Home About us Contact
|
Home About us Contact | ||
|
|
||
Transmembrane
Terms modified by Transmembrane Selected AbstractsTransmembrane 4 L six family member 5 (TM4SF5) enhances migration and invasion of hepatocytes for effective metastasisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2010Sin-Ae Lee Abstract Overexpression of transmembrane 4 L six family member 5 (TM4SF5), a four-transmembrane L6 family member, causes aberrant cell proliferation and angiogenesis, but the roles of TM4SF5 in migration, invasion, and tumor metastasis remain unknown. Using in vitro hepatocarcinoma cells that ectopically or endogenously express TM4SF5 and in vivo mouse systems, roles of TM4SF5 in metastatic potentials were examined. We found that TM4SF5 expression facilitated migration, invadopodia formation, MMP activation, invasion, and eventually lung metastasis in nude mice, but suppression of TM4SF5 with its shRNA blocked the effects. Altogether, TM4SF5-mediated migration and invasion suggest that TM4SF5 may be therapeutically targeted to deal with TM4SF5-mediated hepatocellular cancers. J. Cell. Biochem. 111: 59,66, 2010. © 2010 Wiley-Liss, Inc. [source] Transmembrane signaling through phospholipase C-, in the developing human prefrontal cortexJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2006Iñigo Ruiz de Azúa Abstract To investigate changes in muscarinic receptor-stimulated phospholipase C-, (PLC-,) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-,1,4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5,-o-3 thiotriphosphate (GTP,S) or carbachol (in the presence of GTP,S) to modulate prenatal PLC-, was significantly weaker than that associated with postnatal PLC-,. Western blot analysis revealed that the levels of G,q/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-,1,4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-,1,4) and receptors (M1,3,5 subtypes) than by altering the level of G,q/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain. © 2006 Wiley-Liss, Inc. [source] Lipid bilayers: an essential environment for the understanding of membrane proteinsMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2007Richard C. Page Abstract Membrane protein structure and function is critically dependent on the surrounding environment. Consequently, utilizing a membrane mimetic that adequately models the native membrane environment is essential. A range of membrane mimetics are available but none generates a better model of native aqueous, interfacial, and hydrocarbon core environments than synthetic lipid bilayers. Transmembrane ,-helices are very stable in lipid bilayers because of the low water content and low dielectric environment within the bilayer hydrocarbon core that strengthens intrahelical hydrogen bonds and hinders structural rearrangements within the transmembrane helices. Recent evidence from solid-state NMR spectroscopy illustrates that transmembrane ,-helices, both in peptides and full-length proteins, appear to be highly uniform based on the observation of resonance patterns in PISEMA spectra. Here, we quantitate for the first time through simulations what we mean by highly uniform structures. Indeed, helices in transmembrane peptides appear to have backbone torsion angles that are uniform within ± 4° . While individual helices can be structurally stable due to intrahelical hydrogen bonds, interhelical interactions within helical bundles can be weak and nonspecific, resulting in multiple packing arrangements. Some helical bundles have the capacity through their amino acid composition for hydrogen bonding and electrostatic interactions to stabilize the interhelical conformations and solid-state NMR data is shown here for both of these situations. Solid-state NMR spectroscopy is unique among the techniques capable of determining three-dimensional structures of proteins in that it provides the ability to characterize structurally the membrane proteins at very high resolution in liquid crystalline lipid bilayers. Copyright © 2007 John Wiley & Sons, Ltd. [source] One ,,Hairpin after the Other: Exploring Mechanical Unfolding Pathways of the Transmembrane ,-Barrel Protein OmpG,ANGEWANDTE CHEMIE, Issue 44 2009Tanuj Sapra Dr. Sauber auseinandergefädelt: Mithilfe der Einzelmolekül-Rasterkraftspektroskopie wurde erstmals ein äußeres Membranprotein mit ,-Fass-Struktur entfaltet. Überraschenderweise entfalten einzelne ,-Stränge von OmpG aus E. coli nicht individuell, sondern in Form von ,-Haarnadeln. Diese ,-Haarnadeln entfalten eine nach der anderen, bis das gesamte Protein entfaltet ist (siehe Bild). [source] Protein tyrosine phosphatases in Chaetopterus egg activationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Shantá D. Hinton Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTP,, -,, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with ,-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process. [source] Interleukin-5 does not influence differential transcription of transmembrane and soluble isoforms of IL-5R, in vivoEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006Jonas Byström Abstract:, Interleukin-5 (IL-5) promotes signal transduction and expansion of eosinophil colonies in bone marrow via interactions with its heterodimeric receptor (IL-5R). Two variants encoding soluble forms of the alpha subunit (sIL-5R,) have been described, although the signals promoting and/or limiting differential transcription remain to be clarified. Objectives:,Our intent was to explore the role of IL-5 in regulating differential transcription of these splice variants in vivo. Methods:,We have designed a quantitative reverse transcriptase-polymerase chain reaction assay to detect transcripts encoding the transmembrane, soluble 1 and 2 forms of IL-5R, in two strains of wild-type (BALB/c and C57BL/6) and corresponding IL-5 gene-deleted mice. Wild-type mice respond to S. mansoni infection with a gradual increase in serum IL-5 and eosinophilia, which is not observed in IL-5 gene-deleted mice. Results and conclusions:,We find that IL-5 is not necessary for differential splicing to occur in vivo, as all three forms of the IL-5R, are detected in both strains of IL-5 gene-deleted mice, with ratios of transcript expression (transmembrane : soluble 1 : soluble 2) that were indistinguishable from their wild-type counterparts. Differential splicing does vary markedly between strains, potentially because of local effects of strain-specific polymorphisms. [source] Proteolytic cleavage of the voltage-gated Ca2+ channel ,2, subunit: structural and functional featuresEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Arturo Andrade Abstract By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (,1) and three auxiliary (,2,, , and ,) subunits. The ,2, subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, ,2, must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular ,2 from its transmembrane , domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature ,2,, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of ,2, was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. [source] A unique binding epitope for salvinorin A, a non-nitrogenous kappa opioid receptor agonistFEBS JOURNAL, Issue 9 2006Brian E. Kane Salvinorin A is a potent kappa opioid receptor (KOP) agonist with unique structural and pharmacological properties. This non-nitrogenous ligand lacks nearly all the structural features commonly associated with opioid ligand binding and selectivity. This study explores the structural basis to salvinorin A binding and selectivity using a combination of chimeric and single-point mutant opioid receptors. The experiments were designed based on previous models of salvinorin A that locate the ligand within a pocket formed by transmembrane (TM) II, VI, and VII. More traditional sites of opioid recognition were also explored, including the highly conserved aspartate in TM III (D138) and the KOP selectivity site E297, to determine the role, if any, that these residues play in binding and selectivity. The results indicate that salvinorin A recognizes a cluster of residues in TM II and VII, including Q115, Y119, Y312, Y313, and Y320. Based on the position of these residues within the receptor, and prior study on salvinorin A, a model is proposed that aligns the ligand vertically, between TM II and VII. In this orientation, the ligand spans residues that are spaced one to two turns down the face of the helices within the receptor cavity. The ligand is also in close proximity to EL-2 which, based on chimeric data, is proposed to play an indirect role in salvinorin A binding and selectivity. [source] Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomesFEBS JOURNAL, Issue 21 2002François Devesa Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane ,,helices and small ecto- and endodomains. A His6 -tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N -dodecyl-,- d -maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins. [source] Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domainFEBS JOURNAL, Issue 14 2000Seiya Yoshida Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis -dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane. [source] Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region,HUMAN MUTATION, Issue 1 2006Satoshi Yonezawa Abstract BUS/Idr mice carrying a mutant waltzer allele (vbus) are characterized by splayed hair bundles in inner ear sensory cells, providing a mouse homolog of USH1D/DFNB12. RT-PCR-based screening for the presence of mutations in mouse Cdh23, the gene responsible for the waltzer phenotype, has identified a G>A mutation in the donor splice site of intron 67 (Cdh23:c.9633+1G>A: GenBank AF308939.1), indicating that two altered Cdh23 molecules having intron-derived COOH-terminal structures could be generated in BUS mouse tissues. Immunochemical analyses with anti-Cdh23 antibodies showed, however, no clear Cdh23-related proteins in vbus/vbus tissues, while the antibodies immunoreacted with ,350,kDa proteins in control mice. Immunofluorescent experiments revealed considerable weakening of Cdh23 signals in sensory hair cell stereocilia and Reissner's membrane in the vbus/vbus inner ear, and transmission electron microscopy demonstrated abundant autophagosome/autolysosome vesicles, suggesting aberrant Cdh23:c.9633+1G>A-derived protein-induced acceleration of lysosomal bulk degradation of proteins. In transfection experiments, signal sequence-preceded FLAG-tagged transmembrane plus cytoplasmic regions (TMCy) of tissue-specific Cdh23(±68) isoforms were localized to filamentous actin-rich protrusions and the plasma membrane of cultured cells, whereas FLAG-TMCy:c.9633+1G>A proteins were highly insoluble and retained in the cytoplasm. In contrast, FLAG-tagged TMCy:p.Arg3175His and human TMCy:c.9625_9626insC forms were both localized to the plasma membrane in cultured cells, allowing prediction that USH1D-associated CDH23:p.Arg3175His and CDH23:c.9625_9626insC proteins could be transported to the plasma membrane in vivo. The present results thus suggest different fates of CDH23/Cdh23 with mutations affecting the cytoplasmic region. Hum Mutat 27(1), 88,97, 2005. © 2005 Wiley-Liss, Inc. [source] Decay-accelerating factor induction by tumour necrosis factor-,, through a phosphatidylinositol-3 kinase and protein kinase C-dependent pathway, protects murine vascular endothelial cells against complement depositionIMMUNOLOGY, Issue 2 2003Saifur R. Ahmad Summary We have shown that human endothelial cells (EC) are protected against complement-mediated injury by the inducible expression of decay-accelerating factor (DAF). To understand further the importance of DAF regulation, we characterized EC DAF expression on murine EC in vitro and in vivo using a model of glomerulonephritis. Flow cytometry using the monoclonal antibody (mAb) Riko-3 [binds transmembrane- and glycosylphosphatidylinositol (GPI)-anchored DAF], mAb Riko-4 (binds GPI-anchored DAF) and reverse transcription,polymerase chain reaction (RT,PCR), demonstrated that murine EC DAF is GPI-anchored. Tumour necrosis factor-, (TNF-,) increased EC DAF expression, detectable at 6 hr and maximal at 24,48 hr poststimulation. DAF upregulation required increased steady-state DAF mRNA and protein synthesis. In contrast, no increased expression of the murine complement receptor-related protein-Y (Crry) was seen with TNF-,. DAF upregulation was mediated via a protein kinase C (PKC),, phosphoinositide-3 kinase (PI-3 kinase), p38 mitogen-activated protein kinase (MAPK) and nuclear factor-,B (NF-,B)-dependent pathway. The increased DAF was functionally relevant, resulting in a marked reduction in C3 deposition following complement activation. In a nephrotoxic nephritis model, DAF expression on glomerular capillaries was significantly increased 2 hr after the induction of disease. The demonstration of DAF upregulation above constitutive levels suggests that this may be important in the maintenance of vascular integrity during inflammation, when the risk of complement-mediated injury is increased. The mouse represents a suitable model for the study of novel therapeutic approaches by which vascular endothelium may be conditioned against complement-mediated injury. [source] Enhanced efficacy of DNA vaccination against Her-2/neu tumor antigen by genetic adjuvantsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Sun Young Chang Abstract Certain types of malignant tumors overexpress Her-2/neu, a transmembrane glycoprotein of the class I receptor tyrosine kinase erbB family. To develop an effective Her-2/neu vaccine for selective immunotherapy of these malignancies, we prepared Her-2/neu DNA plasmid encoding the transmembrane and extracellular domain (pHM) and tested the ability of this construct to induce antitumor immunity in animal models. In addition, we investigated the effects of cytokine used as a genetic adjuvant. Modulation by factors that affect T-cell function or hematopoiesis, including interleukin-12, interleukin-15, interleukin-18, interleukin-23, Eta-1, Flt3L and GM-CSF, was studied in the forms of monocistronic and bicistronic plasmid. Our results demonstrated that vaccination of pHM could induce successful antitumor immunity against Her-2/neu-expressing murine tumor cells in BALB/c mice. We also showed that the antitumor activity of pHM was augmented by coadministration and coexpression of different cytokines. Despite the similar levels of gene expression, the antitumor effects of bicistronic plasmids coexpressing Her-2/neu antigen and cytokine were improved in comparison with coadministration of separate monocistronic plasmid. In particular, coexpression of interleukin-18 or GM-CSF with Her-2/neu increased antitumor activity in both preventive and therapeutic experiments. These findings can help in the decision concerning which of the various cytokine adjuvants should be used for the development of a Her-2/neu DNA vaccine. In addition, our results from a large panel of cytokine adjuvants in the various tumor models may provide an insight into the important immune components of antitumor immunity. © 2004 Wiley-Liss, Inc. [source] Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR productsINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 6 2003C. Chung Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures. [source] Molecular cloning of feline tumour necrosis factor receptor type I (TNFR I) and expression of TNFR I and TNFR II in lymphoid cells in catsINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2003T. Mizuno Summary Tumour necrosis factor (TNF)-, is a pro-inflammatory cytokine produced by many types of cells. It has been shown that two distinct TNF receptors (TNFRs), TNFR type I (TNFR I) and TNFR type II (TNFR II), have different functions in signal transduction, which is possibly associated with the development of a variety of diseases. In this study, we isolated a feline TNFR I cDNA clone and analysed the expression of TNFR I and TNFR II mRNA in feline lymphoid cells. The deduced amino acid sequence of feline TNFRI cDNA showed 75.8, 62.5 60.9 and 72.1% similarity with those of its human, mouse, rat, and pig counterparts, respectively. The feline TNFR I cDNA was shown to encode extracellular, transmembrane and intracellular domains fundamentally conserved in the homologues of other species. Expression of TNFR I and TNFR II mRNAs was shown to be up-regulated in feline peripheral blood mononuclear cells (PBMC) by stimulation with concanavalin A. Five of six feline lymphoma cell lines were shown to express both TNFR I and TNFR II mRNAs. The expression of TNFR I in PBMC was up-regulated in cats infected with feline immunodeficiency virus (FIV), whereas the expression of TNFR II in PBMC was not different between FIV-infected cats and uninfected cats. The present study indicate that expression of TNFR I and TNFR II may be associated with disease progression, especially in retrovirus infections in cats. [source] Effects of Secreted Frizzled-Related Protein 3 on Osteoblasts In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004Yoon-Sok Chung Abstract To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. Introduction: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. Materials and Methods: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on ,-catenin levels using Western immunoblot analyses. Results: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular ,-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular ,-catenin levels. Treatment with endostatin, an agent known to degrade ,-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. Conclusions: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a ,-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's. [source] Transmembrane 4 L six family member 5 (TM4SF5) enhances migration and invasion of hepatocytes for effective metastasisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2010Sin-Ae Lee Abstract Overexpression of transmembrane 4 L six family member 5 (TM4SF5), a four-transmembrane L6 family member, causes aberrant cell proliferation and angiogenesis, but the roles of TM4SF5 in migration, invasion, and tumor metastasis remain unknown. Using in vitro hepatocarcinoma cells that ectopically or endogenously express TM4SF5 and in vivo mouse systems, roles of TM4SF5 in metastatic potentials were examined. We found that TM4SF5 expression facilitated migration, invadopodia formation, MMP activation, invasion, and eventually lung metastasis in nude mice, but suppression of TM4SF5 with its shRNA blocked the effects. Altogether, TM4SF5-mediated migration and invasion suggest that TM4SF5 may be therapeutically targeted to deal with TM4SF5-mediated hepatocellular cancers. J. Cell. Biochem. 111: 59,66, 2010. © 2010 Wiley-Liss, Inc. [source] Novel free energy calculations to explore mechanisms and energetics of membrane protein structure and functionJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 11 2009Wonpil Im Abstract Understanding the delicate balance of forces governing helix or ,-hairpin interactions in transmembrane (TM) proteins is central to understanding membrane structure and function. These membrane constituent interactions play an essential role in determining the structure and function of membrane proteins, and protein interactions in membranes, and thus form the basis for many vital processes, including TM signaling, transport of ions and small molecules, energy transduction, and cell,cell recognition. "Why does a single-pass TM helix or ,-hairpin have specific orientations in membranes?" "What are the roles of hydrogen bonds, close packing, and helix-lipid or ,-hairpin-lipid interactions in helix or ,-hairpin associations in membranes?" "How do these interactions change the membrane structures?" "How do TM domains transmit signals across membranes?" These are important membrane biophysical questions that can be addressed by understanding the delicate balance of forces governing helix or ,-hairpin interactions with/in membranes. In this work, we summarize a series of helix/,-hairpin restraint potentials that we have developed, and illustrate their applications that begin to address the complicated energetics and molecular mechanisms of these interactions at the atomic level by calculating the potentials of mean force (PMFs) along reaction coordinates relevant to helix/,-hairpin motions in membranes and dissecting the total PMF into the contributions arising from physically important microscopic forces. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009 [source] Neural network-based prediction of transmembrane ,-strand segments in outer membrane proteinsJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 5 2004M. Michael Gromiha Abstract Prediction of transmembrane ,-strands in outer membrane proteins (OMP) is one of the important problems in computational chemistry and biology. In this work, we propose a method based on neural networks for identifying the membrane-spanning ,-strands. We introduce the concept of "residue probability" for assigning residues in transmembrane ,-strand segments. The performance of our method is evaluated with single-residue accuracy, correlation, specificity, and sensitivity. Our predicted segments show a good agreement with experimental observations with an accuracy level of 73% solely from amino acid sequence information. Further, the predictive power of N- and C-terminal residues in each segments, number of segments in each protein, and the influence of cutoff probability for identifying membrane-spanning ,-strands will be discussed. We have developed a Web server for predicting the transmembrane ,-strands from the amino acid sequence, and the prediction results are available at http://psfs.cbrc.jp/tmbeta-net/. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 762,767, 2004 [source] HTLV-1 infection in blood donors from the Western Brazilian Amazon region: Seroprevalence and molecular study of viral isolatesJOURNAL OF MEDICAL VIROLOGY, Issue 11 2008Aline Cristina Mota-Miranda Abstract To determine the seroprevalence of HTLV-1 in Brazil, and to review the virus molecular epidemiology in this Amazon population (Rio Branco-Acre), 219 blood donors were screened for HTLV-1. Only one case of infection (0.46% seroprevalence) was detected during July 2004 screening at the Acre Hospital Foundation (FUNDACRE). Neighbor-joining and Maximum Likelihood phylogenetic analyses of two (n,=,2) complete LTR region sequences were performed with the PAUP* software. Since the HTLV-1 envelope surface (gp46) and transmembrane (gp21) glycoproteins are important for virus fitness, three envelope glycoproteins sequences (n,=,3) were analyzed using the Prosite tool to determinate potential protein sites. Phylogenetic analysis demonstrated that the new isolate described in this study, and the unpublished LTR strain described in a previous report belong to the Transcontinental subgroup of the Cosmopolitan subtype, inside the Latin American cluster. A similar result was obtained when submitting, to the Automated Genotyping System, three LTR partial sequences from a previous study of the seroprevalence of HTLV-1 in the same Amazon population. In all analyzed env sequences, the potential protein site was found: two PKC phosphorylation sites at amino acid (aa) positions 310,312 and 342,344, one CK2 phosphorylation site at 194,197aa, three N -glycosylation sites at 222,225aa, 244,247aa and 272,275aa, and a single N -myristylation site at 327,338aa. In conclusion, potential protein sites described in HTLV-1 gp46 and gp21 confirm the presence of conserved sites in the HTLV-1 envelope proteins, likewise phylogenetic analysis suggests a possible recent introduction of the virus into North Brazil. J. Med. Virol. 80:1966,1971, 2008. © 2008 Wiley-Liss, Inc. [source] Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib,IX complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2007S.-Z. LUO Summary.,Background:,The glycoprotein (GP) Ib,IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIb, and GPIb, subunits interact and participate in complex assembly. Objectives and methods:,Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb,IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells. Results:,Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb,IX complex. In contrast, replacing the GPIX TM domain (residues 132,153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIb,, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues. Conclusions:,The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb,IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex. [source] Lipid bilayers: an essential environment for the understanding of membrane proteinsMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2007Richard C. Page Abstract Membrane protein structure and function is critically dependent on the surrounding environment. Consequently, utilizing a membrane mimetic that adequately models the native membrane environment is essential. A range of membrane mimetics are available but none generates a better model of native aqueous, interfacial, and hydrocarbon core environments than synthetic lipid bilayers. Transmembrane ,-helices are very stable in lipid bilayers because of the low water content and low dielectric environment within the bilayer hydrocarbon core that strengthens intrahelical hydrogen bonds and hinders structural rearrangements within the transmembrane helices. Recent evidence from solid-state NMR spectroscopy illustrates that transmembrane ,-helices, both in peptides and full-length proteins, appear to be highly uniform based on the observation of resonance patterns in PISEMA spectra. Here, we quantitate for the first time through simulations what we mean by highly uniform structures. Indeed, helices in transmembrane peptides appear to have backbone torsion angles that are uniform within ± 4° . While individual helices can be structurally stable due to intrahelical hydrogen bonds, interhelical interactions within helical bundles can be weak and nonspecific, resulting in multiple packing arrangements. Some helical bundles have the capacity through their amino acid composition for hydrogen bonding and electrostatic interactions to stabilize the interhelical conformations and solid-state NMR data is shown here for both of these situations. Solid-state NMR spectroscopy is unique among the techniques capable of determining three-dimensional structures of proteins in that it provides the ability to characterize structurally the membrane proteins at very high resolution in liquid crystalline lipid bilayers. Copyright © 2007 John Wiley & Sons, Ltd. [source] Coupling of Protonation Switches During Rhodopsin Activation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2007Reiner Vogel Recent studies of the activation mechanism of rhodopsin involving Fourier-transform infrared spectroscopy and a combination of chromophore modifications and site-directed mutagenesis reveal an allosteric coupling between two protonation switches. In particular, the ring and the 9-methyl group of the all- trans retinal chromophore serve to couple two proton-dependent activation steps: proton uptake by a cytoplasmic network between transmembrane (TM) helices 3 and 6 around the conserved ERY (Glu-Arg-Tyr) motif and disruption of a salt bridge between the retinal protonated Schiff base (PSB) and a protein counterion in the TM core of the receptor. Retinal analogs lacking the ring or 9-methyl group are only partial agonists,the conformational equilibrium between inactive Meta I and active Meta II photoproduct states is shifted to Meta I. An artificial pigment was engineered, in which the ring of retinal was removed and the PSB salt bridge was weakened by fluorination of C14 of the retinal polyene. These modifications abolished allosteric coupling of the proton switches and resulted in a stabilized Meta I state with a deprotonated Schiff base (Meta ISB). This state had a partial Meta II-like conformation due to disruption of the PSB salt bridge, but still lacked the cytoplasmic proton uptake reaction characteristic of the final transition to Meta II. As activation of native rhodopsin is known to involve deprotonation of the retinal Schiff base prior to formation of Meta II, this Meta ISB state may serve as a model for the structural characterization of a key transient species in the activation pathway of a prototypical G protein-coupled receptor. [source] Bridging the gap: A GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-terminiPROTEIN SCIENCE, Issue 4 2010Jennifer M. Hsieh Abstract Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (Cin) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (Cout) to Cin ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted Cout topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with Cout topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging. [source] The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C-terminal juxtamembrane residuesPROTEIN SCIENCE, Issue 2 2009Weng Chuan Peng Abstract The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of , and , integrin TM interactions. However, we show herein that in FGFR3-TM, four C-terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3-TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C-terminal residues were present. In the absence of these four residues, A391E was dimer-destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3-TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR-TM. [source] Buried water molecules in helical transmembrane proteinsPROTEIN SCIENCE, Issue 2 2008Robert Renthal Abstract Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules. [source] Expression screening of integral membrane proteins from Helicobacter pylori 26695PROTEIN SCIENCE, Issue 12 2007Georgios Psakis Abstract The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins ,40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions. [source] The transmembrane homotrimer of ADAM 1 in model lipid bilayersPROTEIN SCIENCE, Issue 2 2007Siok Wan Gan Abstract Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin , and fertilin , that plays an important role in sperm,egg fusion. Fertilin , and , are members of the ADAM family, and contain each one transmembrane ,-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane ,-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate. [source] An amino acid "transmembrane tendency" scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: Relationship to biological hydrophobicityPROTEIN SCIENCE, Issue 8 2006Gang Zhao Abstract Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known structure were used to resolve total genomic hydrophobicity profiles into (helical) TM sequences and mainly "subhydrophobic" soluble components. This information was used to define a refined "hydrophobicity"-type TM sequence prediction scale that should approach the theoretical limit of accuracy. The refinement procedure involved adjusting scale values to eliminate differences between the average amino acid composition of populations TM and soluble sequences of equal hydrophobicity, a required property of a scale having maximum accuracy. Application of this procedure to different hydrophobicity scales caused them to collapse to essentially a single TM tendency scale. As expected, when different scales were compared, the TM tendency scale was the most accurate at predicting TM sequences. It was especially highly correlated (r = 0.95) to the biological hydrophobicity scale, derived experimentally from the percent TM conformation formed by artificial sequences passing though the translocon. It was also found that resolution of total genomic sequence data into TM and soluble components could be used to define the percent probability that a sequence with a specific hydrophobicity value forms a TM segment. Application of the TM tendency scale to whole genomic data revealed an overlap of TM and soluble sequences in the "semihydrophobic" range. This raises the possibility that a significant number of proteins have sequences that can switch between TM and non-TM states. Such proteins may exist in moonlighting forms having properties very different from those of the predominant conformation. [source] Asymmetric amino acid compositions of transmembrane ,-strandsPROTEIN SCIENCE, Issue 8 2004Aaron K. Chamberlain Abstract In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane ,-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a ,-strand. This new characteristic of ,-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane ,-barrels. [source] | ||||||||||||||||