Purified Plasma Membranes Research Articles

Human glioblastoma-derived cell membrane nanovesicles: a novel, cell-specific strategy for boron neutron capture therapy of brain tumors

Glioblastoma (GBM), one of the deadliest brain tumors, accounts for approximately 50% of all primary malignant CNS tumors, therefore novel, highly effective remedies are urgently needed. Boron neutron capture therapy, which has recently repositioned as a promising strategy to treat high-grade gliomas, requires a conspicuous accumulation of boron atoms in the cancer cells. With the aim of selectively deliver sodium borocaptate (BSH, a 12 B atoms-including molecule already employed in the clinics) to GBM cells, we developed novel cell membrane-derived vesicles (CMVs), overcoming the limits of natural extracellular vesicles as drug carriers, while maintaining their inherent homing abilities that make them preferable to fully synthetic nanocarriers. Purified cell membrane fragments, isolated from patient-derived GBM stem-like cell cultures, were used to prepare nanosized CMVs, which retained some membrane proteins specific of the GBM parent cells and were devoid of potentially detrimental genetic material. In vitro tests evidenced the targeting ability of this novel nanosystem and ruled out any cytotoxicity. The CMVs were successfully loaded with BSH, by following two different procedures, i.e. sonication and electroporation, demonstrating their potential applicability in GBM therapy.

Abstract Mo042: Poison Peptides May Contribute to Dysregulation of SERCA in Heart Failure

Proteolysis increases in heart disease, resulting in an accumulation of degraded membrane protein fragments in heart cells. Previous studies have shown that diverse hydrophobic alpha helices can bind and non-specifically inhibit the cardiac calcium pump SERCA. Thus, we hypothesized that partially degraded fragments of transmembrane proteins may directly inhibit SERCA or compete with native peptide regulators of the pump, like such as phospholamban. The resulting dysregulation of SERCA may contribute to calcium mishandling in heart failure. To test these hypotheses, purified cell membranes from non-failing or dilated cardiomyopathy human hearts were subjected to SDS-PAGE and mass spectrometry to identify protein fragments smaller than 26 kD. 1800 proteins were detected, of which 92 were upregulated >3 fold in the failing heart samples, consistent with increased production of small fragments from these membrane proteins. 9 of the upregulated fragments containing transmembrane segments were selected for further analysis based on sequence hydrophobicity and structural similarity to endogenous micropeptide regulators of SERCA. Of these 9 fragments, 6 showed ER localization, suggesting potential for a toxic interaction with SERCA. The relative binding of these candidates to SERCA was quantified with FRET microscopy. Fragments of three membrane proteins (SEC61B, VAMP3, VAMP8) showed an avid interaction with SERCA. To measure SERCA inhibition, we utilized a calcium activity assay was performed usingin which HEK293T cells were transfected with SERCA, ryanodine receptor, candidate peptides and rCepia as an ER calcium indicator. ER-calcium level was normalized to minimum fluorescence after caffeine addition and an ionomycin induced maximum fluorescence after addition of ionomycin and calcium and caffeine induced minimum. Unregulated Candidate poison peptides were compared to SERCA alone and SERCA-PLB phospholamban controls were also performed. . SEC61B demonstrated the a strongest inhibition, most (similar to phospholamban, ) while VAMP3 and VAMP8’s effect was more modest. This type of non-nativeThus, we propose accumulation of toxic transmembrane peptide fragments may inhibition could be contributinginhibit SERCA and contribute to the calcium mishandling seen in heart failure. Future experiments will be performed to quantify the relative abundance of the fragments compared to endogenous regulatory micropeptides.

Assaying the Effect of Peptide Treatment on H+-Pumping Activity in Plasma Membranes from Arabidopsis Seedlings.

Extracellular acidification or alkalization is a common response to many plant-signaling peptides and microbial elicitors. This may be a result of peptide-mediated regulation of plasma membrane-localized ion transporters, such as the plasma membrane H+-ATPase. Early responses to some signaling peptides can therefore be analyzed by assaying H+-pumping across the plasma membrane.We describe a set-up suited for the purification of plasma membranes by aqueous two-phase partitioning from a small sample of Arabidopsis seedlings. Seedlings are grown in a liquid culture, suited for the analysis of in vivo peptide treatment. Additionally, we describe how to measure the H+-pumping activity of the plasma membrane H+-ATPase using the fluorescent probe ACMA.

Cell-free, Dendritic Cell-mimicking Extracellular Blebs for Molecularly Controlled Vaccination.

Dendritic cells (DCs) are prime targets for vaccination and immunotherapy. However, limited control over antigen presentation at a desired maturation status in these plastic materials remains a fundamental challenge in efficiently orchestrating a controlled immune response. DC-derived extracellular vesicles (EVs) can overcome some of these issues, but have significant production challenges. Herein, we employ a unique chemically-induced method for production of DC-derived extracellular blebs (DC-EBs) that overcome the barriers of DC and DC-derived EV vaccines. DC-EBs are molecular snapshots of DCs in time, cell-like particles with fixed stimulatory profiles for controlled immune signalling. DC-EBs were produced an order of magnitude more quickly and efficiently than conventional EVs and displayed stable structural integrity and antigen presentation compared to live DCs. Multi-omic analysis confirmed DC-EBs are majorly pure plasma membrane vesicles that are homogeneous at the single-vesicle level, critical for safe and effective vaccination. Immature vs. mature molecular profiles on DC-EBs exhibited molecularly modulated immune responses compared to live DCs, improving remission and survival of tumor-challenged mice via generation of antigen-specific T cells. For the first time, DC-EBs make their case for use in vaccines and for their potential in modulating other immune responses, potentially in combination with other immunotherapeutics.

Cell Membrane Coated Particles.

Nanoformulations are widely considered in the biomedical field for drug delivery, imaging, or detoxification purposes. Cell membrane coatings are a growing concept that aims to camouflage nanomaterials. The cell membranes are the first point of contact for cells to other biological or synthetic materials and nature has established signaling pathways in this context. In contrast to using purified membrane associated proteins, the use of purified cell membranes contains the protein of interest in a very native environment. This report provides an overview over the advances in cell membrane coated (nano)particles from the past 2-3 years. The progress in using cell membranes from mammalian cells without nuclei, i.e., red blood cells and platelets, as well as nucleus-containing cells in particular white blood cell and specific cancer cells is outlined. Additionally, highlights from recent reports considering hybrid cell membrane coating that originate from at least two different cell types are discussed. Finally, a future perspective indicating the challenges and potential of cell membrane coated nanomaterials and biomaterials is provided.

A Detergent-Free Method for Preparation of Lipid Rafts for the Shotgun Lipidomics Study.

Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the postnuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.The method also makes an easy start for lipidomics studies.

Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8-13) analog.

Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8–13) (NT(8–13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8–13). We first examined the binding affinities of this novel NT(8–13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of β-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article “Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog” published in European Journal of Pharmacology[1]. The reader is directed to the associated article for results interpretation, comments, and discussion.

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites.

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.

Content release of extracellular vesicles in a cell-free extract.

Extracellular vesicles (EVs) transfer molecules from donor to acceptor cells. The EV-content delivery process within the acceptor cell is poorly characterized. We developed a new cell-free assay to assess EV-content release invitro. We found that EV-cytosolic cargoes are released from EVs when isolated vesicles are incubated with purified plasma membrane sheets at acidic pH, a characteristic of the endolysosomal environment. This process is protein dependent. Our results suggest that EV-content delivery occurs within the endo/lysosomes of acceptor cells and is triggered by acidification. This process resembles virus content delivery and may require membrane fusion. The assay presented here will facilitate investigations into the core machinery and mechanisms underlying EV content delivery.

Protocol for eukaryotic plasma membrane isolation using superparamagnetic nanoparticles

Abstract Superparamagnetic nanoparticles (SPMNP) are used in cell biology for various applications such as subcellular fractionation and for high pure organelle isolation. We simplified previously established methodology with the use of SPMNP for isolation of high pure and high yield plasma membrane from adherent eukaryotic cell line (Thimiri Govinda Raj et al., 2011, 2012, 2016; Tharkeshwar et al., 2014). Here, we show a step by step protocol for SPMNP based plasma membrane fractionation. Our research thus could be used as an alternative method to isolate plasma membrane fraction for cell biology, system biology and immunology research.

Adenovirus-mediated human aquaporin-1 expression in hepatocytes improves lipopolysaccharide-induced cholestasis.

Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.

Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2•- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2•- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2•- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings

The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

Anti-alpha-actinin antibodies are part of the anti-cell membrane antibody spectrum that characterize patients with lupus nephritis

Anti-membrane autoantibodies (MbA) have been reported in sera from patients with lupus nephritis (LN) but the targets of the MbA remain to be explored, which is the aim of the current study. Sera were collected from 40 patients with LN determined by renal biopsy, and from 30 systemic lupus erythematosus (SLE) patients without clinical evidence of LN. Thirty autoimmune disease control patients (rheumatoid arthritis, Sjögren's syndrome and systemic sclerosis), and 30 healthy controls were also included. Using flow cytometry, the presence of anti-MbA was explored revealing that IgG anti-MbA positivity was associated with LN (62.5% vs 13.3%) when compared to non-LN SLE patients, autoimmune disease patients (6.7%) and healthy controls (0%). Next, using purified plasma membrane fractions from human embryonic kidney (HEK) cells, the more prominent targets and their occurrence rates were located at 50 kDa, 60/65 kDa, 90 kDa, 110 kDa, 180 kDa and 220 kDa. Alpha-actinin (110 kDa) autoAb was characterized as a major target in LN patients positive for anti-MbA, and anti-MbA binding activity was reduced (36.9 ± 13.7%) in the presence of α-actinin. Laminin (200 kDa) was also characterized as a minor target, which was not the case for annexin A2 (36 kDa). Finally, anti-MbA IgG subclass analysis indicated a predominance of IgG2. In conclusion, IgG anti-MbA were detected at high levels in LN patients supporting a primary pathogenic role for anti-MbA and anti-MbA/α-actinin+ in LN that needs further research.

Acid gradient across plasma membrane can drive phosphate bond synthesis in cancer cells: acidic tumor milieu as a potential energy source.

Aggressive cancers exhibit an efficient conversion of high amounts of glucose to lactate accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton-gradient (acid gradient) across the plasma membrane that represents proton-motive energy. Increasing experimental data from physiological relevant models suggest that acid gradient stimulates tumor proliferation, and can also support its energy needs. However, direct biochemical evidence linking extracellular acid gradient to generation of intracellular ATP are missing. In this work, we demonstrate that cancer cells can synthesize significant amounts of phosphate-bonds from phosphate in response to acid gradient across plasma membrane. The noted phenomenon exists in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer. Biochemical assays using viable cancer cells, and purified plasma membrane vesicles utilizing radioactive phosphate, confirmed phosphate-bond synthesis from free phosphate (Pi), and also localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from Pi was also observed when plasma membrane vesicles from cancer cells were subjected to trans-membrane acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles. Acid gradient created through glucose metabolism by cancer cells, as observed in tumors, also proved critical for phosphate-bond synthesis. In brief, these observations reveal a role of acidic tumor milieu as a potential energy source and may offer a novel therapeutic target.

Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

The Sigma1 Receptor Competes with STIM1 to Bind Orai1 to Regulate Store Operated Calcium Entry (SOCE)

The Sigma1 receptor is an ER chaperone protein targeted to mitochondrial associated ER membrane regions (MAMs). An increase in its expression has been observed in a variety of cancer cells and it exerts an anti-apoptotic action, promoting cell proliferation and inhibiting the apoptotic response to tumor suppressor genes such as p53 and Bax. Although its mechanism of action remains unclear, there is evidence to suggest that Sigma1R regulates inter-organelle Ca2+ signaling thereby controlling cellular bioenergetics, and is also a regulator of plasma membrane ion channels. We have investigated its role in the regulation of store operated Ca2+ entry (SOCE) triggered by ER Ca2+ store depletion. Increased expression of Sigma1R in HEK293 cells profoundly inhibited SOCE, whereas knockdown of endogenous Sigma1R in CHO cells increased SOCE. An agonist at Sigma1R, SKF10047, further inhibited SOCE whereas the antagonists, BD1047, relieved this inhibition.Co-immunoprecipitation experiments showed a novel interaction between Sigma1R and STIM1 which is ligand dependent, SKF10047 promoted the interaction and BD1047 reduced it. Total internal reflection fluorescence (TIRF) microscopy showed the recruitment of Sigma1R to ER-plasma membrane junctions was STIM1 dependent, whereas elevated Sigma1R expression slowed STIM1 recruitment. Plasma membrane biotinylation experiments also showed that STIM1 promoted Sigma1R recruitment to a surface complex while Sigma1R reduced the levels of STIM1 in the complex. Increased expression of STIM1 but not Orai1 partially rescued the inhibition of SOCE in cells over-expressing Sigma1R. Imaging of purified plasma membrane Orai1 channels complexes by atomic force microscopy (AFM) showed hexameric Orai1 decorated by both Sigma1R and STIM1. Overall our results suggest that Sigma1R inhibits SOCE in two ways; first, by binding STIM1 and reducing its recruitment to surface Orai1 channels and second, by directly modulating the STIM1-Orai1 complex.

Abstract B34: Signaling in the tumor microenvironment: Proteomics analyses of stromal-tumor interaction in oral cancers

Abstract Paracrine signaling between cancer-associated fibroblasts (CAFs) and cancer cells creates a bidirectional and cooperative network that drives cancer growth and progression. During the development of oral squamous cell carcinoma (OSCC), alterations in the tumor microenvironment and secretion of soluble proteins from subpopulations of CAFs generate a niche that has functional implications on tumor progression. To investigate stromal heterogeneity in OSCC and identify fibroblast-associated proteins that actively contribute to oral cancer carcinogenesis, we employed a proteomics approach to uncover the secretome of patient-derived oral CAFs. Communication in the tumor microenvironment can be mediated by classically secreted molecules, as well as by the release of extracellular vesicles, which are involved in the transfer of oncogenic factors to recipient cells. Hence, we comprehensively characterized the protein content of fibroblasts-derived conditioned media (CM) and exosomes (Exo), to identify secreted, cytoplasmic or membrane-associated molecules that could function as regulators of OSCC progression. Furthermore, to investigate how secreted signals from the surrounding stroma interact with target surface receptors, we also generated a comprehensive proteomic database of highly purified plasma membrane proteins isolated from two established tongue cancer cell lines (SCC4 and SCC25). The aim of the current study is to investigate the molecular crosstalk between CAF-derived factors and epithelial oral cancer cells to improve our understanding of the complex tumor-stroma interactions. Matched pairs of human primary fibroblasts were isolated from resected tongue cancers (CAFs) and tumor adjacent tissue (AFs), characterized according to morphology, expression of myofibroblast markers (α-SMA, tropomyosin), and the ability to degrade collagen. CM and Exo were collected after 48 hours of serum deprivation and Exo were purified by differential ultracentrifugation. Plasma membrane molecules from SCCs were isolated using colloidal silica-beads followed by density gradient ultracentrifugation. Each sample was analyzed by nano-flow ultra-performance liquid chromatography (UHPLC) coupled to a Q-Exactive tandem mass spectrometer. Our proteomic analyses quantified a total of 6638 proteins, 2855 in the CAFs/NAFs secretome (CM and Exo) and 5754 in the SCCs lines (membrane depleted (MD) and plasma membrane (PM) fractions) using the MaxQuant pipeline. CM was highly enriched in fibroblast-secreted proteins such as MMPs, VIM, IGFBPs, SPARC and Gene Ontology (GO) terms related to cytoplasmic and extracellular components. The quality of the Exo purification was confirmed by the presence of known markers such as CD81, CD63, TSG101 and FLOT1 and GO enrichment for endosomal and cytoplasmic vesicle-related terms. We used a subtractive, quantitative proteomics approach to highlight a CAF-enriched exosomal cluster consisting of 255 proteins groups differentially expressed, compared to patient matched AFs. Comparative Reactome pathway analysis revealed that this cluster is significantly enriched in metabolic enzymes involved in glycolysis and catabolic processes, as well as membrane-bound signaling receptors and proteins involved in transport or vesicles trafficking. Our proteomic analyses provide a detailed overview of signaling factors, receptors and intracellular proteins, associated with the induction of a pro-invasive stroma. These findings highlight differential expression of key signal transduction molecules that have been previously associated with cancer, albeit their precise roles in cancer progression need to be further validated. Our CAF-enriched secretome signature, complemented with the plasma membrane proteomics of tongue cancer cells represent a comprehensive data resource to investigate molecular signaling mechanisms within the tumor microenvironment. Citation Format: Simona Principe, Vladimir Ignatchenko, Alexander Ignatchenko, Ankit Sinha, Keira Pereira, Laurie Ailles, Thomas Kislinger. Signaling in the tumor microenvironment: Proteomics analyses of stromal-tumor interaction in oral cancers. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B34. doi:10.1158/1538-7445.CHTME14-B34