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Membrane Proteins (membrane + protein)
Kinds of Membrane Proteins Terms modified by Membrane Proteins Selected AbstractsIsolation and Characterization of a Porin-Like Outer Membrane Protein from Xanthomonas campestris pv. campestrisIUBMB LIFE, Issue 1 2002Lingyun Wang Abstract Xanthomonas campestris pv. campestris, a plant-associated pathogenic bacterium, is the causal agent of foliar spots and blights in crucifers. The major outer membrane protein, Omp37, of 37 kDa, has been identified, purified to homogeneity, and its characterization has also been carried out. Native Omp37 behaved as a trimer, as revealed by gel filtration and SDS-PAGE. FTIR measurements revealed a high ,-structure content. The pore-forming ability of the purified Omp37 was studied by the liposome swelling assay. Omp37, to our knowledge, is the first porin that has been isolated from Xanthomonas . This study clearly demonstrates that Omp37 is related to the family of trimeric bacterial porins. [source] Overexpression and Characterization of the Rhodobacter sphaeroides PufX Membrane Protein in Escherichia coli,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2007Shiho Onodera Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodobacter sphaeroides was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had ,-helix contents of 29% in organic solvents and 22,26% in 0.8,2.0% (w/v) n -octyl ,- d -glucopyranoside solutions, suggesting that the PufX contains a substantial ,-helical region composed of 18,22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 ,- and ,-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional PufX membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system. [source] Epstein-Barr Virus (EBV) Latent Membrane Protein 1 Induces Interleukin-8 through the Nuclear Factor-,B Signaling Pathway in EBV-Infected Nasopharyngeal Carcinoma Cell LineTHE LARYNGOSCOPE, Issue 5 2004Qingchun Ren MD Abstract Background/Objectives: Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor and is associated with Epstein-Barr virus (EBV) infection that exhibits type II latency. Angiogenesis is essential for tumor growth, invasion, and metastasis. Our previous studies have indicated that interleukin (IL)-8 was over-expressed in many NPC tissues and was found to be significantly correlated with angiogenesis by immunohistochemistry. Study Design: In vitro design. Methods: The influence of the EBV genome for IL-8 gene expression was studied using the EBV,genome-positive and -negative epithelial/NPC hybrid cell line NPC-KT. The EBV-positive and -negative clones were selected by polymerase chain reaction and in situ hybridization. Results: EBV-positive clones expressed abundant IL-8 mRNA compared with EBV-negative clones. This result indicated that over-expression of IL-8 depended on the presence of EBV genomes in NPC-KT cells. Two encoded genes, latent membrane protein (LMP)1 and EBV-encoded small RNAs (EBERs), expressed in NPC were transfected in EBV-negative NPC-KT cells. LMP1 transactivated the IL-8 promoter, whereas EBERs did not. Moreover, the nuclear factor (NF)- ,B binding site in the IL-8 promoter was essential for the response to LMP1, and the activator protein (AP)-1 binding site played only a partial role. Conclusions: LMP1 induces IL-8 mainly through the activation of NF-,B and partly through AP-1 in NPC model cell lines, NPC-KT, and this suggests that LMP1 plays an important role in the angiogenesis of NPC. [source] How Does a Membrane Protein Achieve a Vectorial Proton Transfer Via Water Molecules?CHEMPHYSCHEM, Issue 18 2008Steffen Wolf Abstract We present a detailed mechanism for the proton transfer from a protein-bound protonated water cluster to the bulk water directed by protein side chains in the membrane protein bacteriorhodopsin. We use a combined approach of time-resolved Fourier transform infrared spectroscopy, molecular dynamics simulations, and X-ray structure analysis to elucidate the functional role of a hydrogen bond between Ser193 and Glu204. These two residues seal the internal protonated water cluster from the bulk water and the protein surface. During the photocycle of bacteriorhodopsin, a transient protonation of Glu204 leads to a breaking of this hydrogen bond. This breaking opens the gate to the extracellular bulk water, leading to a subsequent proton release from the protonated water cluster. We show in detail how the protein achieves vectorial proton transfer via protonated water clusters in contrast to random proton transfer in liquid water. [source] In Vitro Selection of Self-Interacting Transmembrane Segments--Membrane Proteins Approached from a Different PerspectiveIUBMB LIFE, Issue 3 2002Dieter Langosch Abstract The principles underlying the folding of integral membrane proteins are uncovered in an increasingly detailed way. Experimental determination of high-resolution structures followed by analysis of packing reveal structural similarities as well as differences to soluble globular proteins. At the same time, protein/protein interactions at the level of membrane-embedded domains have been investigated for different model proteins. More recently, self-interacting transmembrane helices have been selected from combinatorial libraries in vitro to study the mechanistic basis of protein/protein interaction in membranes in a systematic way. With an emphasis on the latter approach, this review discusses insights emerging from an integrated view on the recent advances. [source] LIV-1 Breast Cancer Protein Belongs to New Family of Histidine-Rich Membrane Proteins with Potential to Control Intracellular Zn2+ HomeostasisIUBMB LIFE, Issue 4 2000K. M. Taylor Abstract Investigation of the protein product of the oestrogen-regulated gene LIV-1, implicated in metastatic breast cancer, has revealed 10 protein sequences of unknown function that belong to a new family with potential to control intracellular Zn2+ homeostasis. Sequence alignment highlights the similarity in transmembrane domains and extramembrane charged residues, indicating potential ion-transport ability. This family has a novel highly conserved motif of 66 residues, including a transmembrane domain and a catalytic zinc-binding sequence of zinc metalloproteases, containing conserved (indicated in bold type) proline and glutamine residues, HEXPHEXGD. These proteins contain more plentiful histidine-rich repeats than zinc transporters, suggesting an ability to bind or transport zinc across membranes. I propose that these 11 proteins form a new family with the potential to control intracellular Zn2+ homeostasis. [source] Enhanced solubilization of membrane proteins by alkylamines and polyaminesPROTEIN SCIENCE, Issue 3 2010Kazutoshi Yasui Abstract Around 25% of proteins in living organisms are membrane proteins that perform many critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins are extracted from the lipid bilayer and solubilized with a detergent for biochemical characterization; however, their solubilization is an empirical technique and sometimes insufficient quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid synthase localized at the plant Golgi membrane was enhanced by up to 9.9-fold upon addition of spermidine to the solubilization buffer. These additives also enhanced the solubilization of other plant membrane proteins localized in other organelles such as the endoplasmic reticulum and plasma membrane as well as that of an animal Golgi-localized membrane protein. Thus, addition of alkylamines and polyamines to solubilization buffer is a generally applicable method for effective solubilization of membrane proteins. The mechanism of the enhancement of solubilization is discussed. [source] Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source , a membrane proteome-centric viewPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2009Ute Haußmann Abstract The ability of microorganisms to assimilate aromatic substances as alternative carbon sources is the basis of biodegradation of natural as well as industrial aromatic compounds. In this study, Corynebacterium glutamicum was grown on benzoate as sole carbon and energy source. To extend the scarce knowledge about physiological adaptation processes occurring in this cell compartment, the membrane proteome was investigated under quantitative and qualitative aspects by applying shotgun proteomics to reach a comprehensive survey. Membrane proteins were relatively quantified using an internal standard metabolically labeled with 15N. Altogether, 40 proteins were found to change their abundance during growth on benzoate in comparison to glucose. A global adaptation was observed in the membrane of benzoate-grown cells, characterized by increased abundance of proteins of the respiratory chain, by a starvation response, and by changes in sulfur metabolism involving the regulator McbR. Additional to the relative quantification, stable isotope-labeled synthetic peptides were used for the absolute quantification of the two benzoate transporters of C. glutamicum, BenK and BenE. It was found that both transporters were expressed during growth on benzoate, suggesting that both contribute substantially to benzoate uptake. [source] Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2009Atsushi Intoh Abstract Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self-renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2-D PAGE-based analysis using fluorescently labeled proteins and shotgun-based analysis with isotope-labeled peptides identified 338 proteins, including transmembrane, membrane-binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro. [source] Membrane proteins and membrane proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2008Sandra Tan Abstract Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins. [source] Sub-cellular localization of membrane proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2008Pawel G. Sadowski Abstract In eukaryotes, numerous complex sub-cellular structures exist. The majority of these are delineated by membranes. Many proteins are trafficked to these in order to be able to carry out their correct physiological function. Assigning the sub-cellular location of a protein is of paramount importance to biologists in the elucidation of its role and in the refinement of knowledge of cellular processes by tracing certain activities to specific organelles. Membrane proteins are a key set of proteins as these form part of the boundary of the organelles and represent many important functions such as transporters, receptors, and trafficking. They are, however, some of the most challenging proteins to work with due to poor solubility, a wide concentration range within the cell and inaccessibility to many of the tools employed in proteomics studies. This review focuses on membrane proteins with particular emphasis on sub-cellular localization in terms of methodologies that can be used to determine the accurate location of membrane proteins to organelles. We also discuss what is known about the membrane protein cohorts of major organelles. [source] Novel interactors and a role for supervillin in early cytokinesis,CYTOSKELETON, Issue 6 2010Tara C. Smith Abstract Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle. © 2010 Wiley-Liss, Inc. [source] Dynamic compartmentalization of protein tyrosine phosphatase receptor Q at the proximal end of stereocilia: Implication of myosin VI-based transportCYTOSKELETON, Issue 7 2008Hirofumi Sakaguchi Abstract Hair cell stereocilia are apical membrane protrusions filled with uniformly polarized actin filament bundles. Protein tyrosine phosphatase receptor Q (PTPRQ), a membrane protein with extracellular fibronectin repeats has been shown to localize at the stereocilia base and the apical hair cell surface, and to be essential for stereocilia integrity. We analyzed the distribution of PTPRQ and a possible mechanism for its compartmentalization. Using immunofluorescence we demonstrate that PTPRQ is compartmentalized at the stereocilia base with a decaying gradient from base to apex. This distribution can be explained by a model of transport directed toward the stereocilia base, which counteracts diffusion of the molecules. By mathematical analysis, we show that this counter transport is consistent with the minus end-directed movement of myosin VI along the stereocilia actin filaments. Myosin VI is localized at the stereocilia base, and exogenously expressed myosin VI and PTPRQ colocalize in the perinuclear endosomes in COS-7 cells. In myosin VI-deficient mice, PTPRQ is distributed along the entire stereocilia. PTPRQ-deficient mice show a pattern of stereocilia disruption that is similar to that reported in myosin VI-deficient mice, where the predominant features are loss of tapered base, and fusion of adjacent stereocilia. Thin section and freeze-etching electron microscopy showed that localization of PTPRQ coincides with the presence of a dense cell surface coat. Our results suggest that PTPRQ and myosin VI form a complex that dynamically maintains the organization of the cell surface coat at the stereocilia base and helps maintain the structure of the overall stereocilia bundle. Cell Motil. Cytoskeleton 2008. Published 2008 Wiley-Liss, Inc. [source] Identification and characterization of Xenopus OMP25DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2004Masafumi Inui This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube. [source] Man1, an inner nuclear membrane protein, regulates left,right axis formation by controlling nodal signaling in a node-independent mannerDEVELOPMENTAL DYNAMICS, Issue 12 2008Akihiko Ishimura Abstract Man1, an inner nuclear membrane protein, regulates transforming growth factor , signaling by interacting with receptor-associated Smads. In Man1 -deficient (Man1,/,) embryos, vascular remodeling is perturbed by misregulation of Smad activity. Here, we show that Man1,/, embryos exhibit abnormal heart morphogenesis including the looping abnormality. We searched for the molecular basis underlying the heart abnormalities and found that the left side-specific genes responsible for left,right (LR) asymmetry, Nodal, Lefty2, and Pitx2, were expressed bilaterally in the lateral plate mesoderm and that their expression was enhanced significantly in mutants. Notably, Lefty1, a marker for the midline barrier, was maintained in Man1,/, mutants. Crossing Man1,/+ with Nodal hypomorphs (Nodalneo/+), in which Nodal signaling in the node is disrupted, to generate double homozygous embryos (Man1,/,; Nodalneo/neo) revealed that the bilateral Nodal was retained in Man1,/,; Nodalneo/neo embryos. These results suggest that Man1 regulates LR asymmetry by controlling Nodal signaling in a node-independent manner. Developmental Dynamics 237:3565,3576, 2008. © 2008 Wiley-Liss, Inc. [source] Complementary expression and heterophilic interactions between igLON family members neurotrimin and LAMPDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2002Orlando D. Gil Abstract Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 190,204, 2002 [source] Cytosolic protein-protein interactions that regulate the amyloid precursor proteinDRUG DEVELOPMENT RESEARCH, Issue 2 2002Shasta L. Sabo Abstract Alzheimer disease (AD), a progressive neurodegenerative disease, is the most common cause of dementia in the elderly and is among the leading causes of death in adults. AD is characterized by two major pathological hallmarks, amyloid plaques and neurofibrillary tangles. For a number of reasons, amyloid plaque accumulation is widely thought to be the probable cause of AD. The amyloid plaque core is largely composed of an approximately 4-kDa peptide referred to as A,. A, is derived from its precursor, the Alzheimer amyloid protein precursor (APP), by endoproteolytic processing. APP is a type I integral membrane protein, with a long extracellular domain, one transmembrane domain, and a short (,50 amino acid) cytoplasmic tail. Despite intense efforts to decipher the function of APP, its normal physiological role has remained elusive. The carboxy-terminus of APP contains the sequence YENPTY, which is absolutely conserved across APP homologues and across species. The YENPTY sequence is important for regulation of APP processing and trafficking. Given the importance of the cytoplasmic domain in APP physiology, a number of laboratories have hypothesized that proteins that bind to the YENPTY sequence in the cytoplasmic domain of APP might regulate APP processing, trafficking, and/or function. In this article, we will discuss data revealing which proteins bind to the cytoplasmic domain of APP, how these binding-proteins regulate APP metabolism and function, and why such protein-protein interactions provide an exciting new target for therapeutic intervention in AD. Drug Dev. Res. 56:228,241, 2002. © 2002 Wiley-Liss, Inc. [source] Facilitating the hyphenation of CIEF and MALDI-MS for two-dimensional separation of proteinsELECTROPHORESIS, Issue 15 2010Chang Cheng Abstract Both CIEF and MALDI-MS are frequently used in protein analysis, but hyphenation of the two has not been investigated proportionally. One of the major reasons is that the additives (such as carrier ampholytes and detergent) in CIEF severely suppress the MALDI-MS signal, which hampers the hyphenation of the two. In this paper, we develop a simple means to alleviate the above signal-suppressing effect. We first deposit 1,,L of water onto a MALDI-MS target, deliver a fraction of CIEF-separated protein (,0.1,,L) to the water droplet, evaporate the solvent, add 0.5,,L of MALDI matrix to the sample spot, dry the matrix and move the target plate to a MALDI-TOF-MS for mass spectrum measurement. We optimize the droplet volume and the laser-ablation region. Under the optimized conditions, we improve the S/N by two- to tenfold. We also apply this method for 2-D separations of standard proteins and apolipoprotein A,I, a membrane protein expressed in Escherichia coli cells. [source] Detection of carbonyl-modified proteins in interfibrillar rat mitochondria using N, -aminooxymethylcarbonylhydrazino- D -biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometryELECTROPHORESIS, Issue 6 2008Woon-Gye Chung Abstract There is now a large body of supporting data available that links oxidative modifications of proteins to a large number of diseases, degenerative disorders and aging. However, the detailed analysis of oxidative protein modifications remains challenging. Here, we report a new efficient method for identification of oxidatively modified proteins in complex biological samples which is based on the use of an aldehyde-reactive probe, N,-aminooxymethylcarbonylhydrazino- D -biotin (ARP), in combination with Western-type analyses and MS. The biotinylated hydroxylamine derivative forms a chemically stable oxime derivative with the aldehyde/keto group found in carbonyl-modified proteins. The biotin tag is detected by avidin affinity staining. ARP-positive proteins are subsequently subjected to in-gel trypsinization and MS/MS for protein identification. We demonstrate the usefulness of the method for the analysis of protein extracts obtained from interfibrillar heart mitochondria (IFM) from young and old rats. In this study, we identified as putative major protein targets of oxidative modifications the mitochondrial matrix protein, aconitase, the inner mitochondrial membrane protein, ADP/ATP translocase, and constituents of the electron transport chain complexes IV and V. An age-related increase of carbonyl levels was found for aconitase and ATP synthase. [source] Increased infiltration of Chlamydophila pneumoniae in the vessel wall of human veins after perfusionEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2008K. Kupreishvili ABSTRACT Background Several studies have suggested an association between Chlamydophila pneumoniae (Cp) infection and atherosclerosis. A recent study detected Cp DNA in the saphenous vein of 12% of all patients before bypass grafting and in 38% of failed grafts. We used a system in which human veins were perfused with autologous blood under arterial pressure. Materials and methods, Veins were surplus segments of saphenous veins of coronary artery bypass grafting (CABG) patients. Vein grafts were perfused with the blood of the same patient after CABG procedures. Veins were analysed for Cp -specific membrane protein using immunohistochemical and PCR analysis. Veins were analysed before and after perfusion (up to 4 h). The number of Cp positive cells was then quantified in the vein layers. Results Cp protein was detected within macrophages only. In non-perfused veins, Cp was present in the adventitia in 91% of all patients, in the circular (64%) and longitudinal (23%) layer of the media. No positivity was found in the intima. Perfusion subsequently resulted in a significant increase of Cp positive cells within the circular layer of the media that, however, differed strongly between different patients. Cp DNA was not detected by PCR in those specimens. Conclusion Cp protein was present in 91% of veins, but the number of positive cells differed remarkably between patients. Perfusion of veins resulted in increased infiltration of Cp into the circular layer. These results may point to a putative discriminating role of Cp with respect to graft failure between different patients. [source] Protein deficiency balance as a predictor of clinical outcome in hereditary spherocytosisEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2005S. Rocha Abstract:, Vertical and horizontal interactions between membrane constituents account for integrity, strength and deformability of the erythrocyte. Disruption of vertical interactions caused by membrane protein deficiencies in hereditary spherocytosis (HS), favor membrane vesiculation with development of spherocytic cells. Our aim was to evaluate the hematological and clinical presentation of HS according to the type and amount of protein deficiency. We studied 81 Portuguese individuals, 71 belonging to 21 families plus 10 unrelated subjects, and found that 51 of them were HS patients. Patients were classified as presenting mild, typical or severe HS, according to laboratory results and clinical follow-up. We performed screening tests and the standardized electrophoretic membrane protein analysis to identify and quantify protein deficiencies. We found band 3 and ankyrin deficiencies as the major causes for HS. The ratios between the value of the primary and/or secondary protein deficiencies showed significantly different values according to the severity of HS, and a significant inverse correlation with the severity of HS was observed. In mild HS, the ratios between protein deficiencies reflected equivalent protein deficiencies, while an unbalance was observed in typical HS, which was enhanced in severe HS. Our data suggest that the relative quantification of each major membrane protein and of the ratios between the values of protein deficiencies may be helpful in providing additional data about the clinical outcome of HS. [source] Deletion of the LIME adaptor protein minimally affects T and B cell development and functionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Claude Grégoire Abstract LIME (Lck-interacting membrane protein) is a transmembrane adaptor that associates with the Lck and Fyn protein tyrosine kinases and with the C-terminal Src kinase (Csk). To delineate the role of LIME in vivo, LIME-deficient mice were generated. Although Lime transcripts were expressed in immature and mature B and T cells, the absence of LIME impeded neither the development nor the function of B and T cells. TCR transgenic mice deprived of LIME showed, however, a 1.8-fold enhancement in positive selection. Since B cells and activated T cells express LIME and the related adaptor NTAL, mice lacking both adaptors were generated. Double-deficient mice showed no defect in the development and function of B and T cells, and the lack of LIME had no effect on the autoimmune syndrome that develops in aged NTAL-deficient mice. In contrast to a previous report, we further showed that this autoimmune syndrome develops in the absence of T cells. Therefore, our in vivo results refute all the previous roles postulated for LIME on the basis of studies of transformed B and T cells and demonstrate that LIME has no seminal role in the signaling cassette operated by antigen receptors and coreceptors. [source] Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Heather D. VanGuilder Abstract Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague,Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for ,-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes. [source] Membrane-associated guidance cues direct the innervation of forebrain and midbrain by dorsal raphe-derived serotonergic axonsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Audrey Petit Abstract Unlike many neurons that extend an axon precisely to a single target, individual dorsal raphe 5-HT neurons project to multiple brain regions and their axon terminals often lack classical synaptic specializations. It is not known how 5-HT axon collaterals select between multiple target fields, or even if 5-HT axons require specific guidance cues to innervate their targets. Nor is it known how these axon collaterals are restrained within specific innervation target regions. To investigate this, we challenged explants of dorsal raphe with co-explants, or cell membrane preparations of ventral midbrain, striatum or cerebral cortex. We provide evidence for membrane-associated cues that promote 5-HT axon growth into each of these three target regions. The axon growth-promoting activity was heat-, protease- and phosphatidylinositol-phospholipase-C (PI-PLC)-sensitive. Interestingly, 5-HT axons specifically lost the ability to grow in heterotypic explants, or membrane carpets, following contact with ventral midbrain or striatal, but not cortical, explants or membranes. This inductive activity associated with striatal and ventral midbrain membranes was sensitive to both high salt extraction and PI-PLC treatment. By contrast, the activity that inhibited 5-HT axon growth onto heterotypic membranes was sensitive only to high salt extraction. These results provide evidence that a glycosylphosphatidylinositol (GPI)-linked membrane protein promotes 5-HT axon growth, and that short-range membrane-bound, as well as GPI-linked, molecules contribute to the guidance of 5-HT axon collaterals. These findings suggest that 5-HT axon collaterals acquire a target-induced growth-inhibitory response to alternative targets, increasing their selectivity for the newly innervated field. [source] Translation of an integral membrane protein in distal dendrites of hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Jeffrey C. Grigston Abstract Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons. [source] Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formationFEBS JOURNAL, Issue 16 2010Yuki Moritani Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post-translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone-like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell-free-synthesized water-insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins. Structured digital abstract ,,MINT-7900670: Cx-43 (uniprotkb:P08050) and Cx-43 (uniprotkb:P08050) bind (MI:0407) by cross-linking study (MI:0030) [source] Inhibition of PI3K/Akt partially leads to the inhibition of PrPC -induced drug resistance in gastric cancer cellsFEBS JOURNAL, Issue 3 2009Jie Liang Cellular prion protein (PrPC), a glycosyl-phosphatidylinositol-anchored membrane protein with unclear physiological function, was previous found to be upregulated in adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Overexpression of PrPC in gastric cancer has certain effects on drug accumulation through upregulation of P-glycoprotein (P-gp), which is suggested to play an important role in determining the sensitivity of tumor cells to chemotherapy and is linked to activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. In the present study, we further investigate the role of the PI3K/Akt pathway in PrPC -induced multidrug-resistance (MDR) in gastric cancer. Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrPC and phosphorylated Akt (p-Akt) expression in gastric cancer. Using established stable PrPC transfectant cell lines, we demonstrated that the level of p-Akt was increased in PrPC -transfected cells. Inhibition of PrPC expression by RNA interference resulted in decreased p-Akt expression. Inhibition of the PI3K/Akt pathway by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P-gp induced by PrPC. Taken together, our results suggest that PrPC -induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrPC -induced drug resistance and P-gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrPC regulates gastric cancer cell survival. [source] Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen speciesFEBS JOURNAL, Issue 13 2008Hideki Sumimoto NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc1 complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca2+ -binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin,NADP+ reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1,4 subfamily in animals form a stable heterodimer with the membrane protein p22phox, which functions as a docking site for the SH3 domain-containing regulatory proteins p47phox, p67phox, and p40phox; the small GTPase Rac binds to p67phox (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67phox -like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation. [source] Evaluation of detergents for the soluble expression of ,-helical and ,-barrel-type integral membrane proteins by a preparative scale individual cell-free expression systemFEBS JOURNAL, Issue 23 2005Christian Klammt Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation sttif. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial ,-helical multidrug transporter, EmrE, the ,-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy- sn -glycero-3-[phospho- rac -(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a ,-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent. [source] Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substratesFEBS JOURNAL, Issue 5 2005Linda Nováková Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP- N -acetylglucosamine, an essential common precursor to cell envelope components. 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