Total Membrane Protein Research Articles

The Calcium-Sensing Receptor-Dependent Regulation of Cell–Cell Adhesion and Keratinocyte Differentiation Requires Rho and Filamin A

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Fragile X Mental Retardation Protein Regulates Protein Expression and mRNA Translation of the Potassium Channel Kv4.2

A prominent characteristic of the inherited intellectual impairment disease fragile X syndrome (FXS) is neuronal hyperexcitability, resulting in a variety of symptoms, such as hyperactivity, increased sensitivity to sensory stimuli, and a high incidence of epileptic seizures. These symptoms account for a significant part of the disease pattern, but the underlying molecular mechanisms of neuronal hyperexcitability in FXS remain poorly understood. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), which regulates synaptic protein synthesis and is a key player to limit signaling pathways downstream of metabotropic glutamate receptors 1/5 (mGlu1/5). Recent findings suggest that FMRP might also directly regulate voltage-gated potassium channels. Here, we show that total and plasma membrane protein levels of Kv4.2, the major potassium channel regulating hippocampal neuronal excitability, are reduced in the brain of an FXS mouse model. Antagonizing mGlu5 activity with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) partially rescues reduced surface Kv4.2 levels in Fmr1 knock-out (KO) mice, suggesting that excess mGlu1/5 signal activity contributes to Kv4.2 dysregulation. As an additional mechanism, we show that FMRP is a positive regulator of Kv4.2 mRNA translation and protein expression and associates with Kv4.2 mRNA in vivo and in vitro. Our results suggest that absence of FMRP-mediated positive control of Kv4.2 mRNA translation, protein expression, and plasma membrane levels might contribute to excess neuronal excitability in Fmr1 KO mice, and thus imply a potential mechanism underlying FXS-associated epilepsy.

Phenotypical and genetic characterization of Yersinia ruckeri strains isolated from recent outbreaks in farmed rainbow trout Oncorhynchus mykiss (Walbaum) in Peru

A total of 30 strains of Yersinia ruckeri causing recent outbreaks in Peruvian trout culture systems, were studied by means of biochemical characteristics, serology, lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, and ERIC and REP PCR fingerprinting. All the Peruvian isolates were found to be fermentative, oxidase negative and positive for decarboxylation of lysine and ornithine and utilization of glucose and mannitol, allowing their presumptive identification as Y. ruckeri. Sequencing of the 16S rRNA gene confirmed that isolates were indeed Y. ruckeri (> 99.98% identity). Although most of the strains studied were motile and lipase positive corresponding to the biotype 1 of Y. ruckeri, 5 of these strains were negative from both tests, being identified as biotype 2. In addition, drug susceptibility tests determined high sensitivity to sulfamethoxazole/trimethoprim, oxytetracycline, ampicillin and enrofloxacin in all the isolates. Serologically, all the Peruvian strains studied were identified as belonging to the serotype O1 subgroup a. Analysis of the lipopolysaccharide (LPS) as well as total and outer membrane proteins (OMPs) profiles and the correspondent inmunoblotting, supported these results. Genotyping performed by means of ERIC- and REP-PCR determined major correlation of the Peruvian isolates with the type strain NCIMB 2194 T regardless of the biotype.

Drosophila Photoreceptor Cells Exploited for the Production of Eukaryotic Membrane Proteins: Receptors, Transporters and Channels

BackgroundMembrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies.Principal FindingsWe describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies.SignificanceWe demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.

Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity

The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance. We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates. We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile_Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant. OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.

Cigarette smoke induces oxidative stress and apoptosis in normal term fetal membranes

Cigarette smoking and bacterial infections are two major risk factors associated with preterm prelabor rupture of membranes (pPROM). We hypothesized that exposure of fetal membranes to cigarette smoke extracts might induce oxidative stress (OS) and fetal membrane apoptosis, culminating in an alternate pathway to that commonly activated by infection. To test this, we characterized the production of prostanoids and biomarkers of apoptosis in normal term human fetal membrane explant cultures. Fetal membrane explants collected at term (from cesarean deliveries, not in labor) were stimulated with cigarette smoke extract (CSE) for 24 h. Two classes of prostanoids, F2-Isoprostane (F2-IsoP), a marker of OS and PGF2α, a classical uterotonin, were measured by gas chromatography/mass spectrometry. Western blot analyses of tissue lysates were performed to quantify the anti-apoptotic protein Bcl2 and actin (as a control). Fetal membrane apoptosis was detected by immunohistochemistry for active caspase 3 and confirmed by TUNEL staining for nuclear fragmentation. CSE exposure resulted in significantly more F2-IsoP production from fetal membranes (242.8 ± 79.3 pg/ml/mg of total membrane protein) compared to unstimulated controls (131.5 ± 53.1 pg/ml/mg; p < 0.0001). By contrast, PGF2α was not different in CSE vs. controls (1083 ± 527 vs. 1136 ± 835 pg/ml/mg of protein; p = 0.80). CSE-exposed tissues demonstrated a dose-dependent decrease in Bcl2 expression and increases in active caspase 3 and nuclear fragmentation in both amnion and chorion cells compared to controls. In summary, fetal membranes exposed to CSE manifest evidence of OS and apoptosis. The differential pattern of prostanoid production observed in this study supports the hypothesis that an alternate non-inflammatory pathway mediated by OS and apoptosis in pPROM may promote proteolysis resulting in membrane weakening and rupture.

Quantitative immunofluorescent blotting of the Multidrug Resistance-associated Protein 2 (MRP2)

Introduction Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. Purpose The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-Binding Cassette Transporter Isoform C2/Multidrug Resistance-associated Protein 2 (ABCC2/MRP2). Methods Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotted with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. Results The limits of quantitation for the time- insensitive technique described here were from 0.001 μg to 0.5 μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r 2 values were 0.986 ± 0.012. The utility and sensitivity of this technique were demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. Discussion The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.

Oxygen-linked modulation of erythrocyte metabolism: state of the art.

About fifteen years ago a number of experimental data on the specific interaction of haemoglobin (Hb) and various enzymes of the glycolytic pathway with the cytoplasmatic domain of band 3 protein emerged13. These data induced our research group to formulate the hypothesis of a modulation of the erythrocyte (RBC) metabolism driven by the free energy connected to the R to T Hb transition4. The general scheme of this simple model is reported in figure 1. In this model band 3 protein plays a pivotal role: when the erythrocyte is at high oxygenation state (HOS) band 3 interacts with some glycolytic enzymes (inhibiting their activity) and more glucose is addressed towards the pentose phosphate pathway (PPP). When the erythrocyte is at low oxygenation state (LOS) the high affinity of deoxy-Hb for the N-terminal cytoplasmatic domain of band 3 induces the release of glycolytic enzymes, and thus glucose flux towards the glycolytic pathway increases. Some recent results obtained by our and other groups are in agreement with this simple model, even though they indicate, that other factors might play a significant role in this erythrocyte modulation. The aim of this brief review is to offer to the reader a critical examination of the significance of these recent results in light of the model framework. Figure 1 Simplified scheme representing the modulation of erythrocyte metabolism by the O2 transition of Hb and its competition with glycolytic enzymes (mainly phosphofructokinase, PFK) for the cytoplasmic domain of band 3 (CDB3). Band 3 protein Band 3 is a transmembrane protein that accounts for about 25% of the total RBC membrane proteins. It is characterized by three distinct functional domains: (1) the membrane spanning domain, which transverses 12 times the bilayer and serves to catalyze the exchange of anions (mainly Cl− and HCO3−) across the membrane5; (2) the short C-terminal cytoplasmatic domain that binds carbonic anhydrase II6,7, and (3) the N-terminal cytoplasmatic domain (CDB3) that binds a variety of proteins8 and anchors the RBC membrane to the underlying cytoskeleton via ankyrin and protein 4.29.

Catalytic Reactions of the Homogentisate Prenyl Transferase Involved in Plastoquinone-9 Biosynthesis

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10-20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K(m) value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.

Lipid raft-dependent endocytosis: a new route for hepcidin-mediated regulation of ferroportin in macrophages

Expression of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and is decreased by hepcidin. We previously showed that ferroportin is present in specific cell surface domains suggestive of lipid rafts. Herein, we have clarified the localization of ferroportin in macrophage membranes and tested whether raft-mediated endocytosis plays a role in hepcidin activity. Raft/detergent-resistant membranes from murine bone marrow-derived macrophages and J774a1 cells were analyzed by Western blotting. The effect of lipid raft- or clathrin-dependent endocytosis inhibitors was studied on hepcidin activity. For this purpose, after treatment, ferroportin expression was analyzed by fluorescence microscopy, Western blotting of total protein extracts or plasma membrane protein samples, and by quantitative immunofluorescence assay (In-Cell-Western). Macrophage ferroportin was mostly detected in detergent-resistant membranes containing raft markers (caveolin 1, flotillin 1). Interestingly, iron overload strongly increased the presence of ferroportin in the lightest raft fraction. Moreover, lipid raft breakdown by cholesterol sequestration (filipin) or depletion (methyl-beta-cyclodextrin) decreased hepcidin activity on macrophage ferroportin. Cell surface biotinylation and immunofluorescence studies indicated that the process of both hepcidin mediated endocytosis and degradation of ferroportin were affected. By contrast, the inhibition of clathrin dependent endocytosis did not interfere with hepcidin effect. Macrophage ferroportin is present in lipid rafts which contribute to hepcidin activity. These observations reveal the existence of a new cellular pathway in hepcidin mediated degradation of ferroportin and open a new area of investigation in mammalian iron homeostasis.

Adenosine A1 receptor activation reduces opening of mitochondrial permeability transition pores in hypoxic cardiomyocytes

1. Adenosine A(1) receptors (A(1)R) play an important role in cardioprotection against hypoxic damage and the opening of mitochondrial permeability transition pores (MPTP) is central to the regulation of cell apoptosis and necrosis. However, it is still unclear whether A(1)R open MPTP in hypoxic cardiomyocytes. 2. The present study used primary cardiomyocyte cultures from neonatal rats to investigate the mechanisms of A(1)R activation and the effects of A(1)R on MPTP opening under hypoxic conditions. 3. Hypoxia increased both MPTP opening and the production of reactive oxygen species (ROS), while decreasing cell viability and mitochondrial membrane potential (Deltapsi). The A(1)R agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 500 nmol/L) blocked the increase in MPTP opening and ROS production and maintained cell viability and Deltapsi under hypoxic conditions. 4. The protective effects of CCPA were eliminated by both the protein kinase C (PKC) inhibitor chelerythine (2 micromol/L) and the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) inhibitor 5-hydroxydecanoate (500 micromol/L). Moreover, CCPA significantly increased the PKC content in both total protein and membrane protein of cardiomyocytes. 5-Hydroxydecanoate did not prevent these CCPA-induced increases in PKC. 5. These results demonstrate that CCPA reduces MPTP opening in hypoxic cardiomyocytes, possibly by activating PKC, stabilizing Deltapsi and reducing ROS production following the opening of mitoK(ATP). Consequently, fewer MPTP open.

Erratum to: Proteome Analysis of Aspergillus fumigatus Total Membrane Proteins Identifies Proteins Associated with the Glycoconjugates and Cell Wall Biosynthesis Using 2D LC-MS/MS

Erratum to: Proteome Analysis of Aspergillus fumigatus Total Membrane Proteins Identifies Proteins Associated with the Glycoconjugates and Cell Wall Biosynthesis Using 2D LC-MS/MS

Abstract C32: Proteome analysis of pancreatic cancer tissue for identification of novel biomarkers

Abstract Pancreatic cancer is the 4th largest cancer in US with mortality rate almost equal to incidence. The tumor usually presents at a late stage ruling out any curative surgery. Focus of pancreatic cancer research currently is mainly on early diagnosis and development of novel cancer therapeutics. Identifying sensitive, specific and reliable surrogate markers by genomic and proteomic methods1 in cancer tissue, blood or other bodily secretions can prove promising to reach this goal. We are applying standard proteomic techniques like gel electrophoresis and MS (mass spectrometry) with a goal to identify potential biomarkers and new drug targets for the design and development of effective therapeutics in serum and pancreatic tissue samples. Pancreatic cytosolic and membrane proteins were extracted from the 4 pairs of cancerous and adjacent normal tissue. Cytosolic protein fractions were run on PF-2D, traditional 1D and 2D SDS-PAGE. LC/MS/MS was used to identify differently expressed banding patterns. Multiple proteins were found to be either up (24) or downregulated (11). Compared to cytosolic proteins membrane proteins are priced for its accessibility on tumor tissue making these ideal for diagnosis and therapeutics. Isolating only plasma membrane proteins is very challenging due to low extraction efficiency, extremely low abundancy and poor solubility. Total cellular membrane protein was extracted and analyzed by SDS-PAGE and LC/MS/MS, and significant banding pattern differences were observed with 35 upregulated and 14 downregulated proteins with annexin 1, annexin A2 and annexin 5, tumor rejection antigen 1, prolyl 4-hydroxylase, calreticulin precursor, heat shock 70kDa protein 5, chaperonin and alpha 2 actin being the most significantly overexpressed. Findings were confirmed by probing western blots with anti-annexin 1 and 2 antibodies. Our findings suggest these differently expressed proteins can be explored as potential biomarkers after further validation. In contrast, analysis of serum samples by same techniques was futile due to exceedingly low abundance of protein and peptide fraction containing potential biomarkers due to masking by albumin and globulins as well as enzymatic degradation. At present we are continuing the validation of our preliminary findings on larger sample size by using differential protein profiling technologies, immunohistochemistry and quantitative proteomics. Also we are planning to use affinity bait molecule with nanoparticles2 which can help overcome the limitations faced with serum samples in identification of de novo biomarkers. Citation Information: Cancer Res 2009;69(23 Suppl):C32.

Proteome Analysis of Aspergillus fumigatus Total Membrane Proteins Identifies Proteins Associated with the Glycoconjugates and Cell Wall Biosynthesis Using 2D LC-MS/MS

We attempted to identify membrane proteins associated with the glycoconjugates and cell wall biosynthesis in the total membrane preparations of Aspergillus fumigatus. The total membrane preparations were first run on 1D gels, and then the stained gels were cut and submitted to in-gel digestion followed by 2D LC-MS/MS and database search. A total of 530 proteins were identified with at least two peptides detected with MS/MS spectra. Seventeen integral membrane proteins were involved in N-, O-glycosylation or GPI anchor biosynthesis. Nine membrane proteins were involved in cell wall biosynthesis. Eight proteins were identified as enzymes involved in sphingolipid synthesis. In addition, the proteins involved in cell wall and ergosterol biosynthesis can potentially be used as antifungal drug targets. Our method, for the first time, clearly provided a global view of the membrane proteins associated with glycoconjugates and cell wall biosynthesis in the total membrane proteome of A. fumigatus.

Systematic cyanobacterial membrane proteome analysis by combining acid hydrolysis and digestive enzymes with nano-liquid chromatography–Fourier transform mass spectrometry

The identification of membrane proteins is currently under-represented since the trans-membrane domains of membrane proteins have a hydrophobic property. Membrane proteins have mainly been analyzed by cleaving and identifying exposed hydrophilic domains. We developed the membrane proteomics method for targeting integral membrane proteins by the following sequential process: in-solution acid hydrolysis, reverse phase chromatographic separation, trypsin or chymotrypsin digestion and nano-liquid chromatography–Fourier transform mass spectrometry. When we employed total membrane proteins of Synechocystis sp. PCC 6803, 155 integral membrane proteins out of a predictable 706 were identified in a single application, corresponding to 22% of a genome. The combined methods of acid hydrolysis-trypsin (AT) and acid hydrolysis-chymotrypsin (AC) identified both hydrophilic and hydrophobic domains of integral membrane proteins, respectively. The systematic approach revealed a more concrete data in mapping the repertoire of cyanobacterial membrane and membrane-linked proteome.

Expression and function of VAP-33 in murine dendritic cell sarcoma

To elucidate the expression and function of VAP-33 gene in dendritic cell sarcoma (DCS) cell line. The expression of VAP-33 in DCS cells was investigated by mass spectrum with immunoprecipitation membrane protein. DCS cells were treated with antigens in different dosages (150, 850, and 1500 microl) for 24, 48 and 72 h respectively. Cell morphology and phagocytosis activity of DCS cells were measured. Indirect immunofluorescence, confocal microscopy and Western blotting were used to study the distribution and expression changes of VAP-33. Moreover, DCS cells were treated with 0.5 mol/L insulin for 20 min first and followed by Western blotting to detect changes of VAP-33 and glucose transfer protein 4 (GLUT-4) in the total cellular protein, cytoplasmic protein and membrane protein. Confocal microscopy was used to document the expression and distribution changes of VAP-33 and GLUT-4 in DCS cells. VAP-33 expression was obtained at the cell membrane and in the cytoplasm of DCS cells. Upon antigen stimulation, DCS cells showed more active phagocytosis and morphologically became more elongated with branched protrusions. The expression of VAP-33 was decreased by the antigen stimulation. Upon the insulin stimulation, the expression of VAP-33 and GLUT-4 were increased and co-localized. VAP-33 expression in DCS originated from the dendritic cells (DC) seemed relating to the vesicle transportation during antigen processing in DC. Additionally, VAP-33 and GLUT-4 also take part in the glucose transportation in the cells.

Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt

Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.

AT1a receptor knockout in mice impairs urine concentration by reducing basal vasopressin levels and its receptor signaling proteins in the inner medulla

Angiotensin II plays an important role in the regulation of blood pressure, body salt and fluid balance, and urine concentration. Mice with deletion of the AT(1a) receptor develop polyuria and urine concentration defects. We studied the mechanisms of these urine concentration defects by treating wild-type and AT(1a)-knockout mice with arginine vasopressin (AVP) for 2 weeks, controlling their water intake, or giving them an osmotic diuretic (sucrose) in order to determine whether central or nephrogenic mechanisms were involved. Under basal conditions, AT(1a)-knockout mice were hypotensive, had lower plasma AVP, and excreted more urine with a markedly reduced osmolality compared with wild-type mice. However, basal glomerular filtration rates were similar in both strains of mice. We isolated total lysate and membrane proteins from the inner medulla of wild-type and mutant mouse kidneys, and found that the amounts of aquaporin 2 (AQP2), adenylyl cyclases III and V/VI, and phosphorylated MAP kinases ERK 1/2 proteins were all reduced in the inner medulla of the knockout mice. Infusion of AVP raised plasma levels and blood pressure proportionally in both strains, but polyuria persisted and urine osmolality remained significantly lower in the knockout mice. Although AVP increased urine osmolality slightly in water-deprived knockout mice, this was well below the basal osmolality of wild-type mice. The diuretic response to the hyperosmotic sucrose was also impaired in the knockout mice. Neither AVP nor water rationing restored the levels of the inner medullary signaling proteins and membrane AQP2 proteins in the knockout mice. We suggest that AT(1a) receptor deletion causes polyuria and urine concentration defects by decreasing basal AVP release and impairing AVP-induced receptor signaling in the inner medulla.

The deletion of Ffh in Escherichia coli induces adverse physiological and morphological changes

The Escherichia coli Ffh protein is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) that is involved in targeting and translocation of membrane proteins. The functions of Ffh in E. coli were investigated using the mutant with the Ffh deficiency. The mutant showed lower growth rate at 30°C and rapidly lost viability at the non-permissive temperature of 42°C. In addition, the amount of the total membrane proteins decreased sharply in the mutant. The mutant cells cultured at either 30 or 42°C appeared to have an elongated shape as compared to the wild type cells. Transmission electron microscopy revealed that the membrane layer of the mutant cells was thinner than that of the wild type cells.