interaction and signal transduction are carried out by their proteinaceous components, which make the PM protein repertoire to be diverse and dynamic. Here, we report the systematic analysis of PM proteome of a food legume, chickpea and develop a PM proteome reference map. Proteins were extracted from highly enriched PM fraction of four-week-old seedlings using aqueous two-phase partitioning. To address a population of PM proteins that is as comprehensive as possible, both gel-based and gel-free approaches were employed, which led to the identification of a set of 2732 non-redundant proteins. These included both integral proteins having bilayer spanning domains as well as peripheral proteins associated with PMs through posttranslational modifications or protein-protein interactions. Further, the proteins were subjected to various in-silico analyses and functionally classified based on their gene ontology. Finally an inventory of the complete set of PM proteins, identified in several monocot and dicot species, was created for comparative study with the generated PM protein dataset of chickpea. Biological significanceChickpea, a rich source of dietary proteins, is the second most cultivated legume, which is grown over 10 million hectares of land worldwide. The annual global production of chickpea hovers around 8.5 million metric tons. Recent chickpea genome sequencing effort has provided a broad genetic basis for highlighting the important traits that may fortify other crop legumes. Improvement in chickpea varieties can further strengthen the world food security, which includes food availability, access and utilization. It is known that the phenotypic trait of a cultivar is the manifestation of the orchestrated functions of its proteins. Study of the PM proteome offers insights into the mechanism of communication between the cell and its environment by identification of receptors, signalling proteins and membrane transporters. Knowledge of the PM protein repertoire of a relatively dehydration tolerant chickpea variety, JG-62, can contribute in development of strategies for metabolic reprograming of crop species and breeding applications.

Effect of Erythropoietin, Iron Deficiency and Iron Overload on Liver Matriptase-2 (TMPRSS6) Protein Content in Mice and Rats.

Matriptase-2 (TMPRSS6) is an important negative regulator of hepcidin expression; however, the effects of iron overload or accelerated erythropoiesis on liver TMPRSS6 protein content in vivo are largely unknown. We determined TMPRSS6 protein content in plasma membrane-enriched fractions of liver homogenates by immunoblotting, using a commercial antibody raised against the catalytic domain of TMPRSS6. Plasma membrane-enriched fractions were obtained by centrifugation at 3000 g and washing. TMPRSS6 was detected in the 3000 g fraction as a 120 kDa full-length protein in both mice and rats. Feeding of iron-deficient diet as well as erythropoietin treatment increased TMPRSS6 protein content in rats and mice by a posttranscriptional mechanism; the increase in TMPRSS6 protein by erythropoietin was also observed in Bmp6-mutant mice. Administration of high doses of iron to mice (200, 350 and 700 mg/kg) decreased TMPRSS6 protein content. Hemojuvelin was detected in the plasma membrane-enriched fractions of control animals as a full length protein of approximately 52 kDa; in iron deficient animals, the full length protein was partially cleaved at the N-terminus, resulting in an additional weak band of approximately 47 kDa. In livers from hemojuvelin-mutant mice, TMPRSS6 protein content was strongly decreased, suggesting that intact hemojuvelin is necessary for stable TMPRSS6 expression in the membrane. Overall, the results demonstrate posttranscriptional regulation of liver TMPRSS6 protein by iron status and erythropoietin administration, and provide support for the interaction of TMPRSS6 and hemojuvelin proteins in vivo.