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Integral Membrane Proteins (integral + membrane_protein)
Selected AbstractsCytosolic 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] 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] Modulation of sarcoplasmic reticulum Ca2+ -ATPase by chronic and acute exposure to peroxynitriteFEBS JOURNAL, Issue 13 2004Yolanda Gutiérrez-Martín The Ca2+ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA), an integral membrane protein, becomes irreversibly inactivated in vitro by the addition of a single bolus of peroxynitrite with a K0.5 of 200,300 µm, and this results in a large decrease of the ATP-dependent Ca2+ gradient across the sarcoplasmic reticulum (SR) membranes. The inactivation of SERCA is raised by treatment of SR vesicles with repetitive micromolar pulses of peroxynitrite. The inhibition of the SERCA is due to the oxidation of thiol groups and tyrosine nitration. Scavengers that react directly with peroxynitrite, such as cysteine, reduced glutathione, NADH, methionine, ascorbate or Trolox, a water-soluble analog of ,-tocopherol, afforded significant protection. However, dimethyl sulfoxide and mannitol, two hydroxyl radical scavengers, and ,-tocopherol did not protect SERCA from inactivation. Our results showed that the target of peroxynitrite is the cytosolic globular domain of the SERCA and that major skeletal muscle intracellular reductants (ascorbate, NADH and reduced glutathione) protected against inhibition of this ATPase by peroxynitrite. [source] Membrane orientation of laminin binding proteinFEBS JOURNAL, Issue 18 2003An extracellular matrix bridging molecule of Leishmania donovani Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source] The resident endoplasmic reticulum protein, BAP31, associates with ,-actin and myosin B heavy chainFEBS JOURNAL, Issue 2 2003Analysis by capillary liquid chromatography microelectrospray tandem MS BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731,6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle ,-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis. [source] Crystallizing proteins on the basis of their precipitation diagram determined using a microfluidic formulatorJOURNAL OF SYNCHROTRON RADIATION, Issue 6 2005Morten O. A. Sommer Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10,µL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials. [source] Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene gun deliveryPARASITE IMMUNOLOGY, Issue 4 2002Akram A. Da'dara Summary Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0·03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31,34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique. [source] Changes in red cell ion transport, reduced intratumoral neovascularization, and some mild motor function abnormalities accompany targeted disruption of the Mouse Kell gene (Kel),AMERICAN JOURNAL OF HEMATOLOGY, Issue 8 2009Xiang Zhu Kell (ECE-3), a highly polymorphic blood group glycoprotein, displays more than 30 antigens that produce allo-antibodies and, on red blood cells (RBCs), is complexed through a single disulfide bond with the integral membrane protein, XK. XK is a putative membrane transporter whose absence results in a late onset form of neuromuscular abnormalities known as the McLeod syndrome. Although Kell glycoprotein is known to be an endothelin-3-converting enzyme, the full extent of its physiological function is unknown. To study the functions of Kell glycoprotein, we undertook targeted disruption of the murine Kel gene by homologous recombination. RBCs from Kel(,/,) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin-3-converting enzyme activity and had reduced levels of XK. XK mRNA levels in spleen, brain, and testis were unchanged. In Kel(,/,) mice RBC Gardos channel activity was increased and the normal enhancement by endothelin-3 was blunted. Analysis of the microvessels of tumors produced from LL2 cells indicated that the central portion of tumors from wild-type mice were populated with many mature blood vessels, but that vessels in tumors from Kel(,/,) mice were fewer and smaller. The absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathophysiological functions of Kell glycoprotein. Am. J. Hematol., 2009. © 2009 Wiley-Liss, Inc. [source] Generalized Erythrodermic Pemphigus Foliaceus in a Child and Its Successful Response to Rituximab TreatmentPEDIATRIC DERMATOLOGY, Issue 2 2007Elizabeth Alvarez Connelly M.D. The nonendemic or sporadic form of this entity is rare in children and typically presents with a milder, more localized rash that usually follows a benign course of short duration. We describe an affected patient atypical in both her young age and the severity of skin findings. Our patient presented with a full body exfoliative erythroderma at 21 months of age. After an extensive work-up to determine the etiology of her exfoliative erythroderma, direct and indirect immunofluorescence studies confirmed the diagnosis of pemphigus foliaceus. Rituximab therapy was initiated based on the patient's refractory disease course to multiple immunosuppressive agents. Rituximab is a therapeutic monoclonal antibody targeting CD20, an integral membrane protein highly expressed on the surface of pre-B lymphocytes and activated mature B lymphocytes. The patient's skin exhibited marked clinical improvement after the start of rituximab infusions over 12 weeks. Her initial desmoglein 1 antibody level was greater than 1:1280, which decreased to 1:16 after seven rituximab treatments. She has had no skin flares since initiating treatment with rituximab therapy. Based on this clinical and serologic response, the use of rituximab may be helpful in the treatment of pediatric pemphigus foliaceus refractory to mainstays of therapy. [source] Backbone structure of a small helical integral membrane protein: A unique structural characterizationPROTEIN SCIENCE, Issue 1 2009Richard C. Page Abstract The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three-dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C-terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 Å rmsd backbone structure with a transmembrane domain defined at 0.40 Å. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long-range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins. [source] Walls are thin 1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibersTHE PLANT JOURNAL, Issue 3 2010Philippe Ranocha Summary By combining Zinnia elegans in vitro tracheary element genomics with reverse genetics in Arabidopsis, we have identified a new upstream component of secondary wall formation in xylary and interfascicular fibers. Walls are thin 1 (WAT1), an Arabidopsis thaliana homolog of Medicago truncatula NODULIN 21 (MtN21), encodes a plant-specific, predicted integral membrane protein, and is a member of the plant drug/metabolite exporter (P-DME) family (transporter classification number: TC 2.A.7.3). Although WAT1 is ubiquitously expressed throughout the plant, its expression is preferentially associated with vascular tissues, including developing xylem vessels and fibers. WAT1:GFP fusion protein analysis demonstrated that WAT1 is localized to the tonoplast. Analysis of wat1 mutants revealed two cell wall-related phenotypes in stems: a defect in cell elongation, resulting in a dwarfed habit and little to no secondary cell walls in fibers. Secondary walls of vessel elements were unaffected by the mutation. The secondary wall phenotype was supported by comparative transcriptomic and metabolomic analyses of wat1 and wild-type stems, as many transcripts and metabolites involved in secondary wall formation were reduced in abundance. Unexpectedly, these experiments also revealed a modification in tryptophan (Trp) and auxin metabolism that might contribute to the wat1 phenotype. Together, our data demonstrate an essential role for the WAT1 tonoplast protein in the control of secondary cell wall formation in fibers. [source] Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Georgios Skretas Abstract The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production. Biotechnol. Bioeng. 2009;102: 357,367. © 2008 Wiley Periodicals, Inc. [source] Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation,,BIRTH DEFECTS RESEARCH, Issue 7 2008Janee Gelineau-van Waes Abstract BACKGROUND: The reduced folate carrier (RFC1) is a ubiquitously expressed integral membrane protein that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. In this study, embryonic/fetal development is characterized in an RFC1 knockout mouse model in which pregnant dams receive different levels of folate supplementation. METHODS:RFC1+/, males were mated to RFC1+/, females, and pregnant dams were treated with vehicle (control) or folic acid (25 or 50 mg/kg) by daily subcutaneous injection (0.1 mL/10 g bwt), beginning on E0.5 and continuing throughout gestation until the time of sacrifice. RESULTS: Without maternal folate supplementation, RFC1 nullizygous embryos die shortly postimplantation. Supplementation of pregnant dams with 25 mg/kg/day folic acid prolongs survival of mutant embryos until E9.5,E10.5, but they are developmentally delayed relative to wild-type littermates, display a marked absence of erythropoiesis, severe neural tube and limb bud defects, and failure of chorioallantoic fusion. Fgfr2 protein levels are significantly reduced or absent in the extraembryonic membranes of RFC1 nullizygous embryos. Maternal folate supplementation with 50 mg/kg/day results in survival of 22% of RFC1 mutants to E18.5, but they develop with multiple malformations of the eyelids, lungs, heart, and skin. CONCLUSIONS: High doses of daily maternal folate supplementation during embryonic/fetal development are necessary for early postimplantation embryonic viability of RFC1 nullizygous embryos, and play a critical role in chorioallantoic fusion, erythropoiesis, and proper development of the neural tube, limbs, lungs, heart, and skin. Birth Defects Research (Part A), 2008. © 2008 Wiley-Liss, Inc. [source] A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potatoCELLULAR MICROBIOLOGY, Issue 11 2008Anna O. Avrova Summary Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans -membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans. [source] Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosisCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2005E. Janas Summary Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca2+ levels and downstream apoptotic signalling. [source] Association between particular polymorphic residues on apical membrane antigen 1 (AMA-1) and platelet levels in patients with vivax malariaCLINICAL MICROBIOLOGY AND INFECTION, Issue 11 2007P. Grynberg Abstract Apical membrane antigen 1 (AMA-1) is an immunogenic type 1 integral membrane protein, present in all Plasmodium spp., that probably has a role in the initiation of the invasion process of the erythrocyte. The DNA sequence of variable domain I of the Plasmodium vivax ama1 gene was sequenced in Brazilian isolates obtained from thrombocytopenic patients (n = 32) and patients with normal platelet counts (n = 22). There was a significant negative correlation between parasite density and platelet counts. It was concluded that there is an additional effect of sequence on platelet counts. The presence of amino-acid residues Y193 and S210 was associated significantly with normal platelet counts in P. vivax malaria, independent of the level of parasitaemia (p <0.0001). These data have implications for AMA-1-based vaccine design and suggest the possible use of this molecule as a marker of morbidity. [source] Analysis of integral membrane proteins by heat gel-embedment combined with improved in-gel digestionsELECTROPHORESIS, Issue 23 2009Jian Zhou Abstract Analysis of integral membrane proteins (IMPs) presents a special challenge because of their hydrophobic nature and low abundance. Here, a new method was developed, which involved heat gel-embedment and improved in-gel digestion of the proteins. Membrane protein lysate containing detergents was mixed with acrylamide solution and the proteins were embedded when the gel polymerized. For comparison, the protein embedment was made at different temperatures (25, 35 or 45°C), and the in-gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS), 0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. Compared with that at 25°C, gel-embedment at 45°C improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%. 0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel-embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel-embedment combined with improved in-gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of integral membrane proteome. [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] 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] Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpeterseniiFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2001Alejandro de la Peña-Moctezuma Abstract The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5, end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity. [source] The tripartite ATP-independent periplasmic (TRAP) transporters of bacteria and archaeaFEMS MICROBIOLOGY REVIEWS, Issue 4 2001David J Kelly Abstract Until recently, extracytoplasmic solute receptor (ESR)-dependent uptake systems were invariably found to possess a conserved ATP-binding protein (the ATP-binding cassette protein or ABC protein), which couples ATP hydrolysis to the translocation of the solute across the cytoplasmic membrane. While it is clear that this class of ABC transporter is ubiquitous in prokaryotes, it is now firmly established that other, unrelated types of membrane transport systems exist which also have ESR components. These systems have been designated tripartite ATP-independent periplasmic (TRAP) transporters, and they form a distinct class of ESR-dependent secondary transporters where the driving force for solute accumulation is an electrochemical ion gradient and not ATP hydrolysis. Currently, the most well characterised TRAP transporter at the functional and molecular level is the high-affinity C4-dicarboxylate transport (Dct) system from Rhodobacter capsulatus. This consists of three proteins; an ESR (DctP) and small (DctQ) and large (DctM) integral membrane proteins. The characteristics of this system are discussed in detail. Homologues of the R. capsulatus DctPQM proteins are present in a diverse range of prokaryotes, both bacteria and archaea, but not in eukaryotes. The deduced structures and possible functions of these homologous systems are described. In addition to the DctP family, other types of ESRs can be associated with TRAP transporters. A conserved family of immunogenic extracytoplasmic proteins is shown to be invariably associated with TRAP systems that contain a large DctQM fusion protein. All of the currently known archaeal systems are of this type. It is concluded that TRAP transporters are a widespread and ancient type of solute uptake system that transport a potentially diverse range of solutes and most likely evolved by the addition of auxiliary proteins to a single secondary transporter. [source] Mutations of the RDX gene cause nonsyndromic hearing loss at the DFNB24 locus,,HUMAN MUTATION, Issue 5 2007Shahid Y. Khan Abstract Ezrin, radixin, and moesin are paralogous proteins that make up the ERM family and function as cross-linkers between integral membrane proteins and actin filaments of the cytoskeleton. In the mouse, a null allele of Rdx encoding radixin is associated with hearing loss as a result of the degeneration of inner ear hair cells as well as with hyperbilirubinemia due to hepatocyte dysfunction. Two mutant alleles of RDX [c.1732G>A (p.D578N) and c.1404_1405insG (p.A469fsX487)] segregating in two consanguineous Pakistani families are associated with neurosensory hearing loss. Both of these mutant alleles are predicted to affect the actin-binding motif of radixin. Sequence analysis of RDX in the DNA samples from the original DFNB24 family revealed a c.463C>T transition substitution that is predicted to truncate the protein in the FERM domain (F for 4.1, E for ezrin, R for radixin, and M for moesin) (p.Q155X). We also report a more complete gene and protein structure of RDX, including four additional exons and five new isoforms of RDX that are expressed in human retina and inner ear. Further, high-resolution confocal microscopy in mouse inner ear demonstrates that radixin is expressed along the length of stereocilia of hair cells from both the organ of Corti and the vestibular system. Hum Mutat 28(5), 417,423, 2007. Published 2007 Wiley-Liss, Inc. [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] Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glandsJOURNAL OF ANATOMY, Issue 4 2004M. Peppi Abstract Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague,Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels. [source] Prediction of integral membrane protein type by collocated hydrophobic amino acid pairsJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2009Ke Chen Abstract A computational model, IMP-TYPE, is proposed for the classification of five types of integral membrane proteins from protein sequence. The proposed model aims not only at providing accurate predictions but most importantly it incorporates interesting and transparent biological patterns. When contrasted with the best-performing existing models, IMP-TYPE reduces the error rates of these methods by 19 and 34% for two out-of-sample tests performed on benchmark datasets. Our empirical evaluations also show that the proposed method provides even bigger improvements, i.e., 29 and 45% error rate reductions, when predictions are performed for sequences that share low (40%) identity with sequences from the training dataset. We also show that IMP-TYPE can be used in a standalone mode, i.e., it duplicates significant majority of correct predictions provided by other leading methods, while providing additional correct predictions which are incorrectly classified by the other methods. Our method computes predictions using a Support Vector Machine classifier that takes feature-based encoded sequence as its input. The input feature set includes hydrophobic AA pairs, which were selected by utilizing a consensus of three feature selection algorithms. The hydrophobic residues that build up the AA pairs used by our method are shown to be associated with the formation of transmembrane helices in a few recent studies concerning integral membrane proteins. Our study also indicates that Met and Phe display a certain degree of hydrophobicity, which may be more crucial than their polarity or aromaticity when they occur in the transmembrane segments. This conclusion is supported by a recent study on potential of mean force for membrane protein folding and a study of scales for membrane propensity of amino acids. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009 [source] Catenin dislocation in oral pemphigus vulgarisJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 5 2001Michele Davide Mignogna Abstract: Cell-to-cell adhesion is mediated by cadherins (integral membrane proteins), which form a complex with catenins (cytoplasmatic proteins). While E-cadherin expression has been extensively studied in many human skin diseases, less is known about the expression levels of catenins in oral blistering diseases. The purpose of this study was to evaluate the role of these proteins in the pathogenesis of acantholysis in oral pemphigus vulgaris. We evaluated by immunohistochemistry beta- and gamma-catenin expression in 7 cases of oral pemphigus vulgaris (PV) at various stages of the disease and, as controls, in 18 healthy patients. Healthy cases showed, as reported in the literature, a strong reactivity with both beta- and gamma-catenins, with the intensity of staining progressively decreasing from the spinous to the keratinised layers of epithelium, which had a prevalent cellular membrane expression. In PV patients, we detected a loss of membrane expression of these molecules with a progressive displacement of the signal toward the cytosol and, for gamma-catenin, nuclear dislocation, particularly in areas with intense acantholysis. [source] Tetraspanins and Intercellular InteractionsMICROCIRCULATION, Issue 3 2001MARÍA YÁÑEZ-MÓ ABSTRACT The superfamily of tetraspanins comprises a group of polypeptides with four transmembrane domains that form large supramolecular structures in the plasma membrane through their associations to multiple integral membrane proteins. They are involved in homo- and heterotypic intercellular interactions in different processes such as hematopoiesis, lymphocyte activation, cancer metastasis, and fertilization. Intercellularly located tetraspanins regulate the juxtacrine activity of growth factors, cell fusion, and myelin formation. On the other hand, in motile cells they relocalize from cell-cell junctions to actin-based structures such as filopodia or growth cones and regulate cell motility in wound healing and angiogenesis processes. [source] A subset of bacterial inner membrane proteins integrated by the twin-arginine translocaseMOLECULAR MICROBIOLOGY, Issue 5 2003Kostas Hatzixanthis Summary A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK ,twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or ,C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins. [source] Sequential model of phage PRD1 DNA delivery: active involvement of the viral membraneMOLECULAR MICROBIOLOGY, Issue 5 2002A. Marika Grahn Summary DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32. [source] | ||||||||||||||||||||||||||||