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Sample records for associating transmembrane domains

  1. Requirement of transmembrane domain for CD154 association to lipid rafts and subsequent biological events.

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    Nadir Benslimane

    Full Text Available Interaction of CD40 with CD154 leads to recruitment of both molecules into lipid rafts, resulting in bi-directional cell activation. The precise mechanism by which CD154 is translocated into lipid rafts and its impact on CD154 signaling remain largely unknown. Our aim is to identify the domain of CD154 facilitating its association to lipid rafts and the impact of such association on signaling events and cytokine production. Thus, we generated Jurkat cell lines expressing truncated CD154 lacking the cytoplasmic domain or chimeric CD154 in which the transmembrane domain was replaced by that of transferrin receptor I, known to be excluded from lipid rafts. Our results show that cell stimulation with soluble CD40 leads to the association of CD154 wild-type and CD154-truncated, but not CD154-chimera, with lipid rafts. This is correlated with failure of CD154-chimera to activate Akt and p38 MAP kinases, known effectors of CD154 signaling. We also found that CD154-chimera lost the ability to promote IL-2 production upon T cell stimulation with anti-CD3/CD28 and soluble CD40. These results demonstrate the implication of the transmembrane domain of CD154 in lipid raft association, and that this association is necessary for CD154-mediated Akt and p38 activation with consequent enhancement of IL-2 production.

  2. The Immunogenicity of the Tumor-Associated Antigen α-Fetoprotein Is Enhanced by a Fusion with a Transmembrane Domain

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    Lucile Tran

    2012-01-01

    Full Text Available Aim. To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA vector to induce an immune response against a well-tolerated self-antigen. Methods. rMVA vectors expressing different form of α-fetoprotein (AFP were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection. Results. Vaccination with the membrane-associated form of AFP induced a stronger CD8+ T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies. Conclusions. The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.

  3. Localized lipid packing of transmembrane domains impedes integrin clustering.

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    Mehrdad Mehrbod

    Full Text Available Integrin clustering plays a pivotal role in a host of cell functions. Hetero-dimeric integrin adhesion receptors regulate cell migration, survival, and differentiation by communicating signals bidirectionally across the plasma membrane. Thus far, crystallographic structures of integrin components are solved only separately, and for some integrin types. Also, the sequence of interactions that leads to signal transduction remains ambiguous. Particularly, it remains controversial whether the homo-dimerization of integrin transmembrane domains occurs following the integrin activation (i.e. when integrin ectodomain is stretched out or if it regulates integrin clustering. This study employs molecular dynamics modeling approaches to address these questions in molecular details and sheds light on the crucial effect of the plasma membrane. Conducting a normal mode analysis of the intact αllbβ3 integrin, it is demonstrated that the ectodomain and transmembrane-cytoplasmic domains are connected via a membrane-proximal hinge region, thus merely transmembrane-cytoplasmic domains are modeled. By measuring the free energy change and force required to form integrin homo-oligomers, this study suggests that the β-subunit homo-oligomerization potentially regulates integrin clustering, as opposed to α-subunit, which appears to be a poor regulator for the clustering process. If α-subunits are to regulate the clustering they should overcome a high-energy barrier formed by a stable lipid pack around them. Finally, an outside-in activation-clustering scenario is speculated, explaining how further loading the already-active integrin affects its homo-oligomerization so that focal adhesions grow in size.

  4. Structural and dynamic study of the transmembrane domain of the amyloid precursor protein.

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    Nadezhdin, K D; Bocharova, O V; Bocharov, E V; Arseniev, A S

    2011-01-01

    Alzheimer's disease affects people all over the world, regardless of nationality, gender or social status. An adequate study of the disease requires essential understanding of the molecular fundamentals of the pathogenesis. The amyloid β-peptide, which forms amyloid plaques in the brain of people with Alzheimer's disease, is the product of sequential cleavage of a single-span membrane amyloid precursor protein (APP). More than half of the APP mutations found to be associated with familial forms of Alzheimer's disease are located in its transmembrane domain. The pathogenic mutations presumably affect the structural-dynamic properties of the APP transmembrane domain by changing its conformational stability and/or lateral dimerization. In the present study, the structure and dynamics of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined in the membrane mimetic environment composed of detergent micelles using NMR spectroscopy. The structure obtained in dodecylphosphocholine micelles consists of two α-helices: a short surface-associated juxtamembrane helix (Lys687-Asp694) and a long transmembrane helix (Gly700-Leu723), both connected via a mobile loop region. A minor bend of the transmembrane α-helix is observed near the paired residues Gly708-Gly709. A cholesterol-binding hydrophobic cavity is apparently formed under the loop region, where the juxtamembrane α-helix comes into contact with the membrane surface near the N-terminus of the transmembrane α-helix. PMID:22649674

  5. The transmembrane domain of TACE regulates protein ectodomain shedding

    Institute of Scientific and Technical Information of China (English)

    Xiaojin Li; Liliana Pérez; Zui Pan; Huizhou Fan

    2007-01-01

    Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme (TACE), which causes the release of their ectodomains. An ADAM (a disintegrin and metalloprotease domain) family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain (TM) of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylino-sitol (GPI)-binding polypeptide failed to restore shedding of transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α) and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor (PDGFR) also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-a with that of L-selectin enabled TGF-a shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.

  6. Isolated Toll-like receptor transmembrane domains are capable of oligomerization.

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    James I Godfroy

    Full Text Available Toll-like receptors (TLRs act as the first line of defense against bacterial and viral pathogens by initiating critical defense signals upon dimer activation. The contribution of the transmembrane domain in the dimerization and signaling process has heretofore been overlooked in favor of the extracellular and intracellular domains. As mounting evidence suggests that the transmembrane domain is a critical region in several protein families, we hypothesized that this was also the case for Toll-like receptors. Using a combined biochemical and biophysical approach, we investigated the ability of isolated Toll-like receptor transmembrane domains to interact independently of extracellular domain dimerization. Our results showed that the transmembrane domains had a preference for the native dimer partners in bacterial membranes for the entire receptor family. All TLR transmembrane domains showed strong homotypic interaction potential. The TLR2 transmembrane domain demonstrated strong heterotypic interactions in bacterial membranes with its known interaction partners, TLR1 and TLR6, as well as with a proposed interaction partner, TLR10, but not with TLR4, TLR5, or unrelated transmembrane receptors providing evidence for the specificity of TLR2 transmembrane domain interactions. Peptides for the transmembrane domains of TLR1, TLR2, and TLR6 were synthesized to further study this subfamily of receptors. These peptides validated the heterotypic interactions seen in bacterial membranes and demonstrated that the TLR2 transmembrane domain had moderately strong interactions with both TLR1 and TLR6. Combined, these results suggest a role for the transmembrane domain in Toll-like receptor oligomerization and as such, may be a novel target for further investigation of new therapeutic treatments of Toll-like receptor mediated diseases.

  7. A unique phenylalanine in the transmembrane domain strengthens homodimerization of the syndecan-2 transmembrane domain and functionally regulates syndecan-2.

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    Kwon, Mi-Jung; Choi, Youngsil; Yun, Ji-Hye; Lee, Weontae; Han, Inn-Oc; Oh, Eok-Soo

    2015-02-27

    The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe(167)) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe(167) was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe(167) in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.

  8. Neuregulin 1 expression and electrophysiological abnormalities in the Neuregulin 1 transmembrane domain heterozygous mutant mouse.

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    Leonora E Long

    Full Text Available The Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile.Using exonic probes spanning the cysteine-rich, epidermal growth factor (EGF-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine.In both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice.We found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function.

  9. Research Advances in CKLFSF-like MARVEL Transmembrane Domain Containing Member 3.

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    2016-06-10

    CKLF-like MARVEL transmembrane domain containing member/chemokine-like factor super family member (CKLFSF/CMTM) is a novel tumor suppressor gene. CMTM3 is broadly expressed in normal human tissues and evolutionary conserved,especially in testis,spleen,and some cells of peripheral blood mononuclear cells. However,its expression is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM3 may inhibit the proliferation,migration,and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear,CKLFSF3/CMTM3 is closely connected with immune system and associated with sex during tumorigenesis. The study advances of CKLFSF3/CMTM3 are elaborated in this review as CMTM3 may be a new target in the gene therapies for tumors,especially genitourinary tumors,while further studies on CMTM3 and its anti-tumor mechanisms are warranted. PMID:27469927

  10. Species-specific activity of HIV-1 Vpu and positive selection of tetherin transmembrane domain variants.

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    Matthew W McNatt

    2009-02-01

    Full Text Available Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh, African green monkeys (agm and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins.

  11. Membrane-spanning domain of bovine foamy virus transmembrane protein having cytotoxicity

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    MA Yonggang; YU Hong; WANG Jinzhong; CHEN Qimin; GENG Yunqi

    2006-01-01

    Foamy viruses (FVs) have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane (TM) proteins include a region of hydrophobic domain called membrane-spanning domain (MSD),which contains several stretches of hydrophobic amino acids.To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli,a series of mutants were constructed and expressed in the E.coli BL21 (DE3) using pET-32a (+) as expressing vector.The results showed that only TM3 without MSD was expressed in E.coli,whereas the other two containing full or part of the MSD (TM1 and TM2) could not be expressed.Furthermore,the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD,especially on the stretches of hydrophobic amino acids.Western blotting analysis showed that TM3 protein was purified with affinity purification.

  12. Achondroplastic dog breeds have no mutations in the transmembrane domain of the FGFR-3 gene.

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    Martínez, S; Valdés, J; Alonso, R A

    2000-10-01

    One of the most common skeletal affections in humans is achondroplasia, a short-limbed dwarfism that is, in most cases, caused by mutations in the transmembrane domain of the fibroblast growth factor receptor-3 (FGFR-3) gene. Due to the lack of sufficient radiological, genetic, and molecular studies, most types of skeletal anomalies in dogs are classified as achondroplasia. To initiate the molecular characterization of some osteochondrodysplastic dog breeds, we obtained the DNA sequence of the transmembrane domain of the FGFR-3 gene from the dachshund, basset hound, bulldog, and German shepherd dogs. All 4 breeds showed no mutation in the evaluated region. This indicates that the mutation responsible for the osteochondrodysplastic phenotype in the tested dog breeds lies either elsewhere in the FGFR-3 gene or in other ones involved in the formation and development of endochondral bone. PMID:11041504

  13. Achondroplastic dog breeds have no mutations in the transmembrane domain of the FGFR-3 gene.

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    S. Martínez; Valdés, J; R. A. Alonso

    2000-01-01

    One of the most common skeletal affections in humans is achondroplasia, a short-limbed dwarfism that is, in most cases, caused by mutations in the transmembrane domain of the fibroblast growth factor receptor-3 (FGFR-3) gene. Due to the lack of sufficient radiological, genetic, and molecular studies, most types of skeletal anomalies in dogs are classified as achondroplasia. To initiate the molecular characterization of some osteochondrodysplastic dog breeds, we obtained the DNA sequence of th...

  14. Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex

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    Lin Chi-Hui

    2008-01-01

    Full Text Available Abstract Background To study the organization and interaction with the fusion domain (or fusion peptide, FP of the transmembrane domain (TMD of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. Results The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. Conclusion The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

  15. Striated domains: self-organizing ordered assemblies of transmembrane alpha-helical peptides and lipids in bilayers.

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    de Kruijff, Ben; Killian, J Antoinette; Ganchev, Dragomir N; Rinia, Hilde A; Sparr, Emma

    2006-03-01

    This review summarizes the knowledge on striated domains, which are ordered assemblies of transmembrane peptides and lipids under gel-state conditions. The structure, mechanism of function and utility of this system as a model for domain formation is described, resulting in a molecular description of the domains and a discussion on the relevance of these insights for the function/formation and structure of similar domains in biological membranes. PMID:16542143

  16. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    International Nuclear Information System (INIS)

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function

  17. Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

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    Montserrat Samsó

    2009-04-01

    Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

  18. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

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    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  19. Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains.

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    Lu, Xiaohui; Gross, Alec W; Lodish, Harvey F

    2006-03-17

    In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.

  20. Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers.

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    Sparr, Emma; Ganchev, Dragomir N; Snel, Margot M E; Ridder, Anja N J A; Kroon-Batenburg, Loes M J; Chupin, Vladimir; Rijkers, Dirk T S; Killian, J Antoinette; de Kruijff, Ben

    2005-01-11

    Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions. PMID:15628840

  1. Packing Density of the Erythropoietin Receptor Transmembrane Domain Correlates with Amplification of Biological Responses

    Energy Technology Data Exchange (ETDEWEB)

    Becker, Verena [German Cancer Research Center, Heidelberg; Sengupta, D [University of Heidelberg; Ketteler, Robin [German Cancer Research Center, Heidelberg; Ullmann, G. Matthias [University of Bayreuth; Smith, Jeremy C [ORNL; Klingmuller, Ursula [German Cancer Research Center, Heidelberg

    2008-10-01

    The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions.

  2. v-SNARE transmembrane domains function as catalysts for vesicle fusion.

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    Dhara, Madhurima; Yarzagaray, Antonio; Makke, Mazen; Schindeldecker, Barbara; Schwarz, Yvonne; Shaaban, Ahmed; Sharma, Satyan; Böckmann, Rainer A; Lindau, Manfred; Mohrmann, Ralf; Bruns, Dieter

    2016-01-01

    Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion. PMID:27343350

  3. The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans.

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    Kwon, Mi-Jung; Park, Jisu; Jang, Sinae; Eom, Chi-Yong; Oh, Eok-Soo

    2016-01-01

    The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.

  4. Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature.

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    Ho, Chian Sing; Khadka, Nawal K; She, Fengyu; Cai, Jianfeng; Pan, Jianjun

    2016-07-01

    Targeting host cell membranes by M2 of influenza A virus is important for virus invasion and replication. We study the transmembrane domain of M2 (M2TM) interacting with mica-supported planar bilayers and free-standing giant unilamellar vesicles (GUVs). Using solution atomic force microscopy (AFM), we show that the size of M2TM oligomers is dependent on lipid composition. The addition of M2TM to lipid bilayers containing liquid-ordered (Lo) and liquid-disordered (Ld) phases reveals that M2TM preferentially partitions into the Ld phase; phase-dependent partitioning results in a larger rigidity of the Ld phase. We next use fluorescence microscopy to study the effects of M2TM on phase-coexisting GUVs. In particular, M2TM is found to increase GUVs' miscibility transition temperature Tmix. The augmented thermodynamic stability can be accounted for by considering an enhanced energy barrier of lipid mixing between coexisting phases. Our GUV study also shows that M2TM can elicit an array of vesicle shapes mimicking virus budding. M2TM enhanced membrane curvature is consistent with our AFM data, which show altered membrane rigidity and consequently line tension at domain edges. Together, our results highlight that in addition to conducting protons, M2TM can actively regulate membrane heterogeneity and augment membrane curvature. PMID:27285399

  5. Domain Bridging Associations Support Creativity

    OpenAIRE

    Kötter, Tobias; Thiel, Kilian; Berthold, Michael R

    2010-01-01

    This paper proposes a new approach to support creativity through assisting the discovery of unexpected associations across different domains. This is achieved by integrating information from heterogeneous domains into a single network, enabling the interactive discovery of links across the corresponding information resources. We discuss three different pattern of domain crossing associations in this context.

  6. The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction.

    Science.gov (United States)

    Gur, H; Geppert, T D; Wacholtz, M C; Lipsky, P E

    1999-02-01

    Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.

  7. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

  8. An empirical energy function for structural assessment of protein transmembrane domains.

    Science.gov (United States)

    Postic, Guillaume; Ghouzam, Yassine; Gelly, Jean-Christophe

    2015-08-01

    Knowing the structure of a protein is essential to characterize its function and mechanism at the molecular level. Despite major advances in solving structures experimentally, most membrane protein native conformations remain unknown. This lack of available structures, along with the physical constraints imposed by the lipid bilayer environment, constitutes a difficulty for the modeling of membrane protein structures. Assessing the quality of membrane protein models is therefore critical. Using a non-redundant set of 66 membrane protein structures (41 alpha and 25 beta), we have developed an empirical energy function for the structural assessment of alpha-helical and beta-sheet transmembrane domains. This statistical potential quantifies the interatomic distance between residues located in the lipid bilayer. To minimize the problem of insufficient sampling, we have used kernel density estimations of the distance distributions. Following a leave-one-out cross-validation procedure, we show that our method outperforms current statistical potentials in discriminating correct from incorrect membrane protein models. Furthermore, the comparison of our distance-dependent statistical potential with one optimized on globular proteins provides insights into the rules by which residues interact within the lipid bilayer. PMID:26044650

  9. v-SNARE transmembrane domains function as catalysts for vesicle fusion

    Science.gov (United States)

    Dhara, Madhurima; Yarzagaray, Antonio; Makke, Mazen; Schindeldecker, Barbara; Schwarz, Yvonne; Shaaban, Ahmed; Sharma, Satyan; Böckmann, Rainer A; Lindau, Manfred

    2016-01-01

    Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion. DOI: http://dx.doi.org/10.7554/eLife.17571.001 PMID:27343350

  10. Solution structure of the transmembrane domain of the insulin receptor in detergent micelles.

    Science.gov (United States)

    Li, Qingxin; Wong, Ying Lei; Kang, CongBao

    2014-05-01

    The insulin receptor (IR) binds insulin and plays important roles in glucose homeostasis by regulating the tyrosine kinase activity at its C-terminus. Its transmembrane domain (TMD) is shown to be important for transferring conformational changes induced by insulin across the cell membrane to regulate kinase activity. In this study, a construct IR(940-988) containing the TMD was expressed and purified for structural studies. Its solution structure in dodecylphosphocholine (DPC) micelles was determined. The sequence containing residues L962 to Y976 of the TMD of the IR in micelles adopts a well-defined helical structure with a kink formed by glycine and proline residues present at its N-terminus, which might be important for its function. Paramagnetic relaxation enhancement (PRE) and relaxation experimental results suggest that residues following the TMD are flexible and expose to aqueous solution. Although purified IR(940-988) in micelles existed mainly as a monomeric form verified by gel filtration and relaxation analysis, cross-linking study suggests that it may form a dimer or oligomers under micelle conditions.

  11. Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels.

    Science.gov (United States)

    Gebhardt, Manuela; Henkes, Leonhard M; Tayefeh, Sascha; Hertel, Brigitte; Greiner, Timo; Van Etten, James L; Baumeister, Dirk; Cosentino, Cristian; Moroni, Anna; Kast, Stefan M; Thiel, Gerhard

    2012-07-17

    Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K(+) channels. Experimentally, snorkeling activity is not mandatory for Kcv(PBCV-1) because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv(ATCV-1) and Kcv(MT325), lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv(PBCV-1) and N-terminally truncated mutants; the truncated mutants mimic Kcv(ATCV-1) and Kcv(MT325). Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K(+) channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains.

  12. Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

    OpenAIRE

    Melikyan, Grigory B.; Lin, Sasa; Roth, Michael G.; Cohen, Fredric S.

    1999-01-01

    The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity w...

  13. Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

    Directory of Open Access Journals (Sweden)

    Balda Maria S

    2009-12-01

    Full Text Available Abstract Background Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties.

  14. Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.)

    Science.gov (United States)

    Satheesh, Viswanathan; Chidambaranathan, Parameswaran; Jagannadham, Prasanth Tejkumar; Kumar, Vajinder; Jain, Pradeep K.; Chinnusamy, Viswanathan; Bhat, Shripad R.; Srinivasan, R.

    2016-01-01

    Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response. PMID:26445326

  15. Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters.

    Science.gov (United States)

    Wen, Po-Chao; Tajkhorshid, Emad

    2011-08-01

    Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree

  16. Transmembrane domain V plays a stabilizing role in the function of human bile acid transporter SLC10A2.

    Science.gov (United States)

    Moore, Robyn H; Chothe, Paresh; Swaan, Peter W

    2013-07-30

    The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2), primarily expressed in the ileum, is involved in both the recycling of bile acids and cholesterol homeostasis. In this study, the structure-function relationship of transmembrane domain 5 (TM5) residues involved in transport is elucidated. Cysteine scanning mutagenesis of each consecutive residue on TM5 resulted in 96% of mutants having a significantly decreased transport activity, although each was expressed at the cell surface. Specifically, G197 and I208 were no longer functional, and G201 and G212 functioned at a level of Conservative alanine mutations of the four residues displayed a higher activity in all but G197A, indicating its functional importance. G197 and G201 form a GxxxG motif, which has been found to be important in helix-helix interactions. According to our model, G197 and G201 face transmembrane domain 4 (TM4) residues G179 and P175, respectively. Similarly, G212 faces G237, which forms part of a GxxxG domain in transmembrane domain 6 (TM6). It is possible that these GxxxG domains and their interacting partners are responsible for maintaining the structure of the helices and their interactions with one another. I205 and I208 are both in positions to anchor the GxxxG domains and direct the change in interaction of TM5 from TM4 to TM6. Combined, the results suggest that residues along TM5 are critical for ASBT function but are not directly involved in substrate translocation.

  17. A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

    Directory of Open Access Journals (Sweden)

    Haga Christopher L

    2007-09-01

    Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

  18. On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study.

    Science.gov (United States)

    Belmonte, Luca; Moran, Oscar

    2015-04-01

    The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations cause cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process. PMID:25640670

  19. Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups.

    Science.gov (United States)

    Vincent, Nadine; Genin, Christian; Malvoisin, Etienne

    2002-12-23

    A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.

  20. The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

    Energy Technology Data Exchange (ETDEWEB)

    Ghoshal, Kankana [University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 (Canada); Theilmann, Jane; Reade, Ron; Sanfacon, Helene [Agriculture and Agri-Food Canada Pacific Agri-Food Research Centre, 4200 Hwy 97, Summerland, British Columbia, Canada V0H 1Z0 (Canada); Rochon, D’Ann, E-mail: dann.rochon@agr.gc.ca [University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 (Canada); Agriculture and Agri-Food Canada Pacific Agri-Food Research Centre, 4200 Hwy 97, Summerland, British Columbia, Canada V0H 1Z0 (Canada)

    2014-11-15

    Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication.

  1. The intramembrane cleavage site of the amyloid precursor protein depends on the length of its transmembrane domain

    OpenAIRE

    Lichtenthaler, Stefan F.; Beher, Dirk; Heike S Grimm; Wang, Rong; Shearman, Mark S.; Masters, Colin L.; Beyreuther, Konrad

    2002-01-01

    Proteolytic processing of the amyloid precursor protein by β-secretase generates C99, which subsequently is cleaved by γ-secretase, yielding the amyloid β peptide (Aβ). This γ-cleavage occurs within the transmembrane domain (TMD) of C99 and is similar to the intramembrane cleavage of Notch. However, Notch and C99 differ in their site of intramembrane cleavage. The main γ-cleavage of C99 occurs in the middle of the TMD, whereas the cleavage of Notch occurs close to the C-terminal end of the TM...

  2. Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

    DEFF Research Database (Denmark)

    Sikder, K. U.; Stone, K. A.; Kumar, P. B. S.;

    2014-01-01

    We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that mic...... that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. (C) 2014 AIP Publishing LLC.......We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find...

  3. Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

    Science.gov (United States)

    Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

    2014-08-01

    We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells.

  4. Assembly of the transmembrane domain of E. coli PhoQ histidine kinase: implications for signal transduction from molecular simulations.

    Directory of Open Access Journals (Sweden)

    Thomas Lemmin

    Full Text Available The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.

  5. Structures of OppA and PstS from Yersinia pestis indicate variability of interactions with transmembrane domains

    DEFF Research Database (Denmark)

    Tanabe, Mikio; Mirza, Osman; Bertrand, Thomas;

    2007-01-01

    Bacterial ATP-binding cassette (ABC) transport systems couple ATP hydrolysis with the uptake and efflux of a wide range of substances across bacterial membranes. These systems are comprised of transmembrane domains, nucleotide binding domains and, in the case of uptake systems, periplasmic bindin...

  6. Transmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation.

    Science.gov (United States)

    Sabit, Hairat; Mallajosyula, Sairam S; MacKerell, Alexander D; Swaan, Peter W

    2013-11-01

    Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln(75), Phe(76), Met(79), Gly(83), Leu(86), Phe(90), and Asp(91) in hASBT function. Computational analysis indicated that Asp(91) may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr(134), Leu(138), and Thr(149)) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln(75), Met(79), Thr(82), and Leu(86) from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.

  7. Graph-Theoretic Models of Mutations in the Nucleotide Binding Domain 1 of the Cystic Fibrosis Transmembrane Conductance Regulator

    Directory of Open Access Journals (Sweden)

    Debra J. Knisley

    2013-01-01

    Full Text Available Cystic fibrosis is one of the most common inherited diseases and is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR. This protein serves as a chloride channel and regulates the viscosity of mucus lining the ducts of a number of organs. Although much has been learned about the consequences of mutations on the energy landscape and the resulting disrupted folding pathway of CFTR, a level of understanding needed to correct the misfolding has not been achieved. The most common mutations of CFTR are located in one of two nucleotide binding domains, namely, the nucleotide binding domain 1 (NBD1. We model NBD1 using a nested graph model. The vertices in the lowest layer each represent an atom in the structure of an amino acid residue, while the vertices in the mid layer each represent the residue. The vertices in the top layer each represent a subdomain of the nucleotide binding domain. We use this model to quantify the effects of a single point mutation on the protein domain. We compare the wildtype structure with eight of the most common mutations. The graph-theoretic model provides insight into how a single point mutation can have such profound structural consequences.

  8. R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

    Science.gov (United States)

    Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

    2016-01-01

    The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport. PMID:26849958

  9. Autonomous Transmembrane Segment S4 of the Voltage Sensor Domain Partitions into the Lipid Membrane

    Science.gov (United States)

    Tiriveedhi, Venkataswarup; Miller, Melissa; Butko, Peter; Li, Min

    2012-01-01

    The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 ± 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 Å. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence. PMID:22465069

  10. Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

    Energy Technology Data Exchange (ETDEWEB)

    Zou Chao [University of Zurich, Institute of Organic Chemistry (Switzerland); Naider, Fred [College of Staten Island, CUNY, Department of Chemistry (United States); Zerbe, Oliver [University of Zurich, Institute of Organic Chemistry (Switzerland)], E-mail: oliver.zerbe@oci.uzh.ch

    2008-12-15

    The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His){sub 6} tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

  11. Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion

    OpenAIRE

    Liu, Tina Y.; Bian, Xin; Sun, Sha; Hu, Xiaoyu; Klemm, Robin W.; Prinz, William A; Rapoport, Tom A.; Hu, Junjie

    2012-01-01

    The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a C-terminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles. The essenti...

  12. Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W., E-mail: n.isaacs@chem.gla.ac.uk [Department of Chemistry and WestChem, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    2007-07-01

    P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.

  13. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  14. Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis.

    Science.gov (United States)

    Chen, Zhongying; Noir, Sandra; Kwaaitaal, Mark; Hartmann, H Andreas; Wu, Ming-Jing; Mudgil, Yashwanti; Sukumar, Poornima; Muday, Gloria; Panstruga, Ralph; Jones, Alan M

    2009-07-01

    Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane-localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gbeta subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.

  15. Short transmembrane domains with high-volume exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex.

    Science.gov (United States)

    Quiroga, Rodrigo; Trenchi, Alejandra; González Montoro, Ayelén; Valdez Taubas, Javier; Maccioni, Hugo J F

    2013-12-01

    It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the Golgi SNARE protein Sft1 and the plasma membrane SNARE protein Sso1 from Saccharomyces cerevisiae as model proteins, we modified the length of their TMDs and the volume of their exoplasmic hemi-TMD, and determined their subcellular localization both in yeast and mammalian cells. We found that short TMDs with high-volume exoplasmic hemi-TMDs confer Golgi membrane residence, whereas TMDs with low-volume exoplasmic hemi-TMDs, either short or long, confer plasma membrane residence to these proteins. Results indicate that the shape of the exoplasmic hemi-TMD, in addition to the length of the entire TMD, determine retention in the Golgi or exit to the plasma membrane of Type II membrane proteins.

  16. Epidermal growth factor, latrophilin, and seven transmembrane domain-containing protein 1 marker, a novel angiogenesis marker

    Science.gov (United States)

    Serban, Florentina; Artene, Stefan-Alexandru; Georgescu, Ada Maria; Purcaru, Stefana Oana; Tache, Daniela Elise; Alexandru, Oana; Dricu, Anica

    2015-01-01

    Epidermal growth factor, latrophilin, and seven transmembrane domain-containing protein 1 on chromosome 1 (ELTD1), an orphan adhesion G-protein coupled receptor, was reported as a regulator of angiogenesis, also involved in cancer progression and development. More recently, ELTD1 was identified as a potential new tumor marker for high-grade glioma. ELTD1, belongs to the G-protein coupled receptor superfamily that comprises the biggest receptor family in the human genome. Following the discovery of ELTD1 almost a decade ago, only a few research groups have attempted to find its role in normal and tumor cells, important information about this receptor remaining still unknown. The ELTD1 ligand has not currently been identified and intracellular signaling studies have not yet been performed in normal or tumor cells. Although the current published data on ELTD1 function and structure are rather limited, this receptor seems to be very important, not only as biomarker, but also as molecular target in glioblastoma. This review summarizes and discusses the current knowledge on ELTD1 structure, function, and its role in both physiological and tumoral angiogenesis. PMID:26719704

  17. The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

    Institute of Scientific and Technical Information of China (English)

    Tosikazu AMANO; Olivia KWAK; Liezhen FU; Anastasia MARSHAK; Yun-Bo SHI

    2005-01-01

    The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However,like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites,distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demonstrated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior observed during development and pathogenesis.

  18. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    Science.gov (United States)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  19. Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1.

    Science.gov (United States)

    Zhang, Yunlong; Zhang, Xiuping; Qu, Shaogang

    2014-07-01

    Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure-function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd(2+)) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + L-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

  20. VAMP-associated Proteins (VAP) as Receptors That Couple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Proteostasis with Lipid Homeostasis.

    Science.gov (United States)

    Ernst, Wayne L; Shome, Kuntala; Wu, Christine C; Gong, Xiaoyan; Frizzell, Raymond A; Aridor, Meir

    2016-03-01

    Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.

  1. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

    Directory of Open Access Journals (Sweden)

    Steffen Kawelke

    Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

  2. Several novel nuclear envelope transmembrane proteins identified in skeletal muscle have cytoskeletal associations.

    Science.gov (United States)

    Wilkie, Gavin S; Korfali, Nadia; Swanson, Selene K; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R W; Florens, Laurence; Schirmer, Eric C

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  3. Role of the sixth transmembrane segment of domain IV of the cockroach sodium channel in the action of sodium channel-blocker insecticides

    OpenAIRE

    Silver, Kristopher S.; Nomura, Yoshiko; Salgado, Vincent L.; Dong, Ke

    2009-01-01

    Sodium channel-blocker insecticides (SCBIs), such as indoxacarb and metaflumizone, are a new class of insecticides with a mechanism of action different from those of other insecticides that target sodium channels. SCBIs block sodium channels in a manner similar to local anesthetics (LA) such as lidocaine. Several residues, particularly F1579 and Y1586, in the sixth transmembrane segment (S6) of domain IV (IV) of rat Nav1.4 sodium channels are required for the action of LAs and SCBIs and may f...

  4. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    OpenAIRE

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine res...

  5. Graph-Theoretic Models of Mutations in the Nucleotide Binding Domain 1 of the Cystic Fibrosis Transmembrane Conductance Regulator

    OpenAIRE

    Knisley, Debra J.; Knisley, Jeff R; Andrew Cade Herron

    2013-01-01

    Cystic fibrosis is one of the most common inherited diseases and is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR). This protein serves as a chloride channel and regulates the viscosity of mucus lining the ducts of a number of organs. Although much has been learned about the consequences of mutations on the energy landscape and the resulting disrupted folding pathway of CFTR, a level of understanding needed to correct the misfolding ...

  6. Three ways in, one way out: water dynamics in the trans-membrane domains of the inner membrane translocase AcrB.

    Science.gov (United States)

    Fischer, Nadine; Kandt, Christian

    2011-10-01

    Powered by proton-motive force, the inner membrane translocase AcrB is the engine of the AcrAB-TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen-bonded networks of polar residues and water molecules, knowledge of the protein-internal water distribution and water-interacting residues allows drawing conclusions to possible pathways of proton conduction. Here, we report a series of 6× 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment. Simulating each monomer in its proposed protonation state, we calculated for each trans-membrane domain the average water distribution, identified residues interacting with these waters and quantified each residue's frequency of water hydrogen bond contact. Combining this information we find three possible routes of proton transfer connecting a continuously hydrated region of known key residues in the TMD interior to bulk water by one cytoplasmic and up to three periplasm water channels in monomer B and A. We find that water access of the trans-membrane domains is regulated by four groups of residues in a combination of side chain re-orientations and shifts of trans-membrane helices. Our findings support a proton release event via Arg971 during the C intermediate or in the transition to A, and proton uptake occurring in the A or B state or during a so far unknown intermediate in between B and C where cytoplasmic water access is still possible. Our simulations suggest experimentally testable hypotheses, which have not been investigated so far. PMID:21905112

  7. Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Thea Bismo Strøm

    2014-01-01

    Full Text Available More than 1700 mutations in the low density lipoprotein receptor (LDLR gene have been found to cause familial hypercholesterolemia (FH. These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

  8. Valine 738 and lysine 735 in the fifth transmembrane domain of rTas1r3 mediate insensitivity towards lactisole of the rat sweet taste receptor

    Directory of Open Access Journals (Sweden)

    Meyerhof Wolfgang

    2005-04-01

    Full Text Available Abstract Background The sweet taste inhibitor lactisole acts on the human sweet taste receptor heteromer TAS1R2-TAS1R3 but not on its rodent counterpart. Recently, it was shown that the lactisole sensitivity of the human sweet taste receptor involves the part of TAS1R3 encompassing the seven transmembrane regions but not the huge N-terminal domain. Using mutational analysis we investigated which amino acid residues distinguish lactisole insensitive rat from sensitive human T1R3 receptors. Results The functional analysis of specific receptor mutants in HEK293T cells revealed that the exchange of valine 738 in the fifth transmembrane domain of rTas1r3 by an alanine is sufficient to confer lactisole sensitivity to the rat sweet taste receptor. The sensitivity of this receptor mutant is ~2 fold lower than the sensitivity of the human sweet taste receptor. Additional substitution of lysine 735 by phenylalanine in rTas1r3 results in a rat sweet taste receptor that is as sensitive to lactisole as its human counterpart. The exchange of valine 738 to alanine was accompanied by a ~50% reduction in receptor efficacy. This effect was seen with all six different sweet compounds examined. Conclusion The lactisole insensitivity of rat sweet taste receptor is caused by only two amino acids in transmembrane region five, which is critical for the interaction of lactisole with the sweet taste receptor. The observation that the mutant receptor simultaneously displays a generally reduced sensitivity towards all agonists suggests that the lactisole insensitivity of the rodent receptor might be more likely caused by the inaccessibility of the lactisole binding site rather then by its direct disruption.

  9. Transmembrane recognition of the semaphorin co-receptors neuropilin 1 and plexin A1: coarse-grained simulations.

    Directory of Open Access Journals (Sweden)

    Samia Aci-Sèche

    Full Text Available The cancer associated class 3 semaphorins require direct binding to neuropilins and association to plexins to trigger cell signaling. Here, we address the role of the transmembrane domains of neuropilin 1 and plexin A1 for the dimerization of the two receptors by characterizing the assembly in lipid bilayers using coarse-grained molecular dynamics simulations. From experimental evidence using a two-hybrid system showing the biochemical association of the two receptors transmembrane domains, we performed molecular simulations in DOPC and POPC demonstrating spontaneously assembly to form homodimers and heterodimers with a very high propensity for right-handed packing of the helices. Inversely, left-handed packing was observed with a very low propensity. This mode of packing was observed uniquely when the plexin A1 transmembrane domain was involved in association. Potential of mean force calculations were used to predict a hierarchy of self-association for the monomers: the two neuropilin 1 transmembrane domains strongly associated, neuropilin 1 and plexin A1 transmembrane domains associated less and the two plexin A1 transmembrane domains weakly but significantly associated. We demonstrated that homodimerization and heterodimerization are driven by GxxxG motifs, and that the sequence context modulates the packing mode of the plexin A1 transmembrane domains. This work presents major advances towards our understanding of membrane signaling platforms assembly through membrane domains and provides exquisite information for the design of antagonist drugs defining a novel class of therapeutic agents.

  10. Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its correlation with the differentiation status of hepatocellular carcinoma

    OpenAIRE

    Peng, Cong; Zhou, Rou-Li; Shao, Gen-Ze; Rui, Jing-An; Wang, Shao-Bin; Lin, Ming; Zhang, Sha; Gao, Zi-Feng

    2005-01-01

    AIM: To produce high-quality polyclonal antibody to lysosome-associated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC).

  11. MAST205 competes with cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand for binding to CFTR to regulate CFTR-mediated fluid transport.

    Science.gov (United States)

    Ren, Aixia; Zhang, Weiqiang; Yarlagadda, Sunitha; Sinha, Chandrima; Arora, Kavisha; Moon, Chang-Suk; Naren, Anjaparavanda P

    2013-04-26

    The PDZ (postsynaptic density-95/discs large/zona occludens-1) domain-based interactions play important roles in regulating the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR). Several PDZ domain-containing proteins (PDZ proteins for short) have been identified as directly or indirectly interacting with the C terminus of CFTR. To better understand the regulation of CFTR processing, we conducted a genetic screen and identified MAST205 (a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa) as a new CFTR regulator. We found that overexpression of MAST205 increased the expression of CFTR and augmented CFTR-mediated fluid transport in a dose-dependent manner. Conversely, knockdown of MAST205 inhibited CFTR function. The PDZ motif of CFTR is required for the regulatory role of MAST205 in CFTR expression and function. We further demonstrated that MAST205 and the CFTR-associated ligand competed for binding to CFTR, which facilitated the processing of CFTR and consequently up-regulated the expression and function of CFTR at the plasma membrane. More importantly, we found that MAST205 could facilitate the processing of F508del-CFTR mutant and augment its quantity and channel function at the plasma membrane. Taken together, our data suggest that MAST205 plays an important role in regulating CFTR expression and function. Our findings have important clinical implications for treating CFTR-associated diseases such as cystic fibrosis and secretory diarrheas.

  12. The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region.

    Science.gov (United States)

    Apellániz, Beatriz; Rujas, Edurne; Serrano, Soraya; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M M; Jiménez, M Ángeles; Nieva, José L

    2015-05-22

    The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

  13. Rapidly evolving in humans topologically associating domains

    OpenAIRE

    Glinsky, Gennadi

    2015-01-01

    Genome-wide proximity placement analysis of 10,598 HSGRL within the context of the principal regulatory structures of the interphase chromatin, namely topologically-associating domains (TADs) and specific sub-TAD structures termed super-enhancer domains (SEDs) revealed that 0.8%-10.3% of TADs contain more than half of HSGRL. Of the 3,127 TADs in the hESC genome, 24 (0.8%); 53 (1.7%); 259 (8.3%); and 322 (10.3%) harbor 1,110 (52.4%); 1,936 (50.9%); 1,151 (59.6%); and 1,601 (58.3%) HSGRL sequen...

  14. Distinct phenotypes of new transmembrane-domain neuregulin 1 mutant mice and the rescue effects of valproate on the observed schizophrenia-related cognitive deficits.

    Directory of Open Access Journals (Sweden)

    Ju-Chun ePei

    2014-04-01

    Full Text Available Accumulating evidence suggests that neuregulin 1 (NRG1 might be involved in the neurodevelopment, neural plasticity, GABAergic neurotransmission and pathogenesis of schizophrenia. NRG1 is abundantly expressed in the hippocampus, and emerging studies have begun to reveal the link between NRG1 signaling and cognitive deficits in schizophrenic patients. Because the transmembrane domain of NRG1 is vital for both forward and reverse signaling cascades, new Nrg1-deficient mice that carry a truncation of the transmembrane domain of the Nrg1 gene were characterized and used in this study to test a NRG1 loss-of-function hypothesis for schizophrenia. Both male and female Nrg1 heterozygous mutant mice and their wild-type littermates were used in a series of 4 experiments to characterize the impact of Nrg1 on behavioral phenotypes and to determine the importance of Nrg1 in the regulation of hippocampal neuromorphology and local GABAergic interneurons. First, a comprehensive battery of behavioral tasks indicated that male Nrg1-deficient mice exhibited significant impairments in cognitive functions. Second, pharmacological challenges were conducted and revealed that Nrg1 haploinsufficiency altered GABAergic activity in males. Third, although no genotype-specific neuromorphological alterations were found in the hippocampal CA1 pyramidal neurons, significant reductions in the hippocampal expressions of GAD67 and parvalbumin were revealed in the Nrg1-deficient males. Fourth, chronic treatment with valproate rescued the observed behavioral deficits and hippocampal GAD67 reduction in Nrg1-deficient males. Collectively, these results indicate the potential therapeutic effect of valproate and the importance of Nrg1 in the regulation of cognitive functions and hippocampal GABAergic interneurons, especially in males.

  15. Associativity Between Feature Models Across Domains

    OpenAIRE

    Subramani, S; Gurumoorthy, B

    2003-01-01

    Associativity between feature models implies the automatic updating of different feature models of a part after changes are made in one of its feature models. This is an important requirement in a distributed and concurrent design environment, where integrity of part geometry has to be maintained through changes made in different task domains. The proposed algorithm takes multiple feature models of apart as input and modifies other feature models to reflect the changes made to a feature ...

  16. Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

    Institute of Scientific and Technical Information of China (English)

    Yan Cai; Jing Yang; He Huang; Fang Li; Ganqiu Wu; Jing Yang; Xuegang Luo

    2009-01-01

    BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood.OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins.DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008.MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coli (E. Coli) JM109 were purchased from Promega. E. Coil BL21 was provided by Novagen.METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. Coli BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained.MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods.RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then cloned into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass

  17. The interaction between the first transmembrane domain and the thumb of ASIC1a is critical for its N-glycosylation and trafficking.

    Directory of Open Access Journals (Sweden)

    Lan Jing

    Full Text Available Acid-sensing ion channel-1a (ASIC1a, the primary proton receptor in the brain, contributes to multiple diseases including stroke, epilepsy and multiple sclerosis. Thus, a better understanding of its biogenesis will provide important insights into the regulation of ASIC1a in diseases. Interestingly, ASIC1a contains a large, yet well organized ectodomain, which suggests the hypothesis that correct formation of domain-domain interactions at the extracellular side is a key regulatory step for ASIC1a maturation and trafficking. We tested this hypothesis here by focusing on the interaction between the first transmembrane domain (TM1 and the thumb of ASIC1a, an interaction known to be critical in channel gating. We mutated Tyr71 and Trp287, two key residues involved in the TM1-thumb interaction in mouse ASIC1a, and found that both Y71G and W287G decreased synaptic targeting and surface expression of ASIC1a. These defects were likely due to altered folding; both mutants showed increased resistance to tryptic cleavage, suggesting a change in conformation. Moreover, both mutants lacked the maturation of N-linked glycans through mid to late Golgi. These data suggest that disrupting the interaction between TM1 and thumb alters ASIC1a folding, impedes its glycosylation and reduces its trafficking. Moreover, reducing the culture temperature, an approach commonly used to facilitate protein folding, increased ASIC1a glycosylation, surface expression, current density and slowed the rate of desensitization. These results suggest that correct folding of extracellular ectodomain plays a critical role in ASIC1a biogenesis and function.

  18. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  19. RNF185 is a novel E3 ligase of endoplasmic reticulum-associated degradation (ERAD) that targets cystic fibrosis transmembrane conductance regulator (CFTR).

    Science.gov (United States)

    El Khouri, Elma; Le Pavec, Gwenaëlle; Toledano, Michel B; Delaunay-Moisan, Agnès

    2013-10-25

    In the endoplasmic reticulum (ER), misfolded or improperly assembled proteins are exported to the cytoplasm and degraded by the ubiquitin-proteasome pathway through a process called ER-associated degradation (ERAD). ER-associated E3 ligases, which coordinate substrate recognition, export, and proteasome targeting, are key components of ERAD. Cystic fibrosis transmembrane conductance regulator (CFTR) is one ERAD substrate targeted to co-translational degradation by the E3 ligase RNF5/RMA1. RNF185 is a RING domain-containing polypeptide homologous to RNF5. We show that RNF185 controls the stability of CFTR and of the CFTRΔF508 mutant in a RING- and proteasome-dependent manner but does not control that of other classical ERAD model substrates. Reciprocally, its silencing stabilizes CFTR proteins. Turnover analyses indicate that, as RNF5, RNF185 targets CFTR to co-translational degradation. Importantly, however, simultaneous depletion of RNF5 and RNF185 profoundly blocks CFTRΔF508 degradation not only during translation but also after synthesis is complete. Our data thus identify RNF185 and RNF5 as a novel E3 ligase module that is central to the control of CFTR degradation.

  20. A form of mitofusin 2 (Mfn2) lacking the transmembrane domains and the COOH-terminal end stimulates metabolism in muscle and liver cells.

    Science.gov (United States)

    Segalés, Jessica; Paz, José C; Hernández-Alvarez, María Isabel; Sala, David; Muñoz, Juan Pablo; Noguera, Eduard; Pich, Sara; Palacín, Manuel; Enríquez, José Antonio; Zorzano, Antonio

    2013-11-15

    Mitofusin 2 (Mfn2), a protein that participates in mitochondrial fusion, is required to maintain normal mitochondrial metabolism in skeletal muscle and liver. Given that muscle Mfn2 is repressed in obese or type 2 diabetic subjects, this protein may have a potential pathophysiological role in these conditions. To evaluate whether the metabolic effects of Mfn2 can be dissociated from its function in mitochondrial dynamics, we studied a form of human Mfn2, lacking the two transmembrane domains and the COOH-terminal coiled coil (ΔMfn2). This form localized in mitochondria but did not alter mitochondrial morphology in cells or in skeletal muscle fibers. The expression of ΔMfn2 in mouse skeletal muscle stimulated glucose oxidation and enhanced respiratory control ratio, which occurred in the absence of changes in mitochondrial mass. ΔMfn2 did not stimulate mitochondrial respiration in Mfn2-deficient muscle cells. The expression of ΔMfn2 in mouse liver or in hepatoma cells stimulated gluconeogenesis. In addition, ΔMfn2 activated basal and maximal respiration both in muscle and liver cells. In all, we show that a form of Mfn2 lacking mitochondrial fusion activity stimulates mitochondrial function and enhances glucose metabolism in muscle and liver tissues. This study suggests that Mfn2 regulates metabolism independently of changes in mitochondrial morphology.

  1. The SAX-3 receptor stimulates axon outgrowth and the signal sequence and transmembrane domain are critical for SAX-3 membrane localization in the PDE neuron of C. elegans.

    Directory of Open Access Journals (Sweden)

    Jia Li

    Full Text Available SAX-3, a receptor for Slit in C. elegans, is well characterized for its function in axonal development. However, the mechanism that regulates the membrane localization of SAX-3 and the role of SAX-3 in axon outgrowth are still elusive. Here we show that SAX-3::GFP caused ectopic axon outgrowth, which could be suppressed by the loss-of-function mutation in unc-73 (a guanine nucleotide exchange factor for small GTPases and unc-115 (an actin binding protein, suggesting that they might act downstream of SAX-3 in axon outgrowth. We also examined genes related to axon development for their possible involvement in the subcellular localization of SAX-3. We found the unc-51 mutants appeared to accumulate SAX-3::GFP in the neuronal cell body of the posterior deirid (PDE neuron, indicating that UNC-51 might play a role in SAX-3 membrane localization. Furthermore, we demonstrate that the N-terminal signal sequence and the transmembrane domain are essential for the subcellular localization of SAX-3 in the PDE neurons.

  2. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.

    Directory of Open Access Journals (Sweden)

    Zhen Gong

    Full Text Available The membrane proximal region (MPR, residues 649-683 and transmembrane domain (TMD, residues 684-705 of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683 of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705, in Escherichia coli as a fusion protein with maltose binding protein (MBP. MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM. Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

  3. Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5).

    Science.gov (United States)

    Georgieva, Elka R; Borbat, Peter P; Grushin, Kirill; Stoilova-McPhie, Svetla; Kulkarni, Nichita J; Liang, Zhichun; Freed, Jack H

    2016-01-01

    The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21-49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21-49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21-49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21-49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5-8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21-49 through increased motional ordering. In contrast to wild-type M2TMD21-49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain

  4. Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5)

    Science.gov (United States)

    Georgieva, Elka R.; Borbat, Peter P.; Grushin, Kirill; Stoilova-McPhie, Svetla; Kulkarni, Nichita J.; Liang, Zhichun; Freed, Jack H.

    2016-01-01

    The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation

  5. Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain

    International Nuclear Information System (INIS)

    The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation and invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer. - Highlights: • TM-JM1/2 peptides are efficiently delivered into cells by AuNP-Apt-conjugates. • TM-JM1/2 peptides loaded onto AuNP-Apt conjugates inhibit DDR2 activation. • Inhibition of DDR2 activation by TM-JM1/2 peptides decreases tumor progression

  6. Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Daehwan; Yeom, Ji-Hyun; Lee, Boeun; Lee, Kangseok [Department of Life Science, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Bae, Jeehyeon, E-mail: jeehyeon@cau.ac.kr [Department of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Rhee, Sangmyung, E-mail: sangmyung.rhee@cau.ac.kr [Department of Life Science, Chung-Ang University, Seoul 156-756 (Korea, Republic of)

    2015-08-21

    The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation and invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer. - Highlights: • TM-JM1/2 peptides are efficiently delivered into cells by AuNP-Apt-conjugates. • TM-JM1/2 peptides loaded onto AuNP-Apt conjugates inhibit DDR2 activation. • Inhibition of DDR2 activation by TM-JM1/2 peptides decreases tumor progression.

  7. Relationship between G-proteins associated transmembrane cascades and MAPK phosphorylation induced by recombinant interleukin-α in fibroblast-like synoviocytes from rats with adjuvant arthritis

    Institute of Scientific and Technical Information of China (English)

    Yong-qiuZHENG; WeiWEI; LeiZHU; Xiao-yiJIA

    2004-01-01

    AIM: To elucidate the relationship between G protein-associ-ated transmembrane cascade and mitogen-activated protein kinases (MAPKs) phosphorylation in recombinant rat IL-1α(rIL-1α)-induced fibroblast-like synoviocytes (FLS) from rats with adjuvant arthritis (AA). METHODS: The expressions of MAPKs phosphorylation, the stimulatory subunit of G alpha

  8. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    Science.gov (United States)

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  9. Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides.

    Science.gov (United States)

    Du, Yuzhe; Lee, Jung-Eun; Nomura, Yoshiko; Zhang, Tianxiang; Zhorov, Boris S; Dong, Ke

    2009-04-15

    A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNa(v) sodium channel is required for the binding and action of pyrethroids. However, whether or not other residues in IIIS6 participate in the action of pyrethroids remains to be determined. In the present study, we conducted a systematic analysis of 20 residues in IIIS6 of the BgNa(v) channel using alanine-scanning mutagenesis. Our results show that alanine substitutions of four residues, Ile1514, Gly1516, Phe1518 and Asn1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions diminished sodium channel sensitivity to all seven pyrethroids examined, including four type I (lacking the alpha-cyano group at the phenoxybenzyl alcohol) and three type II (containing the alpha-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to four type I and type II pyrethroids that contain the phenoxybenzyl alcohol only. We also show that alanine/lysine substitutions of Leu1521 and Ser1517 affected the action of BTX (batrachotoxin), but not pyrethroids. In the Kv1.2-based homology model of the open sodium channel, side chains of Ile1514, Phe1518 and Asn1522 are exposed towards helix IIS5 and linker IIS4-IIS5, which contain previously identified pyrethroid-interacting residues, whereas Ser1517 and Leu1521 face the inner pore where the BTX receptor is located. Thus the present study provides further evidence for structural models in which pyrethroids bind to the lipid-exposed interface formed by helices IIIS6, IIS5 and linker helix IIS4-IIS5, whereas BTX binds to the pore-exposed side of the IIIS6 helix.

  10. One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.

    Science.gov (United States)

    Lock, Antonia; Forfar, Rachel; Weston, Cathryn; Bowsher, Leo; Upton, Graham J G; Reynolds, Christopher A; Ladds, Graham; Dixon, Ann M

    2014-12-01

    G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs.

  11. Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture.

    Science.gov (United States)

    Gardner, Thomas J; Hernandez, Rosmel E; Noriega, Vanessa M; Tortorella, Domenico

    2016-03-30

    The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

  12. Molecular dynamics simulation of human serum paraoxonase 1 in DPPC bilayer reveals a critical role of transmembrane helix H1 for HDL association.

    Science.gov (United States)

    Patra, Mahesh Chandra; Rath, Surya Narayan; Pradhan, Sukanta Kumar; Maharana, Jitendra; De, Sachinandan

    2014-01-01

    Serum paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-bound mammalian enzyme exhibiting antiatherosclerotic activity. Despite years of research, an accurate model for the binding interaction between PON1 and HDL has not been established. However, it is reported that anchoring of PON1 to HDL is mainly governed by an N-terminal alpha helix H1 and another short helix H2. Here, we studied the molecular association of full-length human PON1 (huPON1) with a HDL-mimetic dipalmitoylphosphatidylcholine (DPPC) bilayer using homology modeling and molecular dynamics simulations. Our results indicate that H1 is the highly dynamic part of huPON1, showing clockwise rotation of up to 30° within the DPPC bilayer. However, without phospholipid molecules, H1 experiences helical distortions, illustrating an incompatible HDL-anchoring conformation. Snorkeling interactions of K3, R18, and R27 together with aromatic locks formed by Y187, Y190, W194, and W202 are highly essential for anchoring of huPON1 to HDL's surface. Molecular mechanics/Poisson-Boltzmann solvent-accessible surface area (MM/PBSA) binding free energy calculation revealed that H1 displays greater binding affinity towards lipid molecules compared with H2 and H3, suggesting that H1 is the most probable HDL-binding domain of PON1. Binding free energy decomposition showed that K3, R18, and R27 interact with polar headgroups of DPPC membrane through electrostatic interaction. Moreover, Y187, Y190, W194, and W202 interact with DPPC lipids mainly through van der Waals interaction. Taken together, these results show that the transmembrane helix H1 along with the interfacial positively charged and aromatic resides were crucial for PON1's association with HDL particle. The current study will be useful towards understanding the antiatherosclerotic and bioscavenging properties of this promiscuous enzyme. PMID:24297451

  13. Structural and functional diversity of Topologically Associating Domains.

    Science.gov (United States)

    Dekker, Job; Heard, Edith

    2015-10-01

    Recent studies have shown that chromosomes in a range of organisms are compartmentalized in different types of chromatin domains. In mammals, chromosomes form compartments that are composed of smaller Topologically Associating Domains (TADs). TADs are thought to represent functional domains of gene regulation but much is still unknown about the mechanisms of their formation and how they exert their regulatory effect on embedded genes. Further, similar domains have been detected in other organisms, including flies, worms, fungi and bacteria. Although in all these cases these domains appear similar as detected by 3C-based methods, their biology appears to be quite distinct with differences in the protein complexes involved in their formation and differences in their internal organization. Here we outline our current understanding of such domains in different organisms and their roles in gene regulation. PMID:26348399

  14. Substitution of the transmembrane domain of Vpu in simian-human immunodeficiency virus (SHIVKU1bMC33) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques

    International Nuclear Information System (INIS)

    The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 μM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 μM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and

  15. Scrambling of the amino acids within the transmembrane domain of Vpu results in a simian-human immunodeficiency virus (SHIVTM) that is less pathogenic for pig-tailed macaques

    International Nuclear Information System (INIS)

    Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques

  16. Chemically modified peptide scaffolds target the CFTR-associated ligand PDZ domain.

    Directory of Open Access Journals (Sweden)

    Jeanine F Amacher

    Full Text Available PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR. Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL, and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

  17. The ectodomains but not the transmembrane domains of the fusion proteins of subtypes A and B avian pneumovirus are conserved to a similar extent as those of human respiratory syncytial virus.

    Science.gov (United States)

    Naylor, C J; Britton, P; Cavanagh, D

    1998-06-01

    The fusion glycoprotein (F(B)) gene of five strains of the B subtype of avian pneumovirus (APV; turkey rhinotracheitis virus) has been sequenced. The length of the F(B) protein was 538 amino acids, identical to that of the F protein of subtype A virus, with which it had 74% and 83% overall nucleotide and deduced amino acid identities, respectively. The F(B) and F(A) ectodomains had 90% amino acid identity, very similar to the 91% identity between the ectodomains of the F proteins of subtype A and B human respiratory syncytial virus (HRSV). As with HRSV, the F2 polypeptide was less conserved (83% identity) than F1 (94%). In contrast to the ectodomain, the transmembrane and cytoplasmic domains of the two APV subtypes were much less conserved (30% and 48% identity, respectively) than those of HRSV (92% and 87%, respectively). Comparisons within all the genera of the Paramyxoviridae (Pneumovirus, Morbillivirus, Paramyxovirus and Rubullavirus) show that low amino acid identity between F protein transmembrane domains is a feature of different species of virus rather than of strain differences. This may indicate that the two subtypes of APV have evolved in different geographical regions and/or different avian species. This is the first report of an F gene sequence from a subtype B APV.

  18. A single base insertion in the putative transmembrane domain of the tyrosinase gene as a cause for tyrosinase-negative oculocutaneous albinism

    Energy Technology Data Exchange (ETDEWEB)

    Chintamaneni, C.D.; Kobayashi, Y.; Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (United States)); Halaban, R. (Yale Univ. School of Medicine, New Haven, CT (United States)); Witkop, C.J. Jr. (Univ. of Minnesota, Minneapolis (United States))

    1991-06-15

    The authors have determined a molecular defect to be the likely basis for inactivity of the tyrosinase from a patient with tyrosinase-negative oculocutaneous albinism. A single base (thymine) was inserted in exon 5 of the tyrosinase gene following codon 471 in the putative transmembrane coding region. This insertion caused a shift in the reading frame of 19 amino acids at the 3{prime} end and introduced a premature termination signal that would be expected to truncate the protein by 21 amino acids at the carboxyl terminus. The albino tyrosinase was not recognized by antibodies directed to the carboxyl terminus of tyrosinase. Furthermore, as shown by gel electrophoresis of the immunoprecipitated protein, the tyrosinase was {approx} 3kDa smaller than normal. Similar immunoprecipitation data were obtained when cloned normal and mutant tyrosinases were expressed in COS-1 cells.

  19. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    Science.gov (United States)

    Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

    2015-08-14

    Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.

  20. ACTN1 rod domain mutation associated with congenital macrothrombocytopenia.

    Science.gov (United States)

    Yasutomi, Motoko; Kunishima, Shinji; Okazaki, Shintaro; Tanizawa, Akihiko; Tsuchida, Shinya; Ohshima, Yusei

    2016-01-01

    Mutations in ACTN1, the gene encoding the actin-crosslinking protein α-actinin-1, cause autosomal dominant macrothrombocytopenia. α-Actinin-1 exists as antiparallel dimers, composed of an N-terminal actin-binding domain (ABD), four spectrin-like repeats (SLRs), which form the spacer rod, and a C-terminal calmodulin-like (CaM) domain. All of the previously reported ACTN1 mutations associated with macrothrombocytopenia reside within the ABD and the CaM domain and not within the SLR domain. In this report, we describe a mutation in SLR2 of α-actinin-1 (p.Leu395Gln) associated with familial macrothrombocytopenia. A 3-year-old boy and his mother both had this mutation. They showed a mild form of thrombocytopenia without severe bleeding, accompanied by an elevated mean platelet volume. Consistent with the previous reports of mutations that reside in the ABD or the CaM domain, immunofluorescence examination revealed disorganization of the actin cytoskeleton in Gln395 mutant-transduced Chinese hamster ovary cells. Our findings suggest a novel mechanism for the pathogenesis of ACTN1-related macrothrombocytopenia that does not involve functional domain mutations. PMID:26453073

  1. Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

    Energy Technology Data Exchange (ETDEWEB)

    Hamann, J. [Univ. of Amsterdam (Netherlands)]|[Max Planck Society, Berlin-Buch (Germany); Hamann, D.; Lier, R.A.W. [Univ. of Amsterdam (Netherlands)] [and others

    1995-08-15

    CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).

  2. A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIVKU-1bMC33) susceptible to rimantadine

    International Nuclear Information System (INIS)

    Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIVKU-1bMC33 in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIVM2) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIVKU-1bMC33) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIVVpuA19H replicated with similar kinetics as the parental SHIVKU-1bMC33 and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIVKU-1bMC33. This SHIVVpuA19H virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIVM2. Electron microscopic examination of SHIVVpuA19H-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIVM2-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide additional evidence

  3. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Ying [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Yunnan Academy of Tobacco Science, Kunming, Yunnan 650106 (China); Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Li, Qihan, E-mail: imbcams.lq@gmail.com [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China)

    2014-09-15

    During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress. - Highlights: • Cellular TMEM140 protein interacts with HSV-1 UL31 protein during viral infection. • Increasing expression of TMEM140 leads to inhibition of HSV-1 proliferation. • Increasing expression of TMEM140 blocks HSV-1 nucleocapsid egress process. • Binding to UL31 of TMEM140 impedes formation of HSV-1 UL31–UL34 complex.

  4. Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

    Directory of Open Access Journals (Sweden)

    Jorge Fernández-Trillo

    Full Text Available A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.

  5. Structure and function of the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    M.M. Morales

    1999-08-01

    Full Text Available Cystic fibrosis (CF is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR. Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs, and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs.

  6. Single-channel SCAM Identifies Pore-lining Residues in the First Extracellular Loop and First Transmembrane Domains of Cx46 Hemichannels

    OpenAIRE

    KRONENGOLD, J.; Trexler, E B; Bukauskas, F.F.; Bargiello, T A; Verselis, V K

    2003-01-01

    Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to ...

  7. Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins.

    Science.gov (United States)

    Duchesne, Laurence; Pellerin, Isabelle; Delamarche, Christian; Deschamps, Stephane; Lagree, Valerie; Froger, Alexandrine; Bonnec, Georgette; Thomas, Daniel; Hubert, Jean-Francois

    2002-06-01

    We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability. PMID:11927589

  8. ACRATA: a novel electron transfer domain associated to apoptosis and cancer

    Directory of Open Access Journals (Sweden)

    Martinez-A Carlos

    2004-12-01

    Full Text Available Abstract Background Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM domain and involved in apoptosis and cancer (e.g. STEAP, STAMP1, TSAP6, have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown. Methods We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search. Results Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox family and the YedZ family of bacterial oxidoreductases. Conclusions This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families.

  9. Robust red FRET sensors using self-associating fluorescent domains

    OpenAIRE

    Lindenburg, Laurens H.; Hessels, Anne M.; Ebberink, Eduard H. T. M.; Arts, Remco; Merkx, Maarten

    2013-01-01

    Elucidation of sub-cellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dyna...

  10. Topologically Associating Domains: An invariant framework or a dynamic scaffold?

    Science.gov (United States)

    Cubeñas-Potts, Caelin; Corces, Victor G

    2015-01-01

    Metazoan genomes are organized into regions of topologically associating domains (TADs). TADs are demarcated by border elements, which are enriched for active genes and high occupancy architectural protein binding sites. We recently demonstrated that 3D chromatin architecture is dynamic in response to heat shock, a physiological stress that downregulates transcription and causes a global redistribution of architectural proteins. We utilized a quantitative measure of border strength after heat shock, transcriptional inhibition, and architectural protein knockdown to demonstrate that changes in both transcription and architectural protein occupancy contribute to heat shock-induced TAD dynamics. Notably, architectural proteins appear to play a more important role in altering 3D chromatin architecture. Here, we discuss the implications of our findings on previous studies evaluating the dynamics of TAD structure during cellular differentiation. We propose that the subset of variable TADs observed after differentiation are representative of cell-type specific gene expression and are biologically significant. PMID:26418477

  11. Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

    OpenAIRE

    Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2014-01-01

    CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal ...

  12. The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

    Directory of Open Access Journals (Sweden)

    Priyadarsini Kumar

    Full Text Available MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB. While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC

  13. Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its correlation with the differentiation status of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Cong Peng; Rou-Li Zhou; Gen-Ze Shao; Jing-An Rui; Shao-Bin Wang; Ming Lin; Sha Zhang; Zi-Feng Gao

    2005-01-01

    AIM: To produce high-quality polydonal antibody to lysosome associated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC).METHODS: The 297 bp 5' end of LAPTM4BcDNA was obtained by PCR and inserted into prokaryotic expression vector pGEXKG. Then the recombinant pGEX-KG-N1-99 was transformedinto E. coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharoseTM 4B agarose. The purified GST-LAPTM4B-N1-99 was characterized by SDSPAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N1-99 was isolated from 1 L of bacteria. The GST-LAPTM4B-N1-99produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4B-N1-99-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely

  14. Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

    Directory of Open Access Journals (Sweden)

    Bryan D Moyer

    Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

  15. Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

    Science.gov (United States)

    Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

    2016-03-29

    Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional

  16. The Role of Domain Satisfaction in Explaining the Paradoxical Association between Life Satisfaction and Age

    Science.gov (United States)

    McAdams, Kimberly K.; Lucas, Richard E.; Donnellan, M. Brent

    2012-01-01

    Although aging is associated with declines in many life domains, overall life satisfaction does not appear to decline sharply with age. One explanation for this paradoxical finding is that several life domains improve with age such that increases in certain domains balance the decreases in others. Because different issues are problematic at…

  17. Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process*

    Science.gov (United States)

    Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2014-01-01

    CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer. PMID:24652284

  18. Hemolytic lectin CEL-III heptamerizes via a large structural transition from α-helices to a β-barrel during the transmembrane pore formation process.

    Science.gov (United States)

    Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2014-05-01

    CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer. PMID:24652284

  19. CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC.

    Science.gov (United States)

    Pelaseyed, Thaher; Hansson, Gunnar C

    2011-09-15

    The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

  20. The tyrosine phosphatase SHP2 associates with CUB domain-containing protein-1 (CDCP1, regulating its expression at the cell surface in a phosphorylation-dependent manner.

    Directory of Open Access Journals (Sweden)

    Leslie Yewakon Gandji

    Full Text Available CUB domain-containing protein-1 (CDCP1 is a transmembrane glycoprotein that is phosphorylated by SRC family kinases (SFK before recruiting and activating PKCδ. CDCP1 is overproduced in many cancers. It promotes metastasis and resistance to anoïkis. The robust production of CDCP1 would be associated with stemness and has been proposed as a novel prognosis marker. The natural transmembrane location of CDCP1 makes it an ideal therapeutic target and treatments based on the use of appropriate antibodies are currently being evaluated. However, we still know very little about the molecular fate of CDCP1 and its downstream signaling events. Improvements in our understanding of the molecular events occurring downstream of CDCP1 are required to make use of changes of CDCP1 production or functions for therapeutic purposes. By the mean of co-immunoprecipitation and affinity precipitation we show here, for the first time, that CDCP1 interacts directly, with the cytosolic tyrosine phosphatase SHP2. Point mutants of CDCP1 show that residues Y734 and Y743 are responsible for its interaction with SHP2. It may therefore compete with SFK. We also demonstrate that a shRNA-mediated down regulation of SHP2 is associated with a stronger CDCP1 phosphorylation and an impairment of antibody-mediated CDCP1 internalization.

  1. Control of phospholipid flip-flop by transmembrane peptides

    Energy Technology Data Exchange (ETDEWEB)

    Kaihara, Masanori; Nakao, Hiroyuki; Yokoyama, Hirokazu [Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501 (Japan); Endo, Hitoshi [Quantum Beam Science Directorate, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Ishihama, Yasushi [Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501 (Japan); Handa, Tetsurou [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minami-Tamagaki-cho, Suzuka, Mie 513-8670 (Japan); Nakano, Minoru, E-mail: mnakano@pha.u-toyama.ac.jp [Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2013-06-20

    Highlights: ► Phospholipid flip-flop in transmembrane peptide-containing vesicles was investigated. ► Peptides that contained polar residues in the center of the transmembrane region promoted phospholipid flip-flop. ► A bioinformatics approach revealed the presence of polar residues in the transmembrane region of ER membrane proteins. ► Polar residues in ER membrane proteins possibly provide flippase-like activity. - Abstract: We designed three types of transmembrane model peptides whose sequence originates from a frequently used model peptide KALP23, and we investigated their effects on phospholipid flip-flop. Time-resolved small-angle neutron scattering and a dithionite fluorescent quenching assay demonstrated that TMP-L, which has a fully hydrophobic transmembrane region, did not enhance phospholipid flip-flop, whereas TMP-K and TMP-E, which have Lys and Glu, respectively, in the center of their transmembrane regions, enhanced phospholipid flip-flop. Introduction of polar residues in the membrane-spanning helices is considered to produce a locally polar region and enable the lipid head group to interact with the polar side-chain inside the bilayers, thereby reducing the activation energy for the flip-flop. A bioinformatics approach revealed that acidic and basic residues account for 4.5% of the central region of the transmembrane domain in human ER membrane proteins. Therefore, polar residues in ER membrane proteins are considered to provide flippase-like activity.

  2. Control of phospholipid flip-flop by transmembrane peptides

    International Nuclear Information System (INIS)

    Highlights: ► Phospholipid flip-flop in transmembrane peptide-containing vesicles was investigated. ► Peptides that contained polar residues in the center of the transmembrane region promoted phospholipid flip-flop. ► A bioinformatics approach revealed the presence of polar residues in the transmembrane region of ER membrane proteins. ► Polar residues in ER membrane proteins possibly provide flippase-like activity. - Abstract: We designed three types of transmembrane model peptides whose sequence originates from a frequently used model peptide KALP23, and we investigated their effects on phospholipid flip-flop. Time-resolved small-angle neutron scattering and a dithionite fluorescent quenching assay demonstrated that TMP-L, which has a fully hydrophobic transmembrane region, did not enhance phospholipid flip-flop, whereas TMP-K and TMP-E, which have Lys and Glu, respectively, in the center of their transmembrane regions, enhanced phospholipid flip-flop. Introduction of polar residues in the membrane-spanning helices is considered to produce a locally polar region and enable the lipid head group to interact with the polar side-chain inside the bilayers, thereby reducing the activation energy for the flip-flop. A bioinformatics approach revealed that acidic and basic residues account for 4.5% of the central region of the transmembrane domain in human ER membrane proteins. Therefore, polar residues in ER membrane proteins are considered to provide flippase-like activity

  3. C2 domain of synaptotagminⅠassociates with lipid rafts of plasma membrane

    Institute of Scientific and Technical Information of China (English)

    L(U) JiHua; HE Li; SUI SenFang

    2008-01-01

    In this paper we report that the C2 domain of synaptotagmin I (syt I) could associate with lipid rafts of plasma membrane. We demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) in the target membrane and Ca2+ are the key factors to enhance the raft association of the C2 domain. We also found that the raft association of the C2 domain could be fulfilled by either C2A or C2B alone, suggesting that their raft association might be complementary. Finally, we indicate that destroying lipid rafts or blocking syt I-raft association could significantly reduce the Ca2+-driven release of glutamates. Our data indicate that the raft association of the C2 domain might play an important role in the regulated exocytosis.

  4. Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Xin Wang

    Full Text Available BACKGROUND: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C to the pathogenic one (PrP(Sc. The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. METHODOLOGY/PRINCIPAL FINDINGS: To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER, the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL and PrP with three amino acids exchange in transmembrane region (PrP-3AV were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K failed. CONCLUSIONS/SIGNIFICANCE: The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them

  5. Hydrophobic pulses predict transmembrane helix irregularities and channel transmembrane units

    Directory of Open Access Journals (Sweden)

    Claustres Mireille

    2011-05-01

    Full Text Available Abstract Background Few high-resolution structures of integral membranes proteins are available, as crystallization of such proteins needs yet to overcome too many technical limitations. Nevertheless, prediction of their transmembrane (TM structure by bioinformatics tools provides interesting insights on the topology of these proteins. Methods We describe here how to extract new information from the analysis of hydrophobicity variations or hydrophobic pulses (HPulses in the sequence of integral membrane proteins using the Hydrophobic Pulse Predictor, a new tool we developed for this purpose. To analyze the primary sequence of 70 integral membrane proteins we defined two levels of analysis: G1-HPulses for sliding windows of n = 2 to 6 and G2-HPulses for sliding windows of n = 12 to 16. Results The G2-HPulse analysis of 541 transmembrane helices allowed the definition of the new concept of transmembrane unit (TMU that groups together transmembrane helices and segments with potential adjacent structures. In addition, the G1-HPulse analysis identified helix irregularities that corresponded to kinks, partial helices or unannotated structural events. These irregularities could represent key dynamic elements that are alternatively activated depending on the channel status as illustrated by the crystal structures of the lactose permease in different conformations. Conclusions Our results open a new way in the understanding of transmembrane secondary structures: hydrophobicity through hydrophobic pulses strongly impacts on such embedded structures and is not confined to define the transmembrane status of amino acids.

  6. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    OpenAIRE

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrat...

  7. Human Immunodeficiency Virus - associated lymphomas: A neglected domain

    Directory of Open Access Journals (Sweden)

    Nagesh Taterao Sirsath

    2013-01-01

    Full Text Available Background: Human immunodeficiency virus (HIV associated lymphoma is an important public health concern; however, the epidemiological data available from India is sparse. Aims: The present study was carried out at a tertiary cancer care center in South India to analyze the scenario of HIV-associated lymphoma. Materials and Methods: This was a retrospective observational study conducted at our center, on consecutive patients diagnosed with HIV-associated lymphoma, from January 2008 to December 2012. Results: A total of 44 patients were diagnosed with HIV-associated lymphoma, of which 18 opted for treatment. There were 11 males and 7 females in the study population. Median interval from the diagnosis of HIV infection to diagnosis of lymphoma was 18 months. Median CD4 count at the time of lymphoma diagnosis was 218/mm 3 . Five patients had Hodgkin′s lymphoma, and the rest had non-Hodgkin′s lymphoma. Five out of 18 (28% patients in the present study expired during treatment. Ten (55.5% patients are alive and lymphoma free, with a median follow up of 18 months. Conclusions: More than half of our treated patients are lymphoma free with a median follow up of 18 months; hence treatment of patients with HIV-associated lymphoma should be encouraged.

  8. Disordered regions in transmembrane proteins.

    Science.gov (United States)

    Tusnády, Gábor E; Dobson, László; Tompa, Peter

    2015-11-01

    The functions of transmembrane proteins in living cells are widespread; they range from various transport processes to energy production, from cell-cell adhesion to communication. Structurally, they are highly ordered in their membrane-spanning regions, but may contain disordered regions in the cytosolic and extra-cytosolic parts. In this study, we have investigated the disordered regions in transmembrane proteins by a stringent definition of disordered residues on the currently available largest experimental dataset, and show a significant correlation between the spatial distributions of positively charged residues and disordered regions. This finding suggests a new role of disordered regions in transmembrane proteins by providing structural flexibility for stabilizing interactions with negatively charged head groups of the lipid molecules. We also find a preference of structural disorder in the terminal--as opposed to loop--regions in transmembrane proteins, and survey the respective functions involved in recruiting other proteins or mediating allosteric signaling effects. Finally, we critically compare disorder prediction methods on our transmembrane protein set. While there are no major differences between these methods using the usual statistics, such as per residue accuracies, Matthew's correlation coefficients, etc.; substantial differences can be found regarding the spatial distribution of the predicted disordered regions. We conclude that a predictor optimized for transmembrane proteins would be of high value to the field of structural disorder. PMID:26275590

  9. Novel predicted RNA-binding domains associated with the translation machinery.

    Science.gov (United States)

    Aravind, L; Koonin, E V

    1999-03-01

    Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.

  10. Ion fluxes through nanopores and transmembrane channels

    Science.gov (United States)

    Bordin, J. R.; Diehl, A.; Barbosa, M. C.; Levin, Y.

    2012-03-01

    We introduce an implicit solvent Molecular Dynamics approach for calculating ionic fluxes through narrow nanopores and transmembrane channels. The method relies on a dual-control-volume grand-canonical molecular dynamics (DCV-GCMD) simulation and the analytical solution for the electrostatic potential inside a cylindrical nanopore recently obtained by Levin [Europhys. Lett.EULEEJ0295-507510.1209/epl/i2006-10240-4 76, 163 (2006)]. The theory is used to calculate the ionic fluxes through an artificial transmembrane channel which mimics the antibacterial gramicidin A channel. Both current-voltage and current-concentration relations are calculated under various experimental conditions. We show that our results are comparable to the characteristics associated to the gramicidin A pore, especially the existence of two binding sites inside the pore and the observed saturation in the current-concentration profiles.

  11. SH4-domain-induced plasma membrane dynamization promotes bleb-associated cell motility.

    Science.gov (United States)

    Tournaviti, Stella; Hannemann, Sebastian; Terjung, Stefan; Kitzing, Thomas M; Stegmayer, Carolin; Ritzerfeld, Julia; Walther, Paul; Grosse, Robert; Nickel, Walter; Fackler, Oliver T

    2007-11-01

    SH4 domains provide bipartite membrane-targeting signals for oncogenic Src family kinases. Here we report the induction of non-apoptotic plasma membrane (PM) blebbing as a novel and conserved activity of SH4 domains derived from the prototypic Src kinases Src, Fyn, Yes and Lck as well as the HASPB protein of Leishmania parasites. SH4-domain-induced blebbing is highly dynamic, with bleb formation and collapse displaying distinct kinetics. These reorganizations of the PM are controlled by Rho but not Rac or Cdc42 GTPase signalling pathways. SH4-induced membrane blebbing requires the membrane association of the SH4 domain, is regulated by the activities of Rock kinase and myosin II ATPase, and depends on the integrity of F-actin as well as microtubules. Endogenous Src kinase activity is crucial for PM blebbing in SH4-domain-expressing cells, active Src and Rock kinases are enriched in SH4-domain-induced PM blebs, and PM blebbing correlates with enhanced cell invasion in 3D matrices. These results establish a novel link between SH4 domains, Src activity and Rho signalling, and implicate SH4-domain-mediated PM dynamization as a mechanism that influences invasiveness of cells transformed by SH4-domain-containing oncoproteins. PMID:17959630

  12. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Junsen; Yang, Huiseon [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Eom, Soo Hyun [School of Life Sciences, Steitz Center for Structural Biology, and Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of); Chun, ChangJu, E-mail: cchun1130@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Im, Young Jun, E-mail: imyoungjun@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2014-09-12

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

  13. A Protein Domain and Family Based Approach to Rare Variant Association Analysis

    Science.gov (United States)

    Richardson, Tom G.; Shihab, Hashem A.; Rivas, Manuel A.; McCarthy, Mark I.; Campbell, Colin; Timpson, Nicholas J.; Gaunt, Tom R.

    2016-01-01

    Background It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Methods Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). Results We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. Conclusion We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals. PMID:27128313

  14. A Protein Domain and Family Based Approach to Rare Variant Association Analysis.

    Directory of Open Access Journals (Sweden)

    Tom G Richardson

    Full Text Available It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit.Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1% together in three different ways 1 variants within single genomic regions which map to individual protein domains 2 variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3 all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families. A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT.We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed.We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals.

  15. ESTIMATION OF ATTRACTION DOMAIN AND EXPONENTIAL CONVERGENCE RATE OF CONTINUOUS FEEDBACK ASSOCIATIVE MEMORY

    Institute of Scientific and Technical Information of China (English)

    周冬明; 曹进德; 李继彬

    2001-01-01

    The attraction domain of memory patterns and exponential convergence rate of the network trajectories to memory patterns for continuous feedback associative memory are estimated again by using of some analysis techniques and Liapunov method, some new re sults are obtained, that can be used for evaluation of fault-tolerance capability and the syn thesis procedures for continuous feedback associative memory neural networks.

  16. Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

    OpenAIRE

    Ulianov, Sergey V; Khrameeva, Ekaterina E.; Gavrilov, Alexey A.; Flyamer, Ilya M.; Kos, Pavel; Mikhaleva, Elena A.; Penin, Aleksey A.; Logacheva, Maria D.; Imakaev, Maxim V.; Chertovich, Alexander; Gelfand, Mikhail S; Shevelyov, Yuri Y.; Razin, Sergey V.

    2016-01-01

    Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophi...

  17. Self-association of TPR domains: Lessons learned from a designed, consensus-based TPR oligomer.

    Science.gov (United States)

    Krachler, Anne Marie; Sharma, Amit; Kleanthous, Colin

    2010-07-01

    The tetratricopeptide repeat (TPR) motif is a protein-protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self-association. In the present work we characterize the trimeric TPR-containing protein YbgF, which is linked to the Tol system in Gram-negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C-terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C-terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open-ended self-association does not occur in full-length YbgF suggesting that the protein's N-terminal coiled-coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled-coil domain of YbgF was engineered onto the N-terminus of CTPR3Y3 and shown to block self-association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self-association and how such self-association can be regulated in TPR domain-containing proteins.

  18. Evolution of vertebrate interferon inducible transmembrane proteins

    Directory of Open Access Journals (Sweden)

    Hickford Danielle

    2012-04-01

    Full Text Available Abstract Background Interferon inducible transmembrane proteins (IFITMs have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. Results Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. Conclusions Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.

  19. NMR Derived Model of GTPase Effector Domain (GED) Self Association: Relevance to Dynamin Assembly

    OpenAIRE

    Chakraborty, Swagata; Pratihar, Supriya; Hosur, Ramakrishna V.

    2012-01-01

    Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its ‘coiled coil’ GTPase Effector Domain (GED), which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the...

  20. Nanocolumnar association and domain formation in porous thin films grown by evaporation at oblique angles

    Science.gov (United States)

    Lopez-Santos, C.; Alvarez, R.; Garcia-Valenzuela, A.; Rico, V.; Loeffler, M.; Gonzalez-Elipe, A. R.; Palmero, A.

    2016-09-01

    Porous thin films grown at oblique angles by evaporation techniques are formed by tilted nanocolumnar structures which, depending on the material type and growth conditions, associate along certain preferential directions, giving rise to large domains. This arrangement, commonly denoted as bundling association, is investigated in the present work by performing fundamental experiments and growth simulations. It is proved that trapping processes of vapor species at the film surface, together with the shadowing mechanism, mediate the anisotropic widening of the nanocolumns and promote their preferential coalescence along certain directions, giving rise to domains with different shape and size. The role of these two processes is thoroughly studied in connection with the formation of these domains in materials as different as SiO2 and TiO2.

  1. Associations among Adolescent Risk Behaviours and Self-Esteem in Six Domains

    Science.gov (United States)

    Wild, Lauren G.; Flisher, Alan J.; Bhana, Arvin; Lombard, Carl

    2004-01-01

    Background: This study investigated associations among adolescents' self-esteem in 6 domains (peers, school, family, sports/athletics, body image and global self-worth) and risk behaviours related to substance use, bullying, suicidality and sexuality. Method: A multistage stratified sampling strategy was used to select a representative sample of…

  2. The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity

    International Nuclear Information System (INIS)

    Research highlights: → We examine the role of a ubiquitin-associated (UBA) domain in an E2 enzyme. → The E2-25K UBA domain directs polyubiquitin chain linkage specificity. → The E2-25K UBA domain regulates length of polyubiquitin chains synthesized. -- Abstract: E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.

  3. Rigidity of transmembrane proteins determines their cluster shape

    CERN Document Server

    Jafarinia, Hamidreza; Jalali, Mir Abbas

    2015-01-01

    Protein aggregation in cell membrane is vital for majority of biological functions. Recent experimental results suggest that transmembrane domains of proteins such as $\\alpha$-helices and $\\beta$-sheets have different structural rigidity. We use molecular dynamics simulation of a coarse-grained model of protein-embedded lipid membranes to investigate the mechanisms of protein clustering. For a variety of protein concentrations, our simulations in thermal equilibrium conditions reveal that the structural rigidity of transmembrane domains dramatically affects interactions and changes the shape of the cluster. We have observed stable large aggregates even in the absence of hydrophobic mismatch which has been previously proposed as the mechanism of protein aggregation. According to our results, semi-flexible proteins aggregate to form two-dimensional clusters while rigid proteins, by contrast, form one-dimensional string-like structures. By assuming two probable scenarios for the formation of a two-dimensional tr...

  4. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  5. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  6. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  7. Transmembrane Signaling Characterized in Bacterial Chemoreceptors by Using Sulfhydryl Cross-Linking in vivo

    Science.gov (United States)

    Lee, Geoffrey F.; Lebert, Michael R.; Lilly, Angela A.; Hazelbauer, Gerald L.

    1995-04-01

    Transmembrane signaling by bacterial chemoreceptors is thought to involve conformational changes within a stable homodimer. We investigated the functional consequences of constraining movement between pairs of helices in the four-helix structure of the transmembrane domain of chemoreceptor Trg. Using a family of cysteine-containing receptors, we identified oxidation treatments for intact cells that catalyzed essentially complete sulfhydryl cross-linking at selected positions and yet left flagellar and sensory functions largely unperturbed. Constraining movement by cross-links between subunits had little effect on tactic response, but constraining movement between transmembrane segments of the monomer drastically reduced function. We deduce that transmembrane signaling requires substantial movement between transmembrane helices of a monomer but not between interacting helices across the interface between subunits.

  8. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    Energy Technology Data Exchange (ETDEWEB)

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. (Wistar Inst. of Anatomy and Biology, Philadelphia, PA (USA))

    1990-09-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

  9. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  10. Responses of Transmembrane Peptide and Lipid Chains to Hydrophobic Mismatch

    Institute of Scientific and Technical Information of China (English)

    YANG Lei; LI Jian-tao; QI Hai-yan; LI Fei

    2012-01-01

    Hydrophobic mismatch between the hydrophobic length of membrane proteins and hydrophobic thickness of membranes is a crucial factor in controlling protein function and assembly.We combined fluorescence with circular dichroism(CD) and attenuated total reflection infrared(ATR-IR) spectroscopic methods to investigate the behaviors of the peptide and lipids under hydrophobic mismatch using a model peptide from the fourth transmembrane domain of natural resistance-associated macrophage protein 1 (Nramp 1),the phosphatidylcholines(PCs) and phosphatidylglycerols(PGs) with different lengths of acyl chains(14:0,16:0 and 18:0).In all PG lipid membranes,the peptide forms stable α-helix structure,and the helix axis is parallel to lipid chains.The helical span and orientation hardly change in varying thickness of PG membranes,while the lipid chains can deform to accommodate to the hydrophobic surface of embedded peptide.By comparison,the helical structures of the model peptide in PC lipid membranes are less stable.Upon incorporation with PC lipid membranes,the peptide can deform itself to accommodate to the hydrophobic thickness of lipid membranes in response to hydrophobic mismatch.In addition,hydrophobic mismatch can increase the aggregation propensity of the peptide in both PC and PG lipid membranes and the peptide in PC membranes has more aggregation tendency than that in PG membranes.

  11. A Novel Type III Endosome Transmembrane Protein, TEMP

    Directory of Open Access Journals (Sweden)

    Rohan D. Teasdale

    2012-11-01

    Full Text Available As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.

  12. NMR derived model of GTPase effector domain (GED self association: relevance to dynamin assembly.

    Directory of Open Access Journals (Sweden)

    Swagata Chakraborty

    Full Text Available Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its 'coiled coil' GTPase Effector Domain (GED, which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the self-association of GED by first dissociating the assembly using Dimethyl Sulfoxide (DMSO and then systematically monitoring the refolding into helix and concomitant re-association using NMR spectroscopy, as DMSO concentration is progressively reduced. The short segment, Arg109 - Met116, acts as the nucleation site for helix formation and self-association. Hydrophobic and complementary charge interactions on the surfaces drive self-association, as the helices elongate in both the directions resulting in an antiparallel stack. A small N-terminal segment remains floppy in the assembly. Following these and other published results on inter-domain interactions, we have proposed a plausible mode of dynamin self assembly.

  13. Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

    DEFF Research Database (Denmark)

    Jensen, R B; Lykke-Andersen, K; Frandsen, G I;

    2000-01-01

    Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2...... and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins...

  14. A novel extracellular metallopeptidase domain shared by animal host-associated mutualistic and pathogenic microbes.

    Directory of Open Access Journals (Sweden)

    Sirintra Nakjang

    Full Text Available The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402, which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described

  15. Structural heterogeneity and functional diversity of topologically associating domains in mammalian genomes

    OpenAIRE

    Wang, Xiao-Tao; Dong, Peng-Fei; Zhang, Hong-Yu; Peng, Cheng

    2015-01-01

    Recent chromosome conformation capture (3C) derived techniques have revealed that topologically associating domain (TAD) is a pervasive element in chromatin three-dimensional (3D) organization. However, there is currently no parameter to quantitatively measure the structural characteristics of TADs, thus obscuring our understanding on the structural and functional differences among TADs. Based on our finding that there exist intrinsic chromatin interaction patterns in TADs, we define a theore...

  16. Ethnic Variation in the Cross-sectional Association between Domains of Depressive Symptoms and Clinical Depression

    OpenAIRE

    Assari, Shervin; Moazen-Zadeh, Ehsan

    2016-01-01

    Background The degree by which depressive symptoms and clinical depression reflect each other may vary across populations. The present study compared Blacks and Whites for the magnitude of the cross-sectional associations between various domains of depressive symptoms and endorsement of clinical disorders of depression. Methods Data came from the National Survey of American Life, 2001–2003. We included 3570 Black (African-Americans) and 891 Non-Hispanic Whites. Predictors were positive affect...

  17. Ethnic Variation in the Cross-sectional Association between Domains of Depressive Symptoms and Clinical Depression

    OpenAIRE

    Shervin eAssari; Ehsan eMoazen Zadeh

    2016-01-01

    BackgroundThe degree by which depressive symptoms and clinical depression reflect each other may vary across populations. The present study compared Blacks and Whites for the magnitude of the cross-sectional associations between various domains of depressive symptoms and endorsement of clinical disorders of depression. MethodsData came from the National Survey of American Life (NSAL), 2001–2003. We included 3,570 Black (African Americans), and 891 Non-Hispanic Whites. Predictors were positive...

  18. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J;

    1993-01-01

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...

  19. Decision support methods for finding phenotype--disorder associations in the bone dysplasia domain.

    Directory of Open Access Journals (Sweden)

    Razan Paul

    Full Text Available A lack of mature domain knowledge and well established guidelines makes the medical diagnosis of skeletal dysplasias (a group of rare genetic disorders a very complex process. Machine learning techniques can facilitate objective interpretation of medical observations for the purposes of decision support. However, building decision support models using such techniques is highly problematic in the context of rare genetic disorders, because it depends on access to mature domain knowledge. This paper describes an approach for developing a decision support model in medical domains that are underpinned by relatively sparse knowledge bases. We propose a solution that combines association rule mining with the Dempster-Shafer theory (DST to compute probabilistic associations between sets of clinical features and disorders, which can then serve as support for medical decision making (e.g., diagnosis. We show, via experimental results, that our approach is able to provide meaningful outcomes even on small datasets with sparse distributions, in addition to outperforming other Machine Learning techniques and behaving slightly better than an initial diagnosis by a clinician.

  20. NMR-based approach to measure the free energy of transmembrane helix-helix interactions.

    Science.gov (United States)

    Mineev, Konstantin S; Lesovoy, Dmitry M; Usmanova, Dinara R; Goncharuk, Sergey A; Shulepko, Mikhail A; Lyukmanova, Ekaterina N; Kirpichnikov, Mikhail P; Bocharov, Eduard V; Arseniev, Alexander S

    2014-01-01

    Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain. PMID:24036227

  1. Expression of PTD-bcr/abl fusion oncoprotein fragment carrying protein transduction domain and its transmembrane transportation%PTD-bcr/abl融合癌蛋白片段的表达及其跨膜转运

    Institute of Scientific and Technical Information of China (English)

    梁英民; 孙强; 蒋姗姗; 陈萍; 王冀姝; 刘利; 韩骅

    2001-01-01

    Aim To explore the method of transfering oncoprotein bcr/abl, mediated by protein transduction domain (PTD), in order to provide the experimental basis for immune therapy. Methods DNA fragment encoding PTD was synthesized and fused with PCR-amplified bcr/abl gene fragment. The fusion protein was expressed in E.coli as His-tagged protein. The purified fusion protein was loaded to cultured HL-60 cells. Whether the fusion protein entered into HL-60 cells was determined by Western blot. Results It is shown that PTD could mediate transportation of bcr/abl protein to enter into cells. Conclusion These results may provide a new approach for loading exogenous proteins to antigen presenting cells.%目的探讨蛋白转导结构域 (PTD)介导的转导 bcr/abl癌蛋白片段的方法,为进行免疫治疗提供实验依据。方法合成编码 PTD的基因片段,并与 PCR扩增的慢性粒细胞白血病癌蛋白 bcr/abl基因片段融合 ,在大肠杆菌中表达。将纯化的融合蛋白 PTD-bcr/abl与 HL-60 细胞共孵育,用 Western blot分析融合蛋白的跨膜转运。结果 PTD基序可介导 bcr/abl蛋白由细胞外跨膜转导进入细胞内。结论这一结果可能为应用外源蛋白负载 (loading)抗原提呈细胞 (APC)提供新的途径。

  2. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    Science.gov (United States)

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  3. Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule

    DEFF Research Database (Denmark)

    Holm, Dorte; Fink, Dorte Rosenbek; Grønlund, Jørn;

    2009-01-01

    by a transmembrane region and a cytoplasmic domain. The cytoplasmic domain contains two putative src kinase consensus substrate sequences, three additional potential phosphorylation sites, and two potential internalization motifs. Two possible secreted forms that lack the transmembrane region arise by alternative...... family of the SRCR superfamily. Finally, a novel human scavenger receptor cysteine-rich molecule with high homology to mSCART1 was identified by searching in the human genomic databases using the mSCART1 cDNA sequence....

  4. Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles.

    Science.gov (United States)

    Engelmann, Katja; Kinlough, Carol L; Müller, Stefan; Razawi, Hani; Baldus, Stephan E; Hughey, Rebecca P; Hanisch, Franz-Georg

    2005-11-01

    The human mucin MUC1 is expressed both as a transmembrane heterodimeric protein complex that recycles via the trans-Golgi network (TGN) and as a secreted isoform. To determine whether differences in cellular trafficking might influence the O-glycosylation profiles on these isoforms, we developed a model system consisting of membrane-bound and secretory-recombinant glycosylation probes. Secretory MUC1-S contains only a truncated repeat domain, whereas in MUC1-M constructs this domain is attached to the native transmembrane and cytoplasmic domains of MUC1 either directly (M0) or via an intermitting nonfunctional (M1) or functional sperm protein-enterokinase-agrin (SEA) module (M2); the SEA module contains a putative proteolytic cleavage site and is associated with proteins receiving extensive O-glycosylation. We showed that MUC1-M2 simulates endogenous MUC1 by recycling from the cell surface of Chinese hamster ovary (CHO) mutant ldlD14 cells through intracellular compartments where its glycosylation continues. The profiles of O-linked glycans on MUC1-S secreted by epithelial EBNA-293 and MCF-7 breast cancer cells revealed patterns dominated by core 2-based oligosaccharides. In contrast, the respective membrane-shed probes expressed in the same cells showed a complete shift to patterns dominated by sialyl core 1. In conclusion, glycan core profiles reflected the subcellular trafficking pathways of the secretory or membranous probes and the modifying activities of the resident glycosyltransferases.

  5. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

    Science.gov (United States)

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-03-18

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.

  6. An Autonomously Reciprocating Transmembrane Nanoactuator.

    Science.gov (United States)

    Watson, Matthew A; Cockroft, Scott L

    2016-01-22

    Biological molecular machines operate far from equilibrium by coupling chemical potential to repeated cycles of dissipative nanomechanical motion. This principle has been exploited in supramolecular systems that exhibit true machine behavior in solution and on surfaces. However, designed membrane-spanning assemblies developed to date have been limited to simple switches or stochastic shuttles, and true machine behavior has remained elusive. Herein, we present a transmembrane nanoactuator that turns over chemical fuel to drive autonomous reciprocating (back-and-forth) nanomechanical motion. Ratcheted reciprocating motion of a DNA/PEG copolymer threaded through a single α-hemolysin pore was induced by a combination of DNA strand displacement processes and enzyme-catalyzed reactions. Ion-current recordings revealed saw-tooth patterns, indicating that the assemblies operated in autonomous, asymmetric cycles of conformational change at rates of up to one cycle per minute. PMID:26661295

  7. Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

    Directory of Open Access Journals (Sweden)

    Koji Mizuhashi

    Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

  8. The C2A domain in dysferlin is important for association with MG53 (TRIM72)

    OpenAIRE

    Matsuda, Chie; Miyake, Katsuya; Kameyama, Kimihiko; Keduka, Etsuko; Takeshima, Hiroshi; Imamura, Toru; Araki, Nobukazu; NISHINO, Ichizo; Hayashi, Yukiko

    2012-01-01

    In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild-type dysferlin ...

  9. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    Energy Technology Data Exchange (ETDEWEB)

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  10. Evolution of a domain conserved in microtubule-associated proteins of eukaryotes

    Directory of Open Access Journals (Sweden)

    Alex S Rajangam

    2008-09-01

    Full Text Available Alex S Rajangam1, Hongqian Yang2, Tuula T Teeri1, Lars Arvestad21KTH Biotechnology, Swedish Center for Biomimetic Fiber Engineering, AlbaNova, Stockholm, Sweden; 2Stockholm Bioinformatics Center and School of Computer Science and Communication, Royal Institute of Technology, AlbaNova, Stockholm, SwedenAbstract: The microtubule network, the major organelle of the eukaryotic cytoskeleton, is involved in cell division and differentiation but also with many other cellular functions. In plants, microtubules seem to be involved in the ordered deposition of cellulose microfibrils by a so far unknown mechanism. Microtubule-associated proteins (MAP typically contain various domains targeting or binding proteins with different functions to microtubules. Here we have investigated a proposed microtubule-targeting domain, TPX2, first identified in the Kinesin-like protein 2 in Xenopus. A TPX2 containing microtubule binding protein, PttMAP20, has been recently identified in poplar tissues undergoing xylogenesis. Furthermore, the herbicide 2,6-dichlorobenzonitrile (DCB, which is a known inhibitor of cellulose synthesis, was shown to bind specifically to PttMAP20. It is thus possible that PttMAP20 may have a role in coupling cellulose biosynthesis and the microtubular networks in poplar secondary cell walls. In order to get more insight into the occurrence, evolution and potential functions of TPX2-containing proteins we have carried out bioinformatic analysis for all genes so far found to encode TPX2 domains with special reference to poplar PttMAP20 and its putative orthologs in other plants.Keywords: TPX2 domain, MAP20, evolution, microtubule, cellulose, bioinformatics

  11. Longitudinal associations between activity and cognition vary by age, activity type, and cognitive domain.

    Science.gov (United States)

    Bielak, Allison A M; Gerstorf, Denis; Anstey, Kaarin J; Luszcz, Mary A

    2014-12-01

    The demonstration of correlated change is critical to understanding the relationship between activity engagement and cognitive functioning in older adulthood. Changes in activity have been shown to be related to changes in cognition, but little attention has been devoted to how this relationship may vary between specific activity types, cognitive domains, and age groups. Participants initially aged 65-98 years (M = 77.46 years) from the Australian Longitudinal Study of Ageing (n = 1,321) completed measurements of activity (i.e., cognitive, group social, one-on-one social, and physical) and cognition (i.e., perceptual speed, and immediate and delayed episodic memory) at baseline, 2, 8, 11, and 15 years later. Bivariate latent growth curve models covarying for education, sex, and baseline age and medical conditions revealed multiple positive-level relations between activity and cognitive performance, but activity level was not related to later cognitive change. Change in perceptual speed over 15 years was positively associated with change in cognitive activity, and change in immediate episodic memory was positively associated with change in one-on-one social activity. Old-old adults showed a stronger change-change covariance for mentally stimulating activity in relation to perceptual speed than did young-old adults. The differentiation by activity type, cognitive domain, and age contributes to the growing evidence that there is variation in the way cognitive ability at different ages is related to activity.

  12. Domain dependent associations between cognitive functioning and regular voluntary exercise behavior.

    Science.gov (United States)

    Swagerman, Suzanne C; de Geus, Eco J C; Koenis, Marinka M G; Hulshoff Pol, Hilleke E; Boomsma, Dorret I; Kan, Kees-Jan

    2015-07-01

    Regular exercise has often been suggested to have beneficial effects on cognition, but empirical findings are mixed because of heterogeneity in sample composition (age and sex); the cognitive domain being investigated; the definition and reliability of exercise behavior measures; and study design (e.g., observational versus experimental). Our aim was to scrutinize the domain specificity of exercise effects on cognition, while controlling for the other sources of heterogeneity. In a population based sample consisting of 472 males and 668 females (aged 10-86 years old) we administered the Computerized Neurocognitive Battery (CNB), which provided accuracy and speed measures of abstraction and mental flexibility, attention, working memory, memory (verbal, face, and spatial), language and nonverbal reasoning, spatial ability, emotion identification, emotion- and age differentiation, sensorimotor speed, and motor speed. Using univariate and multivariate regression models, CNB scores were associated with participants' average energy expenditure per week (weekly METhours), which were derived from a questionnaire on voluntary regular leisure time exercise behavior. Univariate models yielded generally positive associations between weekly METhours and cognitive accuracy and speed, but multivariate modeling demonstrated that direct relations were small and centered around zero. The largest and only significant effect size (β = 0.11, p confidence. PMID:25956142

  13. TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

    Directory of Open Access Journals (Sweden)

    Sharron A N Brown

    Full Text Available The tumor necrosis factor (TNF superfamily member TNF-like weak inducer of apoptosis (TWEAK is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14. TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

  14. Domains and correlates of clinical balance impairment associated with Huntington's disease.

    Science.gov (United States)

    Jacobs, Jesse V; Boyd, James T; Hogarth, Penelope; Horak, Fay B

    2015-03-01

    This study sought to (a) determine the domains of clinical balance impairments associated with Huntington's disease (HD), and (b) evaluate associations between balance test scores and other disease-related impairments. Eighteen subjects with genetically definite HD and 17 age-matched control subjects were evaluated on the Mini-BESTest for their clinical balance impairments as well as the Unified HD Rating Scale (UHDRS) motor and total functional capacity scales, Activity-Specific Balance Confidence (ABC) Scale-short form, Montreal Cognitive Assessment (MoCA), and Symbol Digit Modalities Test (SDMT). Results showed that subjects with HD exhibited significantly lower total Mini-BESTest scores than subjects without HD (mean (95% CI)=76 (64-87)% with HD, 98 (96-99)% without HD; p=0.0011). Mini-BESTest item scores were significantly lower for subjects with HD on one-leg stance, postural responses, standing with eyes closed on foam, and dual-task timed up-and-go. Mini-BESTest scores significantly correlated with UHDRS motor (r(2)=0.68; p=0.00003) and total functional capacity (r(2)=0.75; p=0.000006) scores as well as with scores on the ABC short form (r(2)=0.45; p=0.0024), SDMT (r(2)=0.42; p=0.0036), and MoCA (r(2)=0.23; p=0.046) assessments. This study, therefore, demonstrates that balance impairments associated with HD span domains of anticipatory postural adjustments, postural responses, stance in challenging sensory conditions, and gait. Although preliminary, clinical balance impairment appears to be an efficient proxy evaluation of multiple HD-related factors due to associations with functional capacity, other motor impairments, balance confidence, and cognitive abilities. PMID:25797790

  15. Mutation in the sixth immunoglobulin domain of L1CAM is associated with migrational brain anomalies

    Science.gov (United States)

    Shieh, Christine; Moser, Franklin; Graham, John M.; Watiker, Valerie

    2015-01-01

    Objective: To describe the phenotype of a patient with classical features of X-linked L1 syndrome associated with novel brain malformations. Methods: Diagnostic analysis included physical and dysmorphology examinations, MRI of the brain, and exome sequencing of the family trio. Results: We report a 2.5-year-old boy with developmental delay, dysmorphic facies, and adducted thumbs. MRI of the brain showed a truncated corpus callosum and periventricular heterotopias associated with polymicrogyria (PMG). Variant segregation analysis with exome sequencing discovered a novel maternally derived hemizygous variant in exon 14 of the L1CAM gene (c.1759 G>C; p.G587R). Conclusions: This novel L1CAM mutation was located in the protein's sixth immunoglobin domain and involved glycine-587, a key residue in the structure of L1CAM because of its interactions with lysine-606, which indicates that any mutation at this site would likely affect the secondary structure and function of the protein. The replacement of the small nonpolar glycine residue with a large basic arginine would have an even more dramatic result. The presentation of periventricular nodular heterotopias with overlying PMG is very uncommon, and its association with L1CAM may provide insight into other similar cases. Furthermore, this presentation indicates the important role that L1CAM plays in neuronal migration and brain development and extends the phenotype associated with L1CAM-associated disorders. PMID:27066571

  16. Transmembrane Helix Assembly by Max-Min Ant System Algorithm.

    Science.gov (United States)

    Sujaree, Kanon; Kitjaruwankul, Sunan; Boonamnaj, Panisak; Supunyabut, Chirayut; Sompornpisut, Pornthep

    2015-12-01

    Because of the rapid progress in biochemical and structural studies of membrane proteins, considerable attention has been given on developing efficient computational methods for solving low-to-medium resolution structures using sparse structural data. In this study, we demonstrate a novel algorithm, max-min ant system (MMAS), designed to find an assembly of α-helical transmembrane proteins using a rigid helix arrangement guided by distance constraints. The new algorithm generates a large variety with finite number of orientations of transmembrane helix bundle and finds the solution that is matched with the provided distance constraints based on the behavior of ants to search for the shortest possible path between their nest and the food source. To demonstrate the efficiency of the novel search algorithm, MMAS is applied to determine the transmembrane packing of KcsA and MscL ion channels from a limited distance information extracted from the crystal structures, and the packing of KvAP voltage sensor domain using a set of 10 experimentally determined constraints, and the results are compared with those of two popular used stochastic methods, simulated annealing Monte Carlo method and genetic algorithm. PMID:26058409

  17. Cooperative Transmembrane Penetration of Nanoparticles

    Science.gov (United States)

    Zhang, Haizhen; Ji, Qiuju; Huang, Changjin; Zhang, Sulin; Yuan, Bing; Yang, Kai; Ma, Yu-qiang

    2015-01-01

    Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs’ translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery. PMID:26013284

  18. Biophysical Aspects of Transmembrane Signaling

    CERN Document Server

    Damjanovich, Sandor

    2005-01-01

    Transmembrane signaling is one of the most significant cell biological events in the life and death of cells in general and lymphocytes in particular. Until recently biochemists and biophysicists were not accustomed to thinking of these processes from the side of a high number of complex biochemical events and an equally high number of physical changes at molecular and cellular levels at the same time. Both types of researchers were convinced that their findings are the most decisive, having higher importance than the findings of the other scientist population. Both casts were wrong. Life, even at cellular level, has a number of interacting physical and biochemical mechanisms, which finally build up the creation of an "excited" cell that will respond to particular signals from the outer or inner world. This book handles both aspects of the signalling events, and in some cases tries to unify our concepts and help understand the signals that govern the life and death of our cells. Not only the understanding, bu...

  19. Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains.

    Science.gov (United States)

    Ulianov, Sergey V; Khrameeva, Ekaterina E; Gavrilov, Alexey A; Flyamer, Ilya M; Kos, Pavel; Mikhaleva, Elena A; Penin, Aleksey A; Logacheva, Maria D; Imakaev, Maxim V; Chertovich, Alexander; Gelfand, Mikhail S; Shevelyov, Yuri Y; Razin, Sergey V

    2016-01-01

    Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin. PMID:26518482

  20. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

    Directory of Open Access Journals (Sweden)

    Nicola Vitulo

    2007-01-01

    Full Text Available The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confi rming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals fi ve distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed.

  1. Gene regulation during development in the light of topologically associating domains.

    Science.gov (United States)

    Remeseiro, Silvia; Hörnblad, Andreas; Spitz, François

    2016-01-01

    During embryonic development, complex transcriptional programs govern the precision of gene expression. Many key developmental genes are regulated via cis-regulatory elements that are located far away in the linear genome. How sequences located hundreds of kilobases away from a promoter can influence its activity has been the subject of numerous speculations, which all underline the importance of the 3D-organization of the genome. The recent advent of chromosome conformation capture techniques has put into focus the subdivision of the genome into topologically associating domains (TADs). TADs may influence regulatory activities on multiple levels. The relative invariance of TAD limits across cell types suggests that they may form fixed structural domains that could facilitate and/or confine long-range regulatory interactions. However, most recent studies suggest that interactions within TADs are more variable and dynamic than initially described. Hence, different models are emerging regarding how TADs shape the complex 3D conformations, and thereafter influence the networks of cis-interactions that govern gene expression during development. For further resources related to this article, please visit the WIREs website. PMID:26558551

  2. Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

    International Nuclear Information System (INIS)

    Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of Mr = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind [3H]flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinization of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing [3H]flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated

  3. A Business Intelligence Model to Predict Bankruptcy using Financial Domain Ontology with Association Rule Mining Algorithm

    CERN Document Server

    Martin, A; Venkatesan, Dr V Prasanna

    2011-01-01

    Today in every organization financial analysis provides the basis for understanding and evaluating the results of business operations and delivering how well a business is doing. This means that the organizations can control the operational activities primarily related to corporate finance. One way that doing this is by analysis of bankruptcy prediction. This paper develops an ontological model from financial information of an organization by analyzing the Semantics of the financial statement of a business. One of the best bankruptcy prediction models is Altman Z-score model. Altman Z-score method uses financial rations to predict bankruptcy. From the financial ontological model the relation between financial data is discovered by using data mining algorithm. By combining financial domain ontological model with association rule mining algorithm and Zscore model a new business intelligence model is developed to predict the bankruptcy.

  4. Fas-Associated Protein with Death Domain Regulates Notch Signaling during Muscle Regeneration.

    Science.gov (United States)

    Zhang, Rong; Wang, Lu; He, Liangqiang; Yang, Bingya; Yao, Chun; Du, Pan; Xu, Qiang; Cheng, Wei; Hua, Zi-Chun

    2014-01-01

    Notch signaling plays critical roles during myogenesis by promoting the proliferation and inhibiting the differentiation of myogenic progenitors. However, the mechanism of the temporal regulation of Notch signaling during the myogenic lineage progression remains elusive. In the present study, we show that a constitutively phosphoryl-mimicking mutation of Fas-associated death domain (FADD-D) enhances Notch-1 signaling and compromises Wnt signaling in both cultured myoblasts and regenerating muscles, which results in inhibited myogenic differentiation and muscle regeneration. Inhibition of Notch signaling recovers the regeneration ability in injured FADD-D muscles through rescuing Wnt signaling. Furthermore, we found that protein kinase Cα mediates FADD-D-induced Notch-1 signaling by stabilizing Notch-1. Collectively, these data identify a novel mechanism for the temporal regulation of Notch signaling during myogenic lineage progression and muscle regeneration. PMID:26303234

  5. A Business Intelligence Model to Predict Bankruptcy using Financial Domain Ontology with Association Rule Mining Algorithm

    Directory of Open Access Journals (Sweden)

    A Martin

    2011-05-01

    Full Text Available Today in every organization financial analysis provides the basis for understanding and evaluating the results of business operations and delivering how well a business is doing. This means that the organizations can control the operational activities primarily related to corporate finance. One way that doing this is by analysis of bankruptcy prediction. This paper develops an ontological model from financial information of an organization by analyzing the Semantics of the financial statement of a business. One of the best bankruptcy prediction models is Altman Z-score model. Altman Z-score method uses financial rations to predict bankruptcy. From the financial ontological model the relation between financial data is discovered by using data mining algorithm. By combining financial domain ontological model with association rule mining algorithm and Z-score model a new business intelligence model is developed to predict the bankruptcy.

  6. The PAM domain, a multi-protein complex-associated module with an all-alpha-helix fold

    Directory of Open Access Journals (Sweden)

    Izaurralde Elisa

    2003-12-01

    Full Text Available Abstract Background Multimeric protein complexes have a role in many cellular pathways and are highly interconnected with various other proteins. The characterization of their domain composition and organization provides useful information on the specific role of each region of their sequence. Results We identified a new module, the PAM domain (PCI/PINT associated module, present in single subunits of well characterized multiprotein complexes, like the regulatory lid of the 26S proteasome, the COP-9 signalosome and the Sac3-Thp1 complex. This module is an around 200 residue long domain with a predicted TPR-like all-alpha-helical fold. Conclusions The occurrence of the PAM domain in specific subunits of multimeric protein complexes, together with the role of other all-alpha-helical folds in protein-protein interactions, suggest a function for this domain in mediating transient binding to diverse target proteins.

  7. A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

    Directory of Open Access Journals (Sweden)

    Lynch Christopher R

    2007-02-01

    Full Text Available Abstract Background Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. Results This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1. While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. Conclusion Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate

  8. Robust Face Recognition by Hierarchical Kernel Associative Memory Models Based on Spatial Domain Gabor Transforms

    Directory of Open Access Journals (Sweden)

    Bai-ling Zhang

    2006-07-01

    Full Text Available Face recognition can be studied as an associative memory (AM problem and kernel-based AM models have been proven efficient. In this paper, a hierarchical Kernel Associative Memory (KAM face recognition scheme with a multiscale Gabor transform, is proposed. The pyramidal multiscale Gabor decomposition proposed by Nestares, Navarro, Portilla and Tabernero not only provides a very efficient implementation of the Gabor transform in the spatial domain, but also permits a fast reconstruction of images. In our method, face images of each person are first decomposed into their multiscale representations by a quasicomplete Gabor transform, which are then modelled by Kernel Associative Memories. In the recognition stage, a query face image is also represented by a Gabor multiresolution pyramid and the reconstructions from different KAM models corresponding to even Gabor channels are then simply summed to give the recall. The recognition scheme was thoroughly tested using several benchmarking face datasets, including the AR faces, UMIST faces, JAFFE faces and Yale A faces, which include different kind of face variations from occlusions, pose, expression and illumination. The experiment results show that the proposed method demonstrated strong robustness in recognizing faces under different conditions, particularly under occlusions, pose alterations and expression changes.

  9. Association of Patient-Provider Communication Domains with Lung Cancer Treatment

    Science.gov (United States)

    Lin, Jenny J.; Lake, Jessica; Wall, Melanie M.; Berman, Andrew R.; Salazar-Schicchi, John; Powell, Charles; Keller, Steven M.; Halm, Ethan A.; Leventhal, Howard; Wisnivesky, Juan P.

    2014-01-01

    Background Patient-physician communication is critical for helping patients understand and complete the complex steps needed to diagnose stage and treat lung cancer. We assessed which domains of patient-physician communication about lung cancer and its treatment are associated with receipt of disease-directed, stage-appropriate treatment. Methods Patients with recently-diagnosed lung cancer were recruited from four medical centers in New York City from 2008 to 2011. Participants were surveyed about discussions with physicians regarding treatment, symptoms and needs. Multiple regression analysis and structural equation modeling were used to assess which communication factors were associated with disease treatment. Results Of the 352 participants, 191 (54%) received disease-directed, stage-appropriate treatment. Unadjusted associations between communication items and treatment found that participants who felt that their physicians explained the risks and disadvantages of lung cancer treatment (P0.05 for both comparisons). Conclusions These data suggest that treatment information is particularly important for increasing the probability of cancer-directed therapy among lung cancer patients. Clinicians should ensure that they clearly discuss treatment goals and options with patients while maintaining empathy, supporting patient needs, and addressing symptoms. PMID:25122421

  10. Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin

    DEFF Research Database (Denmark)

    Bertin, Gwladys I; Lavstsen, Thomas; Guillonneau, François;

    2013-01-01

    Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi......-conserved types defined by tandem runs of specific domains ("domain cassettes" (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection....

  11. The structure of the CARD8 caspase-recruitment domain suggests its association with the FIIND domain and procaspases through adjacent surfaces.

    Science.gov (United States)

    Jin, Tengchuan; Huang, Mo; Smith, Patrick; Jiang, Jiansheng; Xiao, T Sam

    2013-05-01

    CARD8 plays crucial roles in regulating apoptotic and inflammatory signaling pathways through the association of its caspase-recruitment domain (CARD) with those of procaspase-9 and procaspase-1. The CARD8 CARD has also been predicted to form an intramolecular complex with its FIIND domain. Here, the first crystal structure of the CARD8 CARD is reported; it adopts a six-helix bundle fold with a unique conformation of the α6 helix that is described here for the first time. The surface of the CARD8 CARD displays a prominent acidic patch at its α2, α3 and α5 helices that may interact with the procaspase-9 CARD, whereas an adjacent charged surface at its α3 and α4 helices may associate with the CARD8 FIIND domain without interfering with the CARD-CARD interaction. This suggests that the function of CARD8 may be regulated by both intramolecular and intermolecular interactions involving electrostatic attractions. PMID:23695559

  12. Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Steinocher, Helena; Brooks, Andrew J.;

    2015-01-01

    -labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations...... of single-pass TMDs, which are difficult to solve by other means....

  13. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies

    NARCIS (Netherlands)

    Reggiori, Fulvio; Pelham, Hugh R B; Reggiori, Fulvio

    2002-01-01

    Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. W

  14. Fatty acyl chain-dependent but charge-independent association of the SH4 domain of Lck with lipid membranes

    Indian Academy of Sciences (India)

    Anoop Rawat; Avaronnan Harishchandran; Ramakrishnan Nagaraj

    2013-03-01

    The SH4 domain of Src family of nonreceptor protein tyrosine kinases represents the extreme N-terminal 1–16 amino acid region which mediates membrane association of these proteins and facilitates their functions. The SH4 domains among Src members lack well-defined sequence consensus and vary in the net charge. However, they readily anchor to the cytoplasmic face of the plasma membrane upon fatty acid acylation. Here, we report the membrane association of differentially acylated SH4 domain of Lck kinase, which has net negative charge at physiological pH. Our results suggest that despite the net negative charge, the SH4 domain of Lck associates with membranes upon fatty acid acylation. While myristoylation at the N-terminus is sufficient for providing membrane anchorage, multiple acylation determines orientation of the peptide chain with respect to the lipid bilayer. Hence, fatty acylation serves more than just a lipid anchor. It has an important role in regulating the spatial orientation of the peptide domain with respect to the lipid bilayer, which could be important for the interaction of the other domains of these kinases with their partners.

  15. Rigidity of transmembrane proteins determines their cluster shape

    Science.gov (United States)

    Jafarinia, Hamidreza; Khoshnood, Atefeh; Jalali, Mir Abbas

    2016-01-01

    Protein aggregation in cell membrane is vital for the majority of biological functions. Recent experimental results suggest that transmembrane domains of proteins such as α -helices and β -sheets have different structural rigidities. We use molecular dynamics simulation of a coarse-grained model of protein-embedded lipid membranes to investigate the mechanisms of protein clustering. For a variety of protein concentrations, our simulations under thermal equilibrium conditions reveal that the structural rigidity of transmembrane domains dramatically affects interactions and changes the shape of the cluster. We have observed stable large aggregates even in the absence of hydrophobic mismatch, which has been previously proposed as the mechanism of protein aggregation. According to our results, semiflexible proteins aggregate to form two-dimensional clusters, while rigid proteins, by contrast, form one-dimensional string-like structures. By assuming two probable scenarios for the formation of a two-dimensional triangular structure, we calculate the lipid density around protein clusters and find that the difference in lipid distribution around rigid and semiflexible proteins determines the one- or two-dimensional nature of aggregates. It is found that lipids move faster around semiflexible proteins than rigid ones. The aggregation mechanism suggested in this paper can be tested by current state-of-the-art experimental facilities.

  16. Structural Dynamics of Insulin Receptor and Transmembrane Signaling.

    Science.gov (United States)

    Tatulian, Suren A

    2015-09-15

    The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular β-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure-function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling.

  17. Structural heterogeneity and functional diversity of topologically associating domains in mammalian genomes.

    Science.gov (United States)

    Wang, Xiao-Tao; Dong, Peng-Fei; Zhang, Hong-Yu; Peng, Cheng

    2015-09-01

    Recent chromosome conformation capture (3C) derived techniques have revealed that topologically associating domain (TAD) is a pervasive element in chromatin three-dimensional (3D) organization. However, there is currently no parameter to quantitatively measure the structural characteristics of TADs, thus obscuring our understanding on the structural and functional differences among TADs. Based on our finding that there exist intrinsic chromatin interaction patterns in TADs, we define a theoretical parameter, called aggregation preference (AP), to characterize TAD structures by capturing the interaction aggregation degree. Applying this defined parameter to 11 Hi-C data sets generated by both traditional and in situ Hi-C experimental pipelines, our analyses reveal that heterogeneous structures exist among TADs, and this structural heterogeneity is significantly correlated to DNA sequences, epigenomic signals and gene expressions. Although TADs can be stable in genomic positions across cell lines, structural comparisons show that a considerable number of stable TADs undergo significantly structural rearrangements during cell changes. Moreover, the structural change of TAD is tightly associated with its transcription remodeling. Altogether, the theoretical parameter defined in this work provides a quantitative method to link structural characteristics and biological functions of TADs, and this linkage implies that chromatin interaction pattern has the potential to mark transcription activity in TADs. PMID:26150425

  18. Transcription start site associated RNAs (TSSaRNAs are ubiquitous in all domains of life.

    Directory of Open Access Journals (Sweden)

    Livia S Zaramela

    Full Text Available A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq and a primary-transcript enriched (dRNA-seq strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome. Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  19. Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders.

    Science.gov (United States)

    Gómez-Marín, Carlos; Tena, Juan J; Acemel, Rafael D; López-Mayorga, Macarena; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Maeso, Ignacio; Beccari, Leonardo; Aneas, Ivy; Vielmas, Erika; Bovolenta, Paola; Nobrega, Marcelo A; Carvajal, Jaime; Gómez-Skarmeta, José Luis

    2015-06-16

    Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution. PMID:26034287

  20. Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

    Directory of Open Access Journals (Sweden)

    Evangelos Dioletis

    Full Text Available Death-associated protein kinase (DAPk is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD, NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2 and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural

  1. Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.

    Directory of Open Access Journals (Sweden)

    Ryan Richards

    Full Text Available Channelrhodopsin-2 (ChR2 is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.

  2. Solution NMR and X-ray Crystal Structures of Membrane-associated Lipoprotein-17 Domain Reveal a Novel Fold

    Energy Technology Data Exchange (ETDEWEB)

    R Mani; S Vorobiev; G Swapna; H Neely; H Janjua; C Ciccosanti; D Xiao; J Hunt; G Montelione; et al.

    2011-12-31

    The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0{_}UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a 'domain of unknown function' (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0{_}UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 {angstrom}. The two structures are in good agreement with each other. The domain structure features three {alpha}-helices, {alpha}1 through {alpha}3, and five {beta}-strands. Strands {beta}1/{beta}2, {beta}3/{beta}4, {beta}4/{beta}5 are anti-parallel to each other. Strands {beta}1 and {beta}2 are orthogonal to strands {beta}3, {beta}4, {beta}5, while helix {alpha}3 is formed between the strands {beta}3 and {beta}4. One-turn helix {alpha}2 is formed between the strands {beta}1 and {beta}2, while helix {alpha}1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0{_}UREPA, indicating that it is a novel protein fold.

  3. Structure, function and evolution of topologically associating domains (TADs) at HOX loci.

    Science.gov (United States)

    Lonfat, Nicolas; Duboule, Denis

    2015-10-01

    Hox genes encode transcription factors necessary for patterning the major developing anterior to posterior embryonic axis. In addition, during vertebrate evolution, various subsets of this gene family were co-opted along with the emergence of novel body structures, such as the limbs or the external genitalia. The morphogenesis of these axial structures thus relies in part upon the precisely controlled transcription of specific Hox genes, a mechanism involving multiple long-range enhancers. Recently, it was reported that such regulatory mechanisms were largely shared between different developing tissues, though with some specificities, suggesting the recruitment of ancestral regulatory modalities from one tissue to another. The analysis of chromatin architectures at HoxD and HoxA loci revealed the existence of two flanking topologically associating domains (TADs), precisely encompassing the adjacent regulatory landscapes. Here, we discuss the function of these TADs in the control of Hox gene regulation and we speculate about their capacity to serve as structural frameworks for the emergence of novel enhancers. In this view, TADs may have been used as genomic niches to evolve pleiotropic regulations found at many developmental loci. PMID:25913784

  4. Individual and Contextual Parameters Associated with Adolescents' Domain Specific Self-Perceptions

    Science.gov (United States)

    Kokkinos, Constantinos M.; Hatzinikolaou, Stamatia

    2011-01-01

    The present study examined the role of adolescents' self-esteem and perceptions of family and classroom contexts on their domain specific self-perceptions. 345 Greek junior high school adolescents aged 14-16 completed measures of domain specific self-perceptions, self-esteem, parenting styles and classroom climate. Hierarchical regression analyses…

  5. Detecting pore-lining regions in transmembrane protein sequences

    Directory of Open Access Journals (Sweden)

    Nugent Timothy

    2012-07-01

    Full Text Available Abstract Background Alpha-helical transmembrane channel and transporter proteins play vital roles in a diverse range of essential biological processes and are crucial in facilitating the passage of ions and molecules across the lipid bilayer. However, the experimental difficulties associated with obtaining high quality crystals has led to their significant under-representation in structural databases. Computational methods that can identify structural features from sequence alone are therefore of high importance. Results We present a method capable of automatically identifying pore-lining regions in transmembrane proteins from sequence information alone, which can then be used to determine the pore stoichiometry. By labelling pore-lining residues in crystal structures using geometric criteria, we have trained a support vector machine classifier to predict the likelihood of a transmembrane helix being involved in pore formation. Results from testing this approach under stringent cross-validation indicate that prediction accuracy of 72% is possible, while a support vector regression model is able to predict the number of subunits participating in the pore with 62% accuracy. Conclusion To our knowledge, this is the first tool capable of identifying pore-lining regions in proteins and we present the results of applying it to a data set of sequences with available crystal structures. Our method provides a way to characterise pores in transmembrane proteins and may even provide a starting point for discovering novel routes of therapeutic intervention in a number of important diseases. This software is freely available as source code from: http://bioinf.cs.ucl.ac.uk/downloads/memsat-svm/.

  6. Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain

    NARCIS (Netherlands)

    Levasseur, A.; Pagès, S.; Fierobe, H.-P.; Navarro, D.; Punt, P.; Belaïch, J.-P.; Asther, M.; Record, E.

    2004-01-01

    A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergilhis niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produc

  7. Structural and Functional Studies of the Ras-Associating and Pleckstrin Homology Domains of Grb10 and Grb14

    Energy Technology Data Exchange (ETDEWEB)

    Depetris, R.; Wu, J; Hubbard, S

    2009-01-01

    Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

  8. Activation of p115-RhoGEF requires direct association of Gα13 and the Dbl homology domain.

    Science.gov (United States)

    Chen, Zhe; Guo, Liang; Hadas, Jana; Gutowski, Stephen; Sprang, Stephen R; Sternweis, Paul C

    2012-07-20

    RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

  9. Assembly of transmembrane proteins on oil-water interfaces

    Science.gov (United States)

    Yunker, Peter; Landry, Corey; Chong, Shaorong; Weitz, David

    2015-03-01

    Transmembrane proteins are difficult to handle by aqueous solution-based biochemical and biophysical approaches, due to the hydrophobicity of transmembrane helices. Detergents can solubilize transmembrane proteins; however, surfactant coated transmembrane proteins are not always functional, and purifying detergent coated proteins in a micellar solution can be difficult. Motivated by this problem, we study the self-assembly of transmembrane proteins on oil-water interfaces. We found that the large water-oil interface of oil drops prevents nascent transmembrane proteins from forming non-functional aggregates. The oil provides a hydrophobic environment for the transmembrane helix, allowing the ectodomain to fold into its natural structure and orientation. Further, modifying the strength or valency of hydrophobic interactions between transmembrane proteins results in the self-assembly of spatially clustered, active proteins on the oil-water interface. Thus, hydrophobic interactions can facilitate, rather than inhibit, the assembly of transmembrane proteins.

  10. Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore

    Science.gov (United States)

    Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric

    1996-12-01

    The structure of the Staphylococcus aureus α-hemolysin pore has been determined to 1.9 overset{circ}{mathrm A} resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 overset{circ}{mathrm A} in length, that runs along the sevenfold axis and ranges from 14 overset{circ}{mathrm A} to 46 overset{circ}{mathrm A} in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each protomer contributes two β strands, each 65 overset{circ}{mathrm A} long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 overset{circ}{mathrm A} wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.

  11. Virus-Encoded 7 Transmembrane Receptors

    DEFF Research Database (Denmark)

    Mølleskov-Jensen, Ann-Sofie; Oliveira, MarthaTrindade; Farrell, Helen Elizabeth;

    2015-01-01

    Herpesviruses are an ancient group which have exploited gene capture of multiple cellular modulators of the immune response. Viral homologues of 7 transmembrane receptors (v7TMRs) are a consistent feature of beta- and gammaherpesviruses; the majority of the v7TMRs are homologous to cellular chemo...

  12. Advantages and limitations of the Five Domains model for assessing welfare impacts associated with vertebrate pest control.

    Science.gov (United States)

    Beausoleil, N J; Mellor, D J

    2015-01-01

    Many pest control activities have the potential to impact negatively on the welfare of animals, and animal welfare is an important consideration in the development, implementation and evaluation of ethically defensible vertebrate pest control. Thus, reliable and accurate methods for assessing welfare impacts are required. The Five Domains model provides a systematic method for identifying potential or actual welfare impacts associated with an event or situation in four physical or functional domains (nutrition, environment, health or functional status, behaviour) and one mental domain (overall mental or affective state). Here we evaluate the advantages and limitations of the Five Domains model for this purpose and illustrate them using specific examples from a recent assessment of the welfare impacts of poisons used to lethally control possums in New Zealand. The model has a number of advantages which include the following: the systematic identification of a wide range of impacts associated with a variety of control tools; the production of relative rankings of tools in terms of their welfare impacts; the easy incorporation of new information into assessments; and the highlighting of additional information needed. For example, a recent analysis of sodium fluoroacetate (1080) poisoning in possums revealed the need for more information on the period from the onset of clinical signs to the point at which consciousness is lost, as well as on the level of consciousness during or after the occurrence of muscle spasms and seizures. The model is also valuable because it clearly separates physical or functional and affective impacts, encourages more comprehensive consideration of negative affective experiences than has occurred in the past, and allows development and evaluation of targeted mitigation strategies. Caution must be used in interpreting and applying the outputs of the model, most importantly because relative rankings or grades are fundamentally qualitative in

  13. Crystallizing Transmembrane Peptides in Lipidic Mesophases

    Energy Technology Data Exchange (ETDEWEB)

    Höfer, Nicole; Aragão, David; Caffrey, Martin (Trinity)

    2011-09-28

    Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having {le}4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gramicidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in the human genome.

  14. Association of the SPT2 chromatin protein domain containing 1 gene rs17579600 polymorphism and serum lipid traits

    OpenAIRE

    Guo, Tao; Yin, Rui-Xing; Bin, Yuan; Nie, Rong-Jun; Chen, Xia; Pan, Shang-Ling

    2015-01-01

    SPT2 chromatin protein domain containing 1 gene (SPTY2D1) is a candidate gene for dyslipidemia. The single nucleotide polymorphism (SNP) of rs7934205 near SPTY2D1 locus was ethnic- and sex-specific associated with serum lipid levels in our previous study. Whether SPTY2D1 rs17579600 SNP and several environmental factors are associated with serum lipid profiles is unknown. A total of 712 participants of Han and 689 unrelated individuals of Mulao were included. The genotype and allele frequencie...

  15. Designing Inhibitors against HOX domain mutations of PDX-1 and studying its association in Diabetes

    Directory of Open Access Journals (Sweden)

    Allam Appa Rao

    2012-03-01

    Full Text Available Type 1 diabetes mellitus was formally known as IDDM, type I, or juvenile onset diabetes. Type 1 DM can occur at any age. In this study,we analyzed the involvement of HOX domain of PDX-1 protein.The homeodomain transcription factor, pancreas duodenum homeobox (PDX-1, encoded by PDX-1 gene, which is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 and essential for pancreas development, insulin production, and glucose homeostasis.[1,13]. HOX domain has a length of 63aa and control developmental patterns and cell differentiation in vertebrates by acting positive or negative regulators[4,9,16]. Different approached had been applied to identify the mutational hot spot region of HOX domain and calculate mutational frequency of the amino acids which resides in the hotspot region. Binding site of the domain had been identified and found that THR208, GLN246 ,VAL247, ASN253 involved in interaction with ligand. Potential Inhibitors had been screened on the basis of various criteria and bioactivity score had been calculated. Energy optimization was done by applying AMBER force field and steepest descent method. Docking was performed by CCDC GOLD, Molegro, HEX, and Argus lab to find the best potent inhibitor and increase the accuracy of the docking process. Sitagliptin showed satisfactory result on both docking and bioactivity analysis. It showed a GOLD fitness score of 49.8386 and had a moldock score of -122.919 with a ligand efficiency -4.33692. Compound had a bioactivity score of 0.56 for protease inhibitor. Sitagliptin showed good binding affinity to the target, which helps to work the pancreas in proper way and to secret insulin.

  16. Association of astrocytes with neurons and astrocytes derived from distinct progenitor domains in the subpallium

    OpenAIRE

    Makio Torigoe; Kenta Yamauchi; Yan Zhu; Hiroaki Kobayashi; Fujio Murakami

    2015-01-01

    Astrocytes play pivotal roles in metabolism and homeostasis as well as in neural development and function in a manner thought to depend on their region-specific diversity. In the mouse spinal cord, astrocytes and neurons, which are derived from a common progenitor domain (PD) and controlled by common PD-specific transcription factors, migrate radially and share their final positions. However, whether astrocytes can only interact with neurons from common PDs in the brain remains unknown. Here,...

  17. Individual and contextual parameters associated with adolescents' domain specific self-perceptions.

    Science.gov (United States)

    Kokkinos, Constantinos M; Hatzinikolaou, Stamatia

    2011-04-01

    The present study examined the role of adolescents' self-esteem and perceptions of family and classroom contexts on their domain specific self-perceptions. 345 Greek junior high school adolescents aged 14-16 completed measures of domain specific self-perceptions, self-esteem, parenting styles and classroom climate. Hierarchical regression analyses revealed that both family and classroom contexts predicted students' self-perceptions, after students' demographics, academic achievement and self-esteem were controlled for. However, different patterns emerged in the relationship between family, classroom climate and self-esteem depending on domain specific self-perceptions. Academic self-perceptions (scholastic, mathematics and language competences) were predicted by classroom climate dimensions (order and organization, student involvement, rule clarity), whereas self-perceptions regarding relations with parents, close friends and behaviour conduct, were predicted by parenting styles. Given the fact that adolescence is a period of fluctuation in self-understanding which renders self-perceptions particularly malleable, the results support the critical role of the social environments where adolescents operate.

  18. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L;

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells ...

  19. Body image and personality among British men: associations between the Big Five personality domains, drive for muscularity, and body appreciation.

    Science.gov (United States)

    Benford, Karis; Swami, Viren

    2014-09-01

    The present study examined associations between the Big Five personality domains and measures of men's body image. A total of 509 men from the community in London, UK, completed measures of drive for muscularity, body appreciation, the Big Five domains, and subjective social status, and provided their demographic details. The results of a hierarchical regression showed that, once the effects of participant body mass index (BMI) and subjective social status had been accounted for, men's drive for muscularity was significantly predicted by Neuroticism (β=.29). In addition, taking into account the effects of BMI and subjective social status, men's body appreciation was significantly predicted by Neuroticism (β=-.35) and Extraversion (β=.12). These findings highlight potential avenues for the development of intervention approaches based on the relationship between the Big Five personality traits and body image.

  20. SAP-like domain in nucleolar spindle associated protein mediates mitotic chromosome loading as well as interphase chromatin interaction

    Energy Technology Data Exchange (ETDEWEB)

    Verbakel, Werner, E-mail: werner.verbakel@chem.kuleuven.be [Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200G, Bus 2403, 3001 Heverlee (Belgium); Carmeliet, Geert, E-mail: geert.carmeliet@med.kuleuven.be [Laboratory of Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Herestraat 49, Bus 902, 3000 Leuven (Belgium); Engelborghs, Yves, E-mail: yves.engelborghs@fys.kuleuven.be [Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200G, Bus 2403, 3001 Heverlee (Belgium)

    2011-08-12

    Highlights: {yields} The SAP-like domain in NuSAP is a functional DNA-binding domain with preference for dsDNA. {yields} This SAP-like domain is essential for chromosome loading during early mitosis. {yields} NuSAP is highly dynamic on mitotic chromatin, as evident from photobleaching experiments. {yields} The SAP-like domain also mediates NuSAP-chromatin interaction in interphase nucleoplasm. -- Abstract: Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase Nu

  1. Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin.

    Directory of Open Access Journals (Sweden)

    Gwladys I Bertin

    Full Text Available BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1 is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains ("domain cassettes" (DC. The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection. METHODS: Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM. We assessed the transcript level of var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS. RESULTS: Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples. CONCLUSION: This is the first study to show association between PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis.

  2. Carbohydrate Binding Module 74 is a novel starch binding domain associated with large and multi-domain α-amylase enzymes

    NARCIS (Netherlands)

    Valk, Vincent; Lammerts van Bueren, Alicia; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

    2016-01-01

    Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C-terminus (Domain 2). Deletion of D

  3. Transmembrane and Juxtamembrane Structure of αL Integrin in Bicelles.

    Directory of Open Access Journals (Sweden)

    Wahyu Surya

    Full Text Available The accepted model for the interaction of α and β integrins in the transmembrane (TM domain is based on the pair αIIbβ3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively. In the α chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several α integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an α-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in α integrins. We have studied the system αLβ2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in αIIb but also in αL-TM when reconstituted in bicelles. Additionally, titration with β2 TM showed eight residues clustering along one side of αL-TM, forming a plausible interacting face with β2. The latter orientation is consistent with a previously predicted reported polar interaction between αL Ser-1071 and β2 Thr-686.

  4. Transmembrane and Juxtamembrane Structure of αL Integrin in Bicelles.

    Science.gov (United States)

    Surya, Wahyu; Li, Yan; Millet, Oscar; Diercks, Tammo; Torres, Jaume

    2013-01-01

    The accepted model for the interaction of α and β integrins in the transmembrane (TM) domain is based on the pair αIIbβ3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the α chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several α integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an α-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in α integrins. We have studied the system αLβ2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in αIIb but also in αL-TM when reconstituted in bicelles. Additionally, titration with β2 TM showed eight residues clustering along one side of αL-TM, forming a plausible interacting face with β2. The latter orientation is consistent with a previously predicted reported polar interaction between αL Ser-1071 and β2 Thr-686.

  5. Expression and Purification of Functional HuGALNT3 without the Transmembrane Domain (huGALNT3-sol) in Pichia pastoris%重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

    Institute of Scientific and Technical Information of China (English)

    孔蕴; 郜海涛; 李树芳; 王鹏; 顾国锋; 顾黎

    2012-01-01

    目的:为了获得有催化活性的人乙酰半乳糖胺转移酶3(GALNT3),构建了GALNT3可溶性区域(GALNT3-sol)的真核分泌表达载体,在巴斯德毕赤酵母中表达并纯化GALNT3-sol蛋白,体外检测其转糖基活性.方法:以构建好的pET15b/GALNT3-sol为模板进行PCR,扩增编码人GALNT3-sol的cDNA片段(1755 bp),将其克隆至真核表达载体pPIC9K,载体线性化后采用电击法转化毕赤酵母GS115.通过MD平板和G418平板筛选出阳性高拷贝重组菌株.阳性菌株经过甲醇诱导表达人GALNT3-sol重组蛋白,表达上清进行Ni-NAT分离纯化.分别采用SDS-PAGE和Western blot鉴定纯化的重组蛋白,并使用HPLC和MALDI-TOF/MS分析其转糖基化反应的活性.结果:成功构建了能够分泌表达GALNT3-sol的毕赤酵母菌株.阳性表达菌株在BMMY培养基(pH 6.0)中20℃培养,经0.5%甲醇诱导表达96 h,摇瓶表达量可达5mg/L.SDS-PAGE和Western blot结果显示表达重组蛋白为糖基化形式.活性检测显示表达的重组蛋白具有转糖基活性.结论:成功获得可以高效分泌表达具有活性的人GALNT3-sol蛋白的毕赤酵母菌株,为进一步研究人GALNT3的性质及其应用提供了基础.%Objective: In order to research the bioactivity of GALNT3, the truncated part of GALNT3 (huGALNT3-sol) which was deleted of the hydrophobic trans-membrane domain were obtained using Pichia pastoris expression system, and assayed the transferring GalNAc activity of recombinant huGALNT3-sol. Methods; The gene of human GALNT3-sol (1 755 bp)was amplified from pET15b/ GALNTi-sol and cloned into expression vector pPIC9k, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains with high-level huGALNT3-sol production were screened out by MD plate and G418. High level of huGALNT3-sol was obtained in BMMY medium the induction of methanol, and purified from the supernatant with Ni-NAT. The identity of

  6. An α/β hydrolase and associated Per-ARNT-Sim domain comprise a bipartite sensing module coupled with diverse output domains.

    Directory of Open Access Journals (Sweden)

    Eugene V Nadezhdin

    Full Text Available The RsbQ α/β hydrolase and RsbP serine phosphatase form a signaling pair required to activate the general stress factor σ(B of Bacillus subtilis in response to energy limitation. RsbP has a predicted N-terminal Per-ARNT-Sim (PAS domain, a central coiled-coil, and a C-terminal protein phosphatase M (PPM domain. Previous studies support a model in which RsbQ provides an activity needed for PAS to regulate the phosphatase domain via the coiled-coil. RsbQ and the PAS domain (RsbP-PAS therefore appear to form a sensory module. Here we test this hypothesis using bioinformatic and genetic analysis. We found 45 RsbQ and RsbP-PAS homologues encoded by adjacent genes in diverse bacteria, with PAS and a predicted coiled-coil fused to one of three output domains: PPM phosphatase (Gram positive bacteria, histidine protein kinase (Gram negative bacteria, and diguanylate cyclase (both lineages. Multiple alignment of the RsbP-PAS homologues suggested nine residues that distinguish the class. Alanine substitutions at four of these conferred a null phenotype in B. subtilis, indicating their functional importance. The F55A null substitution lay in the Fα helix of an RsbP-PAS model. F55A inhibited interaction of RsbP with RsbQ in yeast two-hybrid and pull-down assays but did not significantly affect interaction of RsbP with itself. We propose that RsbQ directly contacts the PAS domains of an RsbP oligomer to provide the activating signal, which is propagated to the phosphatase domains via the coiled-coil. A similar mechanism would allow the RsbQ-PAS module to convey a common input signal to structurally diverse output domains, controlling a variety of physiological responses.

  7. Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

    DEFF Research Database (Denmark)

    Anders, Nadine; Nielsen, Michael M.; Keicher, Jutta;

    2008-01-01

    The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate...... association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs....

  8. Different domains of P21Cip1/waf1 regulate DNA replication and DNA repair-associated processes after UV

    International Nuclear Information System (INIS)

    Full text: Many genotoxic insults result in p21 up-regulation and p21-dependent cell cycle arrest but UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated to DNA repair. While the role of p21 in Nucleotide Excision Repair (NER) remains controversial, two recent reports explore its effect on Translesion DNA Synthesis (TLS), a process that avoids replication blockage during S phase. The first report shows that p21 degradation is required for efficient PCNA ubiquitination, a post transcriptional modification that is relevant for TLS. The second report demonstrates that p21 (-/-) cells have increased TLS-associated mutagenic rates. Herein we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK binding domain of p21 is required for cell cycle arrest in unstressed cells; neither the CDK- nor the PCNA-binding domains of p21 are able to block early and late steps of NER. Intriguingly, through its PCNA binding domain, p21 inhibited recruitment of the TLS-polymerase, polη to PCNA foci after UV. Moreover, this obstruction correlates with accumulation of γH2AX and increased apoptosis. Taking together, our data emphasizes the link between p21 turnover and efficient TLS. This might also suggest a potential effect of p21 on other activities of polζ, a DNA polymerase with central roles in other biological scenarios such as genetic conversion, homologous recombination and modulation of the cellular response to genotoxic agents

  9. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

    Science.gov (United States)

    Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

    2015-12-01

    Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3' splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein-protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. PMID:26420826

  10. Spectral domain optical coherence tomography morphology in optic disc pit associated maculopathy

    Directory of Open Access Journals (Sweden)

    Janusz Michalewski

    2014-01-01

    Full Text Available Purpose: Our purpose was to study the clinical manifestation and course of optic pit maculopathy using Spectral Domain Optical Coherence Tomography (SD- OCT images. Materials and Methods: We used SD-OCT to examine 20 eyes of 19 patients with a macular detachment in combination with an optic. Results: We observed five different fovea appearances in regard to fluid localization. In five eyes, we recorded changes in the fluid distribution with SD-OCT. In 17/20 eyes, we noted a communication between the perineural and subretinal and/or intraretinal space at the margin of the optic disc. Conclusion: 3-dimensional SD-OCT (3D-SDOCT scans revealed a three-fold connection, between subretinal and intraretinal space, perineural space, and the vitreous cavity. Therefore, we suppose that intraretinal or subretinal fluid in optic pit maculopathy may have both a vitreous and cerebrospinal origin. A membrane, covering the optic nerve was noted in 14 cases. Even if it seems intact in some B-scans, it is not complete in others several micrometers apart. Additionally, we observed fluid accumulation below the margin of the optic disc and hyperreflective porous tissue in the optic disc excavation. Those findings do not influence the course of maculopathy.

  11. The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution.

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    Anne-Laure Chateigner-Boutin

    Full Text Available Cell walls are complex structures surrounding plant cells with a composition that varies among species and even within a species between organs, cell types and development stages. For years, cell walls in wheat grains were described as simple walls consisting mostly of arabinoxylans and mixed-linked beta glucans. Proteomic and transcriptomic studies identified enzyme families involved in the synthesis of many more cell wall polysaccharides in the wheat grains. Here we describe the discovery of pectic domains in wheat grain using monoclonal antibodies and enzymatic treatment to degrade the major cell wall polymers. Distinct spatial distributions were observed for rhamnogalacturonan I present in the endosperm and mostly in the aleurone layer and homogalacturonan especially found in the outer layers, and tight developmental regulations were unveiled. We also uncovered a massive deposition of homogalacturonan via large vesicular bodies in the seed coat (testa beneath a thick cuticle during development. Our findings raise questions about the function of pectin in wheat grain.

  12. The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase and Other Eukaryotic BAM/GIDE Domain Proteins.

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    Jeremy G Wideman

    Full Text Available MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1 is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings

  13. Childhood trauma and dimensions of depression: a specific association with the cognitive domain

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    Edgar A. Vares

    2015-01-01

    Full Text Available Objective: To investigate associations between a history of childhood trauma and dimensions of depression in a sample of clinically depressed patients. Methods: A sample of 217 patients from a mood-disorder outpatient unit was investigated with the Beck Depression Inventory, the Hamilton Depression Rating Scale, the CORE Assessment of Psychomotor Change, and the Childhood Trauma Questionnaire. A previous latent model identifying six depressive dimensions was used for analysis. Path analysis and Multiple Indicators Multiple Causes (MIMIC models were used to investigate associations between general childhood trauma and childhood maltreatment modalities (emotional, sexual, and physical abuse; emotional and physical neglect with dimensions of depression (sexual, cognition, insomnia, appetite, non-interactiveness/retardation, and agitation. Results: The overall childhood trauma index was uniquely associated with cognitive aspects of depression, but not with any other depressive dimension. An investigation of childhood maltreatment modalities revealed that emotional abuse was consistently associated with depression severity in the cognitive dimension. Conclusion: Childhood trauma, and specifically emotional abuse, could be significant risk factors for the subsequent development of cognitive symptoms of major depression. These influences might be specific to this depressive dimension and not found in any other dimension, which might have conceptual and therapeutic implications for clinicians and researchers alike.

  14. Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension.

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    Alan Y Deng

    Full Text Available Multiple quantitative trait loci (QTLs for blood pressure (BP have been detected in rat models of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1/activator of G protein signaling 8 (Ags8 to be the strongest candidate for C1QTL1, since numerous non-synonymous mutations are found. Moreover, the 5' Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evidence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or targeted-gene modifications, it might represent a part of the BP genetic architecture that operates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could validate the concept that a QTL in itself can influence BP 'indirectly' by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 variations will reveal novel insights into the BP modulation via a regulatory hierarchy.

  15. Association of urinary N-domain Angiotensin I-converting enzyme with plasma inflammatory markers and endothelial function.

    Science.gov (United States)

    Fernandes, Fernanda B; Plavnik, Frida L; Teixeira, Andressa Ms; Christofalo, Dejaldo Mj; Ajzen, Sergio A; Higa, Elisa Ms; Ronchi, Fernanda A; Sesso, Ricardo Cc; Casarini, Dulce E

    2008-01-01

    The aim of this study was to investigate the association between urinary 90 kDa N-domain Angiotensin I-converting enzyme (ACE) form with C-reactive protein (CRP) and homocysteine plasma levels (Hcy), urinary nitric oxide (NOu), and endothelial function (EF) in normotensive subjects. Forty healthy subjects were evaluated through brachial Doppler US to test the response to reactive hyperemia and a panel of blood tests to determine CRP and Hcy levels, NOu, and urinary ACE. They were divided into groups according to the presence (ACE90+) or absence (ACE90-) of the 90 kDa ACE, the presence (FH+) or absence (FH-) of family history of hypertension, and the presence or absence of these two variables FH+/ACE90+ and FH-/ACE90-. We found an impaired endothelial dilatation in subjects who presented the 90 kDa N-domain ACE as follows: 11.4% +/- 5.3% in ACE90+ compared with 17.6% +/- 7.1% in ACE90- group and 12.4% +/- 5.6% in FH+/ACE90+ compared with 17.7% +/- 6.2% in FH-/ACE90- group, P < 0.05. Hcy and CRP levels were statistically significantly lower in FH+/ACE90+ than in FH-/ACE90- group, as follows: 10.0 +/- 2.3 microM compared with 12.7 +/- 1.5 microM, and 1.3 +/- 1.8 mg/L compared with 3.6 +/- 2.0 mg/L, respectively. A correlation between flow-mediated dilatation (FMD) and CRP, Hcy, and NOu levels was not found. Our study suggests a reduction in the basal NO production confirmed by NOu analysis in subjects with the 90 kDa N-domain ACE isoform alone or associated with a family history of hypertension. Our data suggest that the presence of the 90 kDa N-domain ACE itself may have a negative impact on flow-mediated dilatation stimulated by reactive hyperemia. PMID:18475311

  16. Molecular dynamics simulation of the transmembrane subunit of BtuCD in the lipid bilayer

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Based on the crystal structure of the vitamin B12 transporter protein of Escherichia coli(BtuCD) a system consisting of the BtuCD transmembrane domain(BtuC) and the palmitoyloleoyl phosphatidylcholine(POPC) lipid bilayer was constructed in silica,and a more-than-57-nanosecond molecular dynamics(MD) simulation was performed on it to reveal the intrinsic functional motions of BtuC.The results showed that a stable protein-lipid bilayer was obtained and the POPC lipid bilayer was able to adjust its thickness to match the embedded BtuC which underwent relatively complicated motions.These results may help to understand the mechanism of transmembrane substrate transport at the atomic level.

  17. Childhood trauma and dimensions of depression: a specific association with the cognitive domain

    OpenAIRE

    Edgar A. Vares; Salum, Giovanni A; Lucas Spanemberg; Marco A. Caldieraro; Lívia H. de Souza; Roberta de P. Borges; Marcelo P. Fleck

    2015-01-01

    Objective: To investigate associations between a history of childhood trauma and dimensions of depression in a sample of clinically depressed patients. Methods: A sample of 217 patients from a mood-disorder outpatient unit was investigated with the Beck Depression Inventory, the Hamilton Depression Rating Scale, the CORE Assessment of Psychomotor Change, and the Childhood Trauma Questionnaire. A previous latent model identifying six depressive dimensions was used for analysis. Path analysis ...

  18. Novel polymerase gamma (POLG1) gene mutation in the linker domain associated with parkinsonism

    OpenAIRE

    Dolhun, Rachel; Presant, Erin M; Hedera, Peter

    2013-01-01

    Background Mutations in the POLG1 gene have variable phenotypic presentations and a high degree of clinical suspicion is necessary for their recognition. Parkinsonism and ataxia are the most common movement disorders associated with POLG1 mutations but no phenotype-genotype correlation has been established. Case presentation We identified a male patient with progressive external ophthalmoplegia who also developed a progressive bradykinesia, rigidity and camptocormia in the third decade. Parki...

  19. Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression

    Directory of Open Access Journals (Sweden)

    Rivera-Molina Yisel A

    2012-09-01

    Full Text Available Abstract Background DNA viruses, such as herpes simplex virus type 1 (HSV-1, Simian virus 40 (SV40, and Cytomegaloviruses (CMV, start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies. It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. Results Our recent observations provide evidence that a foreign (not endogenous DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor, or from HPV11 (human papillomavirus 11 origin DNA and its binding protein (E2, co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA showed no association with ND10. Furthermore, our examination of PML−/−, Daxx−/−, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. Conclusion Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.

  20. Cancer associated E17K mutation causes rapid conformational drift in AKT1 pleckstrin homology (PH domain.

    Directory of Open Access Journals (Sweden)

    Ambuj Kumar

    Full Text Available BACKGROUND: AKT1 (v-akt murine thymoma viral oncogene homologue 1 kinase is one of the most frequently activated proliferated and survival pathway of cancer. Recently it has been shown that E17K mutation in the Pleckstrin Homology (PH domain of AKT1 protein leads to cancer by amplifying the phosphorylation and membrane localization of protein. The mutant has shown resistance to AKT1/2 inhibitor VIII drug molecule. In this study we have demonstrated the detailed structural and molecular consequences associated with the activity regulation of mutant protein. METHODS: The docking score exhibited significant loss in the interaction affinity to AKT1/2 inhibitor VIII drug molecule. Furthermore, the molecular dynamics simulation studies presented an evidence of rapid conformational drift observed in mutant structure. RESULTS: There was no stability loss in mutant as compared to native structure and the major cation-π interactions were also shown to be retained. Moreover, the active residues involved in membrane localization of protein exhibited significant rise in NHbonds formation in mutant. The rise in NHbond formation in active residues accounts for the 4-fold increase in the membrane localization potential of protein. CONCLUSION: The overall result suggested that, although the mutation did not induce any stability loss in structure, the associated pathological consequences might have occurred due to the rapid conformational drifts observed in the mutant AKT1 PH domain. GENERAL SIGNIFICANCE: The methodology implemented and the results obtained in this work will facilitate in determining the core molecular mechanisms of cancer-associated mutations and in designing their potential drug inhibitors.

  1. Characterization of amyloid-β precursor protein intracellular domain-associated transcriptional complexes in SH-SY5Y neurocytes

    Institute of Scientific and Technical Information of China (English)

    Wulin Yang; Amy Yong Chen Lau; Shuizhong Luo; Qian Zhu3; Li Lu

    2012-01-01

    [Objective] Alzheimer's disease (AD) is one of the major disorders worldwide.Recent research suggests that the amyloid-β precursor protein intracellular domain (AICD) is a potential contributor to AD development and progression.The small AICD is rapidly degraded after processing from the full-length protein.The present study aimed to apply a highly efficient biotinylation approach in vitro to study AICD-associated complexes in neurocytes.[Methods] By coexpressing Escherichia coli biotin ligase with biotinyl-tagged AICD in the SH-SY5Y neuronal cell line,the effects of AICD overexpression on cell proliferation and apoptosis were analyzed.Besides,AICD-associated nuclear transcriptional complexes were purified and then examined by mass spectrometry.[Results] Our data showed that AICD overexpression not only affected cell proliferation but also led to apoptosis in differentiated SH-SY5Y cells.Moreover,biotinylation allowed single-step purification of biotinylated AICD-associated complexes from total nuclear extract via high-affinity biotin-streptavidin binding.Following this by mass spectrometry,we identified physically associated proteins,some reported previously and other novel binding partners,CUX1 and SPT5.[Conclusion]Based on these [Results],a map of theAICD-associated nuclear interactome was depicted.Specifically,AICD can activate CUXI transcriptional activity,which may be associated with AICD-dependent neuronal cell death.This work helps to understand the AICD-associated biologicalevents in AD progression and provides novel insights into the development of AD.

  2. Transmembrane protein sorting driven by membrane curvature

    Science.gov (United States)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  3. Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane

    DEFF Research Database (Denmark)

    Olsen, M; Krog, L; Edvardsen, K;

    1993-01-01

    density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding to......-s1 and NCAM-s2 and the function of soluble NCAM forms were investigated. It was shown that all three soluble forms could be released from brain membranes with M(r) values identical to the three major membrane-associated forms: the large transmembrane 190,000-M(r) form (NCAM-A), the smaller...... intact soluble form from membranes of cells transfected with this isoform. Thus, NCAM-s1 and NCAM-s2 probably represent intact released transmembrane NCAM-A and NCAM-B. The soluble transmembrane forms are likely to exist in vivo, as NCAM-s1 and NCAM-s2 were readily demonstrated in cerebrospinal fluid. By...

  4. Molecular mechanisms for generating transmembrane proton gradients.

    Science.gov (United States)

    Gunner, M R; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side.

  5. Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis.

    Science.gov (United States)

    Neems, Daniel S; Garza-Gongora, Arturo G; Smith, Erica D; Kosak, Steven T

    2016-03-22

    The linear distribution of genes across chromosomes and the spatial localization of genes within the nucleus are related to their transcriptional regulation. The mechanistic consequences of linear gene order, and how it may relate to the functional output of genome organization, remain to be fully resolved, however. Here we tested the relationship between linear and 3D organization of gene regulation during myogenesis. Our analysis has identified a subset of topologically associated domains (TADs) that are significantly enriched for muscle-specific genes. These lineage-enriched TADs demonstrate an expression-dependent pattern of nuclear organization that influences the positioning of adjacent nonenriched TADs. Therefore, lineage-enriched TADs inform cell-specific genome organization during myogenesis. The reduction of allelic spatial distance of one of these domains, which contains Myogenin, correlates with reduced transcriptional variability, identifying a potential role for lineage-specific nuclear topology. Using a fusion-based strategy to decouple mitosis and myotube formation, we demonstrate that the cell-specific topology of syncytial nuclei is dependent on cell division. We propose that the effects of linear and spatial organization of gene loci on gene regulation are linked through TAD architecture, and that mitosis is critical for establishing nuclear topologies during cellular differentiation. PMID:26957603

  6. Protein domain prediction

    NARCIS (Netherlands)

    Ingolfsson, Helgi; Yona, Golan

    2008-01-01

    Domains are considered to be the building blocks of protein structures. A protein can contain a single domain or multiple domains, each one typically associated with a specific function. The combination of domains determines the function of the protein, its subcellular localization and the interacti

  7. Regulatory elements associated with paternally-expressed genes in the imprinted murine Angelman/Prader-Willi syndrome domain.

    Directory of Open Access Journals (Sweden)

    Sara Rodriguez-Jato

    Full Text Available The Angelman/Prader-Willi syndrome (AS/PWS domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain - such as MKRN3 and NDN - are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.

  8. Lack of the lectin-like domain of thrombomodulin worsens Shiga toxin-associated hemolytic uremic syndrome in mice.

    Science.gov (United States)

    Zoja, Carlamaria; Locatelli, Monica; Pagani, Chiara; Corna, Daniela; Zanchi, Cristina; Isermann, Berend; Remuzzi, Giuseppe; Conway, Edward M; Noris, Marina

    2012-10-01

    Shiga toxin (Stx)-producing Escherichia coli is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. The pathophysiology of renal microvascular thrombosis in Stx-HUS is still ill-defined. Based on evidence that abnormalities in thrombomodulin (TM), an anticoagulant endothelial glycoprotein that modulates complement and inflammation, predispose to atypical HUS, we assessed whether impaired TM function may adversely affect evolution of Stx-HUS. Disease was induced by coinjection of Stx2/LPS in wild-type mice (TM(wt/wt)) and mice that lack the lectin-like domain of TM (TM(LeD/LeD)), which is critical for its anti-inflammatory and cytoprotective properties. After Stx2/LPS, TM(LeD/LeD) mice exhibited more severe thrombocytopenia and renal dysfunction than TM(wt/wt) mice. Lack of lectin-like domain of TM resulted in a stronger inflammatory reaction after Stx2/LPS with more neutrophils and monocytes/macrophages infiltrating the kidney, associated with PECAM-1 and chemokine upregulation. After Stx2/LPS, intraglomerular fibrin(ogen) deposits were detected earlier in TM(LeD/LeD) than in TM(wt/wt) mice. More abundant fibrin(ogen) deposits were also found in brain and lungs. Under basal conditions, TM(LeD/LeD) mice exhibited excess glomerular C3 deposits, indicating impaired complement regulation in the kidney that could lead to local accumulation of proinflammatory products. TM(LeD/LeD) mice with HUS had a higher mortality rate than TM(wt/wt) mice. If applicable to humans, these findings raise the possibility that genetic or acquired TM defects might have an impact on the severity of microangiopathic lesions after exposure to Stx-producing E. coli infections and raise the potential for using soluble TM in the treatment of Stx-HUS. PMID:22942429

  9. Evidence for distinct sodium-, dopamine-, and cocaine-dependent conformational changes in transmembrane segments 7 and 8 of the dopamine transporter

    DEFF Research Database (Denmark)

    Norregaard, Lene; Loland, Claus Juul; Gether, Ulrik

    2003-01-01

    Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted...... cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely....... Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M...

  10. Domains and domain loss

    DEFF Research Database (Denmark)

    Haberland, Hartmut

    2005-01-01

    The domain concept, originally suggested by Schmidt-Rohr in the 1930’s (as credited in Fishman’s writings in the 1970s), was an attempt to sort out different areas of language use in multilingual societies, which are relevant for language choice. In Fishman’s version, domains were considered...... not described in terms of domains, and recent research e.g. about the multilingual communities in the Danish-German border area seems to confirm this....

  11. Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

    Science.gov (United States)

    Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

    2016-01-22

    In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

  12. Modeling the structure of SARS 3a transmembrane protein using a minimum unfavorable contact approach

    Indian Academy of Sciences (India)

    S Ramakrishna; Siladitya Padhi; U Deva Priyakumar

    2015-12-01

    3a is an accessory protein from SARS coronavirus that is known to play a significant role in the proliferation of the virus by forming tetrameric ion channels. Although the monomeric units are known to consist of three transmembrane (TM) domains, there are no solved structures available for the complete monomer. The present study proposes a structural model for the transmembrane region of the monomer by employing our previously tested approach, which predicts potential orientations of TM -helices by minimizing the unfavorable contact surfaces between the different TM domains. The best model structure comprising all three -helices has been subjected to MD simulations to examine its quality. The TM bundle was found to form a compact and stable structure with significant intermolecular interactions. The structural features of the proposed model of 3a account for observations from previous experimental investigations on the activity of the protein. Further analysis indicates that residues from the TM2 and TM3 domains are likely to line the pore of the ion channel, which is in good agreement with a recent experimental study. In the absence of an experimental structure for the protein, the proposed structure can serve as a useful model for inferring structure-function relationships about the protein.

  13. A negatively charged transmembrane aspartate residue controls activation of the relaxin-3 receptor RXFP3.

    Science.gov (United States)

    Liu, Yu; Zhang, Lei; Shao, Xiao-Xia; Hu, Meng-Jun; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2016-08-15

    Relaxin-3 is an insulin/relaxin superfamily neuropeptide involved in the regulation of food intake and stress response via activation of its cognate receptor RXFP3, an A-class G protein-coupled receptor (GPCR). In recent studies, a highly conserved ExxxD motif essential for binding of relaxin-3 has been identified at extracellular end of the second transmembrane domain (TMD2) of RXFP3. For most of the A-class GPCRs, a highly conserved negatively charged Asp residue (Asp(2.50) using Ballesteros-Weinstein numbering and Asp128 in human RXFP3) is present at the middle of TMD2. To elucidate function of the conserved transmembrane Asp128, in the present work we replaced it with other residues and the resultant RXFP3 mutants all retained quite high ligand-binding potency, but their activation and agonist-induced internalization were abolished or drastically decreased. Thus, the negatively charged transmembrane Asp128 controlled transduction of agonist-binding information from the extracellular region to the intracellular region through maintaining RXFP3 in a metastable state for efficient conformational change induced by binding of an agonist. PMID:27353281

  14. A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.

    Science.gov (United States)

    Capuano, Christopher M; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N; Desai, Keshal V; Coombs, Gavin; McCaffery, J Michael; Desai, Prashant J

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction. PMID:24824860

  15. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.

    Directory of Open Access Journals (Sweden)

    William Pao

    2005-03-01

    Full Text Available BACKGROUND: Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa or erlotinib (Tarceva usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR. Despite initial responses, patients eventually progress by unknown mechanisms of "acquired" resistance. METHODS AND FINDINGS: We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec. CONCLUSION: In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.

  16. Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

    Science.gov (United States)

    Valk, Vincent; Lammerts van Bueren, Alicia; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

    2016-06-01

    Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract. PMID:27101946

  17. The Origins of Transmembrane Ion Channels

    Science.gov (United States)

    Pohorille, Andrew; Wilson, Michael A.

    2012-01-01

    Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

  18. Penetration of three transmembrane segments of Slc11a1 in lipid bilayers.

    Science.gov (United States)

    Qi, Haiyan; Wang, Ying; Chu, Hongtao; Wang, Wenhua; Mao, Qidong

    2014-03-25

    Slc11a1 is a divalent metal cation transporter with 12 putative transmembrane domains (TM) and plays a role in host defense. In present work, we investigated the secondary structure and topology of the peptides associated to Slc11a1-TM2, TM3 and TM4 (wildtype peptides and function-relating mutants) in the phospholipid vesicles (DMPC, DMPG and their mixtures) using circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. We found that TM3 is obviously different in secondary structure and topology from TM2 to TM4 in the lipid membranes. The peptide TM3 is less structured and embedded in the lipid membranes less deeply than TM2 and TM4 at pH 5.5 and 7. The insertion position of TM3 in the lipid membranes is adjusted by pH, more deeply at more acidic pH environment, whereas the locations of TM2 and TM4 in the lipid membranes are less changed with pH. The E139A substitution of TM3 significantly impairs the pH dependence of the buried depth of TM3 and causes a pronounced increase in helicity in all DMPG-containing lipid vesicles at pH 5.5 and 7 and in DMPC at pH 4. In contrast, TM2 and TM4 are similar in topology. The G169D mutation has little effect on the topological arrangement of TM4 in membranes. The property of headgroups of the phospholipids has an effect on the secondary structure and topology of the peptides. All peptides could be structured with more helicity and embedded more deeply in DMPG-containing lipid vesicles than in DMPC membrane at pH 5.5 and 7.

  19. Structural determinants of protein partitioning into ordered membrane domains and lipid rafts.

    Science.gov (United States)

    Lorent, Joseph Helmuth; Levental, Ilya

    2015-11-01

    Increasing evidence supports the existence of lateral nanoscopic lipid domains in plasma membranes, known as lipid rafts. These domains preferentially recruit membrane proteins and lipids to facilitate their interactions and thereby regulate transmembrane signaling and cellular homeostasis. The functionality of raft domains is intrinsically dependent on their selectivity for specific membrane components; however, while the physicochemical determinants of raft association for lipids are known, very few systematic studies have focused on the structural aspects that guide raft partitioning of proteins. In this review, we describe biophysical and thermodynamic aspects of raft-mimetic liquid ordered phases, focusing on those most relevant for protein partitioning. Further, we detail the variety of experimental models used to study protein-raft interactions. Finally, we review the existing literature on mechanisms for raft targeting, including lipid post-translational modifications, lipid binding, and transmembrane domain features. We conclude that while protein palmitoylation is a clear raft-targeting signal, few other general structural determinants for raft partitioning have been revealed, suggesting that many discoveries lie ahead in this burgeoning field.

  20. Confirmatory factor analysis of the Adult Asperger Assessment: The association of symptom domains within a clinical population

    OpenAIRE

    Kuenssberg, Renate; McKenzie, Karen

    2011-01-01

    Autism Spectrum Disorder (ASD) is a behaviourally defined disorder characterised by impairments in three domains of social interaction, communication, and repetitive, stereotyped behaviours and activities. Proposed changes to diagnostic criteria suggest that the diagnostic triad may no longer fit as the best way to conceptualise ASD, and that social and communication impairments should be considered as a single domain. The aim of this study was to examine the structure of symptom domains with...

  1. Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association

    Science.gov (United States)

    Park, Pil Whan; Ahn, Jeong Yeal

    2016-01-01

    Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

  2. Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

    Directory of Open Access Journals (Sweden)

    Spaan Willy JM

    2009-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. Results A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH. The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. Conclusion Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

  3. Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

    Science.gov (United States)

    Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

    2012-06-01

    The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. PMID:22426196

  4. PorcineLEM domain-containing 3:Molecular cloning, functional characterization, and polymorphism associated with ear size

    Institute of Scientific and Technical Information of China (English)

    LIANG Jing; SHI Hui-bi; ZHANG Qin; WANGLi-xian; LI Na; ZHANG Long-chao; WANGLi-gang; LIU Xin; ZHAO Ke-bin; YAN Hua; PU Lei; ZHANG Yue-bo

    2016-01-01

    Ear size exhibits remarkable diversity in pig breeds.LEM domain-containing 3 (LEMD3) on chromosome 5 is considered as an important candidate for porcine ear size. This is the ifrst study on cloning and characterization ofLEMD3 cDNA. The complete cDNA contains 4843 bp, including a 2736-bp open reading frame (ORF), a 37-bp 5´-untranslated region (UTR) and a 2070-bp 3´-UTR. The completeLEMD3 gene is 126241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter ofLEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms (SNPs): L964C>A in the complete coding region, L4625A>G in the 3´ UTR, and L-394T>C in the promoter region. Genome-wide association study (GWAS) revealed that al of SNPs were shown signiifcant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log10(P-value) decreased by more than 80% when each of three SNPs was included as a ifxed effect. These results suggested direct involvement ofLEMD3 or close linkage to the causative mutation for ear size. The ifndings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.

  5. TSSOM:Transmembrane Segments Prediction by Self—Organizing Map

    Institute of Scientific and Technical Information of China (English)

    LIUQi; ZHUYisheng; WANGBaohua; LIYixue

    2003-01-01

    A novel method ealled TSSOM(Transmembrane segments prediction by self-organizing map)is presented in the paper.The main idea of the method lies in the application of self-organizing feature map together with special visualization techniques to classify the multivariate "time" series of transmembrane proteins into flve classes.Through the analysis of resulting trajectories on the map,frequent patterns of transmembrane segments are detected and even some kind of "new"knowledge about membrane insertion mechanism is acquired.The discovered patterns and the knowledge are then used to predict transmembrane segments for auery sequence.The prediction results not only show that the method is powerful,but also prove that the patterns and the knowledge about the interaction bwtween the patterns are effective and acceptable.

  6. Lipid bilayer microarray for parallel recording of transmembrane ion currents.

    Science.gov (United States)

    Le Pioufle, Bruno; Suzuki, Hiroaki; Tabata, Kazuhito V; Noji, Hiroyuki; Takeuchi, Shoji

    2008-01-01

    This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

  7. Unassisted translocation of large polypeptide domains across phospholipid bilayers

    OpenAIRE

    Brambillasca, Silvia; Yabal, Monica; Makarow, Marja; Borgese, Nica

    2006-01-01

    Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533–2542). In the present study...

  8. A novel COCH mutation associated with autosomal dominant nonsyndromic hearing loss disrupts the structural stability of the vWFA2 domain

    NARCIS (Netherlands)

    Cho, H.J.; Park, H.J.; Trexler, M.; Venselaar, H.; Lee, K.Y.; Robertson, N.G.; Baek, J.I.; Kang, B.S.; Morton, C.C.; Vriend, G.; Patthy, L.; Kim, U.K.

    2012-01-01

    Mutations in COCH have been associated with autosomal dominant nonsyndromic hearing loss (DFNA9) and are frequently accompanied by vestibular hypofunction. Here, we report identification of a novel missense mutation, p.F527C, located in the vWFA2 domain in members of a Korean family with late-onset

  9. Copper Metabolism Domain-Containing 1 Represses Genes That Promote Inflammation and Protects Mice From Colitis and Colitis-Associated Cancer

    NARCIS (Netherlands)

    Li, Haiying; Chan, Lillienne; Bartuzi, Paulina; Melton, Shelby D.; Weber, Axel; Ben-Shlomo, Shani; Varol, Chen; Raetz, Megan; Mao, Xicheng; Starokadomskyy, Petro; van Sommeren, Suzanne; Mokadem, Mohamad; Schneider, Heike; Weisberg, Reid; Westra, Harm-Jan; Esko, Tonu; Metspalu, Andres; Magadi Gopalaiah, Vinod Kumar; Faubion, William A.; Yarovinsky, Felix; Hofker, Marten; Wijmenga, Cisca; Kracht, Michael; Franke, Lude; Aguirre, Vincent; Weersma, Rinse K.; Gluck, Nathan; van de Sluis, Bart; Burstein, Ezra

    2014-01-01

    BACKGROUND & AIMS: Activation of the transcription factor nuclear factor-kappa B (NF-kappa B) has been associated with the development of inflammatory bowel disease (IBD). Copper metabolism MURR1 domain containing 1 (COMMD1), a regulator of various transport pathways, has been shown to limit NF-kapp

  10. Transmembrane adaptor proteins in the high-affinity IgE receptor signaling

    Directory of Open Access Journals (Sweden)

    Petr eDraber

    2012-01-01

    Full Text Available Aggregation of the high-affinity IgE receptor (FcεRI initiates a cascade of signaling events leading to release of preformed inflammatory and allergy mediators and de novo synthesis and secretion of cytokines and other compounds. The first biochemically well defined step of this signaling cascade is tyrosine phosphorylation of the FcεRI subunits by Src family kinase Lyn, followed by recruitment and activation of Syk kinase. Activity of Syk is decisive for the formation of multicomponent signaling assemblies, the signalosomes, in the vicinity of the receptors. Formation of the signalosomes is dependent on the presence of transmembrane adaptor proteins (TRAPs. These proteins are characterized by a short extracellular domain, a single transmembrane domain and a cytoplasmic tail with various motifs serving as anchors for cytoplasmic signaling molecules. In mast cells five TRAPs have been identified (LAT, NTAL, LAX, PAG and GAPT; engagement of four of them (LAT, NTAL, LAX and PAG in FcεRI signaling has been documented. Here we discuss recent progress in the understanding of how TRAPs affect FcεRI-mediated mast cell signaling. The combined data indicate that individual TRAPs have irreplaceable roles in important signaling events such as calcium response, degranulation, cytokines production and chemotaxis.

  11. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane.

    Science.gov (United States)

    Vijayaraghavan, Balaje; Jafferali, Mohammed Hakim; Figueroa, Ricardo A; Hallberg, Einar

    2016-07-01

    Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct.Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea. PMID:27541860

  12. Structural context of disease-associated mutations and putative mechanism of autoinhibition revealed by X-ray crystallographic analysis of the EZH2-SET domain.

    Directory of Open Access Journals (Sweden)

    Stephen Antonysamy

    Full Text Available The enhancer-of-zeste homolog 2 (EZH2 gene product is an 87 kDa polycomb group (PcG protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2. PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.

  13. Domains and domain loss

    DEFF Research Database (Denmark)

    Haberland, Hartmut

    2005-01-01

    politicians and in the media, especially in the discussion whether some languages undergo ‘domain loss’ vis-à-vis powerful international languages like English. An objection that has been raised here is that domains, as originally conceived, are parameters of language choice and not properties of languages...... theoretical constructs that can explain language choice which were supposed to be a more powerful explanatory tool than more obvious (and observable) parameters like topic, place (setting) and interlocutor. In the meantime, at least in Scandinavia, the term ‘domain’ has been taken up in the debate among...

  14. Two memory associated genes regulated by amyloid precursor protein intracellular domain ovel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Chuandong Zheng; Xi Gu; Zhimei Zhong; Rui Zhu; Tianming Gao; Fang Wang

    2012-01-01

    In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.

  15. Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Yufeng; Hota, Prasanta K.; Penachioni, Junia Y.; Hamaneh, Mehdi B.; Kim, SoonJeung; Alviani, Rebecca S.; Shen, Limin; He, Hao; Tempel, Wolfram; Tamagnone, Luca; Park, Hee-Won; Buck, Matthias; (Torino); (Toronto); (Case Western U.-Med)

    2010-02-11

    Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 {angstrom} crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120{sup GAP} {center_dot}H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.

  16. Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca2+.

    Directory of Open Access Journals (Sweden)

    Paola eVenco

    2015-05-01

    Full Text Available Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA, a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type C19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER and MAM (Mitochondria Associated Membrane, while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls.C19orf12 protein is a 17kDa mitochondrial membrane-associated protein whose function is still unknown. Our in silico investigation suggests that, the glycine zipper motifs of C19orf12 form helical regions spanning the membrane. The N- and C-terminal regions with respect to the transmembrane portion, on the contrary, are predicted to rearrange in a structural domain, which is homologues to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs, which are involved in dimerization of transmembrane helices and predicted to impair the correct localization of the protein into the membranes, and one residue present in the regulatory domain, which is important for protein-protein interaction.

  17. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    International Nuclear Information System (INIS)

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism

  18. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    Energy Technology Data Exchange (ETDEWEB)

    Bradshaw, William J. [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Kirby, Jonathan M. [Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Thiyagarajan, Nethaji [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Chambers, Christopher J.; Davies, Abigail H. [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Roberts, April K.; Shone, Clifford C. [Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Acharya, K. Ravi, E-mail: bsskra@bath.ac.uk [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom)

    2014-07-01

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.

  19. Recent improvements to the SMART domain-based sequence annotation resource.

    Science.gov (United States)

    Letunic, Ivica; Goodstadt, Leo; Dickens, Nicholas J; Doerks, Tobias; Schultz, Joerg; Mott, Richard; Ciccarelli, Francesca; Copley, Richard R; Ponting, Chris P; Bork, Peer

    2002-01-01

    SMART (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de) is a web-based resource used for the annotation of protein domains and the analysis of domain architectures, with particular emphasis on mobile eukaryotic domains. Extensive annotation for each domain family is available, providing information relating to function, subcellular localization, phyletic distribution and tertiary structure. The January 2002 release has added more than 200 hand-curated domain models. This brings the total to over 600 domain families that are widely represented among nuclear, signalling and extracellular proteins. Annotation now includes links to the Online Mendelian Inheritance in Man (OMIM) database in cases where a human disease is associated with one or more mutations in a particular domain. We have implemented new analysis methods and updated others. New advanced queries provide direct access to the SMART relational database using SQL. This database now contains information on intrinsic sequence features such as transmembrane regions, coiled-coils, signal peptides and internal repeats. SMART output can now be easily included in users' documents. A SMART mirror has been created at http://smart.ox.ac.uk. PMID:11752305

  20. Recent improvements to the SMART domain-based sequence annotation resource

    Science.gov (United States)

    Letunic, Ivica; Goodstadt, Leo; Dickens, Nicholas J.; Doerks, Tobias; Schultz, Joerg; Mott, Richard; Ciccarelli, Francesca; Copley, Richard R.; Ponting, Chris P.; Bork, Peer

    2002-01-01

    SMART (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de) is a web-based resource used for the annotation of protein domains and the analysis of domain architectures, with particular emphasis on mobile eukaryotic domains. Extensive annotation for each domain family is available, providing information relating to function, subcellular localization, phyletic distribution and tertiary structure. The January 2002 release has added more than 200 hand-curated domain models. This brings the total to over 600 domain families that are widely represented among nuclear, signalling and extracellular proteins. Annotation now includes links to the Online Mendelian Inheritance in Man (OMIM) database in cases where a human disease is associated with one or more mutations in a particular domain. We have implemented new analysis methods and updated others. New advanced queries provide direct access to the SMART relational database using SQL. This database now contains information on intrinsic sequence features such as transmembrane regions, coiled-coils, signal peptides and internal repeats. SMART output can now be easily included in users’ documents. A SMART mirror has been created at http://smart.ox.ac.uk. PMID:11752305

  1. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

    Directory of Open Access Journals (Sweden)

    Sofie Ignoul

    Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

  2. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

    Directory of Open Access Journals (Sweden)

    Kwang-Hyung Kim

    2009-11-01

    Full Text Available The regulation of intracellular levels of reactive oxygen species (ROS is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we

  3. Prediction of three-dimensional transmembrane helical protein structures

    Science.gov (United States)

    Barth, Patrick

    Membrane proteins are critical to living cells and their dysfunction can lead to serious diseases. High-resolution structures of these proteins would provide very valuable information for designing eficient therapies but membrane protein crystallization is a major bottleneck. As an important alternative approach, methods for predicting membrane protein structures have been developed in recent years. This chapter focuses on the problem of modeling the structure of transmembrane helical proteins, and describes recent advancements, current limitations, and future challenges facing de novo modeling, modeling with experimental constraints, and high-resolution comparative modeling of these proteins. Abbreviations: MP, membrane protein; SP, water-soluble protein; RMSD, root-mean square deviation; Cα RMSD, root-mean square deviation over Cα atoms; TM, transmembrane; TMH, transmembrane helix; GPCR, G protein-coupled receptor; 3D, three dimensional; NMR, nuclear magnetic resonance spectroscopy; EPR, electron paramagnetic resonance spectroscopy; FTIR, Fourier transform infrared spectroscopy.

  4. The protein kinase C-responsive inhibitory domain of CARD11 functions in NF-kappaB activation to regulate the association of multiple signaling cofactors that differentially depend on Bcl10 and MALT1 for association.

    Science.gov (United States)

    McCully, Ryan R; Pomerantz, Joel L

    2008-09-01

    The activation of NF-kappaB by T-cell receptor (TCR) signaling is critical for T-cell activation during the adaptive immune response. CARD11 is a multidomain adapter that is required for TCR signaling to the IkappaB kinase (IKK) complex. During TCR signaling, the region in CARD11 between the coiled-coil and PDZ domains is phosphorylated by protein kinase Ctheta (PKCtheta) in a required step in NF-kappaB activation. In this report, we demonstrate that this region functions as an inhibitory domain (ID) that controls the association of CARD11 with multiple signaling cofactors, including Bcl10, TRAF6, TAK1, IKKgamma, and caspase-8, through an interaction that requires both the caspase recruitment domain (CARD) and the coiled-coil domain. Consistent with the ID-mediated control of their association, we demonstrate that TRAF6 and caspase-8 associate with CARD11 in T cells in a signal-inducible manner. Using an RNA interference rescue assay, we demonstrate that the CARD, linker 1, coiled-coil, linker 3, SH3, linker 4, and GUK domains are each required for TCR signaling to NF-kappaB downstream of ID neutralization. Requirements for the CARD, linker 1, and coiled-coil domains in signaling are consistent with their roles in the association of CARD11 with Bcl10, TRAF6, TAK1, caspase-8, and IKKgamma. Using Bcl10- and MALT1-deficient cells, we show that CARD11 can recruit signaling cofactors independently of one another in a signal-inducible manner.

  5. Confirmatory Factor Analysis of the Adult Asperger Assessment: The Association of Symptom Domains within a Clinical Population

    Science.gov (United States)

    Kuenssberg, Renate; McKenzie, Karen

    2011-01-01

    Autism Spectrum Disorder (ASD) is a behaviourally defined disorder characterised by impairments in three domains of social interaction, communication, and repetitive, stereotyped behaviours and activities. Proposed changes to diagnostic criteria suggest that the diagnostic triad may no longer fit as the best way to conceptualise ASD, and that…

  6. The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

    Science.gov (United States)

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2012-11-01

    Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.

  7. Optimizing an emperical scoring function for transmembrane protein structure determination.

    Energy Technology Data Exchange (ETDEWEB)

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  8. Involvement of the heterodimeric interface region of the nucleotide binding domain-2 (NBD2) in the CFTR quaternary structure and membrane stability.

    Science.gov (United States)

    Micoud, Julien; Chauvet, Sylvain; Scheckenbach, Klaus Ernst Ludwig; Alfaidy, Nadia; Chanson, Marc; Benharouga, Mohamed

    2015-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the ATP-binding cassette (ABC) superfamily that functions as a chloride channel. The predicted structure of CFTR protein contains two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD1 and NBD2). The opening of the Cl- channel is directly linked to ATP-driven tight dimerization of CFTR's NBD1 and NBD2 domains. The presence of a heterodimeric interfaces (HI) region in NBD1 and NBD2 generated a head to tail orientation necessary for channel activity. This process was also suggested to promote important conformational changes in the associated transmembrane domains of CFTR, which may impact the CFTR plasma membrane stability. To better understand the role of the individual HI region in this process, we generated recombinant CFTR protein with suppressed HI-NBD1 and HI-NBD2. Our results indicate that HI-NBD2 deletion leads to the loss of the dimerization profile of CFTR that affect its plasma membrane stability. We conclude that, in addition to its role in Cl- transport, HI-NBD2 domain confers membrane stability of CFTR by consolidating its quaternary structure through interactions with HI-NBD1 region.

  9. Structure and function of the Juxta membrane domain of the human epidermal growth factor receptor by NMR spectroscopy

    International Nuclear Information System (INIS)

    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxta membrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking since both basolateral sorting in polarized epithelial cells and lysosomal sorting signals are identified in this region. In order to understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in dodecyl phosphocholine detergent (DPC) by nuclear magnetic resonance (NMR) spectroscopy. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. Two equivalent average structural models on the surface of micelles were obtained that differ only in the relative orientation between the first and second helices. Our data suggests that the activity of sorting signals may be regulated by their membrane association and restricted accessibility in the intact receptor

  10. Functional characterization in Caenorhabditis elegans of transmembrane worm-human orthologs

    Directory of Open Access Journals (Sweden)

    Baillie David L

    2004-11-01

    Full Text Available Abstract Background The complete genome sequences for human and the nematode Caenorhabditis elegans offer an opportunity to learn more about human gene function through functional characterization of orthologs in the worm. Based on a previous genome-wide analysis of worm-human orthologous transmembrane proteins, we selected seventeen genes to explore experimentally in C. elegans. These genes were selected on the basis that they all have high confidence candidate human orthologs and that their function is unknown. We first analyzed their phylogeny, membrane topology and domain organization. Then gene functions were studied experimentally in the worm by using RNA interference and transcriptional gfp reporter gene fusions. Results The experiments gave functional insights for twelve of the genes studied. For example, C36B1.12, the worm ortholog of three presenilin-like genes, was almost exclusively expressed in head neurons, suggesting an ancient conserved role important to neuronal function. We propose a new transmembrane topology for the presenilin-like protein family. sft-4, the worm ortholog of surfeit locus gene Surf-4, proved to be an essential gene required for development during the larval stages of the worm. R155.1, whose human ortholog is entirely uncharacterized, was implicated in body size control and other developmental processes. Conclusions By combining bioinformatics and C. elegans experiments on orthologs, we provide functional insights on twelve previously uncharacterized human genes.

  11. Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

    Science.gov (United States)

    Fayadat, Laurence; Kopito, Ron R.

    2003-01-01

    To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane. PMID:12631739

  12. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Kelly J Culhane

    2015-11-01

    Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  13. Activation of p115-RhoGEF Requires Direct Association of G[alpha subscript 13] and the Dbl Homology Domain

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhe; Guo, Liang; Hadas, Jana; Gutowski, Stephen; Sprang, Stephen R.; Sternweis, Paul C. (IIT); (UTSMC); (Montana)

    2012-09-05

    RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G{sub 12} class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated {alpha} subunits of G{sub 12} and G{sub 13}. Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by G{alpha}{sub 13}, the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated G{alpha}{sub 13} in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of G{alpha}{sub 13} docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the {alpha}3b helix of DH reduces binding to activated G{alpha}{sub 13} and ablates the stimulation of p115 by G{alpha}{sub 13}. Complementary mutations at the predicted DH-binding site in the {alpha}B-{alpha}C loop of the helical domain of G{alpha}{sub 13} also affect stimulation of p115 by G{alpha}{sub 13}. Although the GAP activity of p115 is not required for stimulation by G{alpha}{sub 13}, two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of G{alpha}{sub 13} to the RH domain facilitates direct association of G{alpha}{sub 13} to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

  14. Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

    Science.gov (United States)

    Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

    2016-05-01

    Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation. PMID:26961431

  15. High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae.

    Science.gov (United States)

    Wang, Renjie; Normand, Christophe; Gadal, Olivier

    2016-01-01

    Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines. PMID:27576709

  16. High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae.

    Science.gov (United States)

    Wang, Renjie; Normand, Christophe; Gadal, Olivier

    2016-01-01

    Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.

  17. Structure and function of IQ-domain GTPase-activating protein 1 and its association with tumor progression (Review)

    Science.gov (United States)

    WU, YAN; CHEN, YONG-CHANG

    2014-01-01

    IQ-domain GTPase-activating proteins (IQGAPs) are evolutionary conserved multidomain proteins that are found in numerous organisms, from yeast to mammals. To date, three IQGAP proteins have been identified in humans, of which IQGAP1 is the best characterized. As a scaffold protein, IQGAP1 contains multiple protein-interacting domains, which modulate binding to target proteins. Recent mounting studies demonstrated a role for IQGAP1 in tumor progression, supported by the altered expression and subcellular distribution of IQGAP1 in tumors. The contribution of IQGAP1 to tumor progression appears to involve a complex interplay of cell functions by integrating diverse signal transduction pathways and coordinating activities, such as cell adhesion, migration, invasion, proliferation and angiogenesis. PMID:24649059

  18. Hibernation-associated gene regulation of plasma proteins with a collagen-like domain in mammalian hibernators.

    OpenAIRE

    Takamatsu, N; Ohba, K; Kondo, J; Kondo, N; Shiba, T

    1993-01-01

    In mammals, hibernation is expressed by only a limited number of species, and the molecular mechanisms underlying hibernation are not well understood. Recently, we have found plasma proteins which disappear from blood specifically during hibernation in a mammalian hibernator, the chipmunk. Here, we report the cDNA cloning of these chipmunk hibernation-related proteins, HP-20, -25, and -27, and analyses of their expression. All three proteins contain a collagen-like domain near the N terminus ...

  19. Modelling of a transmembrane evaporation module for desalination of seawater

    NARCIS (Netherlands)

    Guijt, Caroliene M.; Rácz, Imre G.; Heuven, van Jan Willem; Reith, Tom; Haan, de André B.

    1999-01-01

    Transmembrane evaporation (often called membrane distillation) carried out in a countercurrent flow module, in which incoming cold seawater is heated by the condensing product water flow, is a promising technology for low-cost seawater desalination. This paper presents a model for preliminary design

  20. A hidden Markov model for prediction transmembrane helices in proteinsequences

    DEFF Research Database (Denmark)

    Sonnhammer, Erik L.L.; von Heijne, Gunnar; Krogh, Anders Stærmose

    1998-01-01

    constraints involved. Models were estimated both by maximum likelihood and a discriminative method, and a method for reassignment of the membrane helix boundaries were developed. In a cross validated test on single sequences, our transmembrane HMM, TMHMM, correctly predicts the entire topology for 77% of the...

  1. The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid

    NARCIS (Netherlands)

    Moll, Gert N.; Konings, Wil N.; Driessen, Arnold J.M.

    1998-01-01

    Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]ca

  2. Computational approaches to detect allosteric pathways in transmembrane molecular machines.

    Science.gov (United States)

    Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V; Weinstein, Harel; Shi, Lei

    2016-07-01

    Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26806157

  3. A Novel Approach to the Prediction of Transmembrane Proteins

    Institute of Scientific and Technical Information of China (English)

    Jian Ding QIU; Ru Ping LIANG; Xiao Yong ZOU; Jin Yuan MO

    2004-01-01

    A novel method based on continuous wavelet transform (CWT) for predicting the number and location of helices in membrane proteins is presented. The PDB code of 1yst is chosen as an example to describe the prediction of transmembrane helices (HTM) by using CWT. The results indicate that CWT is a promising approach for the prediction of HTM.

  4. Biochemical properties and catalytic domain structure of the CcmH protein from Escherichia coli.

    Science.gov (United States)

    Zheng, Xue-Ming; Hong, Jing; Li, Hai-Yin; Lin, Dong-Hai; Hu, Hong-Yu

    2012-12-01

    In the Gram-negative bacterium of Escherichia coli, eight genes organized as a ccm operon (ccmABCDEFGH) are involved in the maturation of c-type cytochromes. The proteins encoded by the last three genes ccmFGH are believed to form a lyase complex functioning in the reduction of apocytochrome c and haem attachment. Among them, CcmH is a membrane-associated protein; its N-terminus is a catalytic domain with the active CXXC motif and the C-terminus is predicted as a TPR-like domain with unknown function. By using SCAM (scanning cysteine accessibility mutagenesis) and Gaussia luciferase fusion assays, we provide experimental evidence for the entire topological structure of E. coli CcmH. The mature CcmH is a periplasm-resident oxidoreductase anchored to the inner membrane by two transmembrane segments. Both N- and C-terminal domains are located and function in the periplasmic compartment. Moreover, the N-terminal domain forms a monomer in solution, while the C-terminal domain is a compact fold with helical structures. The NMR solution structure of the catalytic domain in reduced form exhibits mainly a three-helix bundle, providing further information for the redox mechanism. The redox potential suggests that CcmH exhibits a strong reductase that may function in the last step of reduction of apocytochrome c for haem attachment. PMID:22789558

  5. Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms.

    Science.gov (United States)

    Spagnol, Gaëlle; Al-Mugotir, Mona; Kopanic, Jennifer L; Zach, Sydney; Li, Hanjun; Trease, Andrew J; Stauch, Kelly L; Grosely, Rosslyn; Cervantes, Matthew; Sorgen, Paul L

    2016-03-01

    The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function. PMID:26542351

  6. Visualizing latent domain knowledge

    OpenAIRE

    Chen, C.; Kuljis, J; Paul, RJ

    2001-01-01

    Knowledge discovery and data mining commonly rely on finding salient patterns of association from a vast amount of data. Traditional citation analysis of scientific literature draws insights from strong citation patterns. Latent domain knowledge, in contrast to the mainstream domain knowledge, often consists of highly relevant but relatively infrequently cited scientific works. Visualizing latent domain knowledge presents a significant challenge to knowledge discovery and quantitative studies...

  7. Cutting Edge: Molecular Structure of the IL-1R-Associated Kinase-4 Death Domain and Its Implications for TLR Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Lasker, Michael V.; Gajjar, Mark M.; Nair, Satish K. (UIUC)

    2010-07-19

    IL-1R-associated kinase (IRAK) 4 is an essential component of innate immunity. IRAK-4 deficiency in mice and humans results in severe impairment of IL-1 and TLR signaling. We have solved the crystal structure for the death domain of Mus musculus IRAK-4 to 1.7 {angstrom} resolution. This is the first glimpse of the structural details of a mammalian IRAK family member. The crystal structure reveals a six-helical bundle with a prominent loop, which among IRAKs and Pelle, a Drosophila homologue, is unique to IRAK-4. This highly structured loop contained between helices two and three, comprises an 11-aa stretch. Although innate immune domain recognition is thought to be very similar between Drosophila and mammals, this structural component points to a drastic difference. This structure can be used as a framework for future mutation and deletion studies and potential drug design.

  8. The N-terminal domain of the Drosophila retinoblastoma protein Rbf1 interacts with ORC and associates with chromatin in an E2F independent manner.

    Directory of Open Access Journals (Sweden)

    Joseph Ahlander

    Full Text Available BACKGROUND: The retinoblastoma (Rb tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC. PRINCIPAL FINDINGS: We characterized the interaction of Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1-345 is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345-845 interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4. CONCLUSIONS/SIGNIFICANCE: Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery.

  9. Is the LIM-domain Protein HaWLIM1 Associated with Cortical Microtubules in Sunflower Protoplasts?

    OpenAIRE

    Brière, Christian; Bordel, Anne-Claire; Barthou, Henri; Jauneau, Alain; Steinmetz, André; Alibert, Gilbert; Petitprez, Michel

    2003-01-01

    Flowering plants express several LIM-domain proteins related to the animal cystein-rich proteins. The expression of sunflower LIM genes was followed by RTPCR in cultured sunflower protoplasts. A transcript was detected only for HaWLIM1, but not for the other two genes HaPLIM1 and HaPLIM2. Polyclonal antibodies raised against either full length recombinant HaWLIM1 protein or peptides recognized a 27 kDa polypeptide on Western blots. Immunocytolocalization studies showed that HaWLIM1 is located...

  10. Association of the formiminotransferase N-terminal sub-domain containing gene and thrombospondin, type 1, domain-containing 7A gene with the prevalence of vertebral fracture in 2427 consecutive autopsy cases.

    Science.gov (United States)

    Zhou, Heying; Mori, Seijiro; Kou, Ikuyo; Fuku, Noriyuki; Naka Mieno, Makiko; Honma, Naoko; Arai, Tomio; Sawabe, Motoji; Tanaka, Masashi; Ikegawa, Shiro; Ito, Hideki

    2013-02-01

    We previously reported 2 osteoporosis-susceptibility genes--formiminotransferase N-terminal sub-domain containing gene (FONG) and thrombospondin, type 1, domain-containing 7A (THSD7A)--in which we identified two common single-nucleotide polymorphisms, rs7605378 (FONG) and rs12673692 (THSD7A). The former was associated with a predisposition to osteoporosis and the latter with bone mineral density. To further elucidate the importance of these polymorphisms in the pathogenesis of osteoporosis, we examined their association with the incidence of vertebral fracture. DNA extracted from the renal cortex of 2427 consecutive Japanese autopsies (1331 men, mean age: 79 years; 1096 women, mean age: 82 years) were examined in this study. The presence or absence of vertebral fracture during each subject's lifetime was determined by a thorough examination of the clinical records, as well as autopsy reports. After adjustments for sex and age at autopsy, logistic regression analysis revealed that homozygotes for the risk alleles of rs7605378 (A-allele) or rs12673629 (A-allele) possess an increased risk of vertebral fracture. The subjects simultaneously homozygous for both the risk alleles of rs7605378 (AA genotype) and rs12673629 (AA genotype) showed significantly higher risk of vertebral fracture (odds ratio 2.401, 95% confidence interval 1.305-4.416, P = 0.0048) than those who had at least one non-risk allele of either rs7605378 (AC/CC genotypes) or rs12673629 (AG/GG genotypes). The results suggest that Japanese subjects homozygous for the risk alleles of rs7605378 and rs12673629 have a higher risk of vertebral fracture.

  11. External Zn(2+) binding to cysteine-substituted cystic fibrosis transmembrane conductance regulator constructs regulates channel gating and curcumin potentiation.

    Science.gov (United States)

    Wang, Guangyu; Linsley, Rheeann; Norimatsu, Yohei

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by ATP binding-induced dimerization of nucleotide-binding domains, the interaction between the phosphorylated regulatory (R) domain and the curcumin-sensitive interface between intracellular loop (ICL) 1 and ICL4, and the resultant inward-to-'outward' reorientation of transmembrane domains. Although transmembrane helices (TM) 2 and TM11 link the ICL1-ICL4 interface with the interface between extracellular loop (ECL) 1 and ECL6, it is unknown whether both interfaces are gating-coupled during the reorientation. Herein, R334C and T1122C mutations were used to engineer two Zn(2+) bridges near and at the ECL1-ECL6 interface, respectively, and the gating effects of a Zn(2+) disturbance at the ECL1-ECL6 interface on the stimulatory ICL1/ICL4-R interaction were determined. The results showed that both Zn(2+) bridges inhibited channel activity in a dose- and Cl(-) -dependent manner, and the inhibition was reversed by a washout or suppressed by thiol-specific modification. Interestingly, their Cl(-) -dependent Zn(2+) inhibition was weakened at higher Zn(2+) concentrations, their Zn(2+) affinity was stronger in the resting state than in the activated state, and their activation current noises were decreased by external Zn(2+) binding. More importantly, the external Zn(2+) inhibition was reversed by internal curcumin in the R334C construct but not in the T1122C mutant. Therefore, although both Zn(2+) bridges may promote channel closure, external Zn(2+) may disturb the ECL1-ECL6 interface and thus prevent the stimulatory ICL1/ICL4-R interaction and curcumin potentiation via a gating coupling between these two interfaces. PMID:27175795

  12. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  13. Thrombomodulin is a determinant of metastasis through a mechanism linked to the thrombin binding domain but not the lectin-like domain

    NARCIS (Netherlands)

    N.A. Horowitz; E.A. Blevins; W.M. Miller; A.R. Perry; K.E. Talmage; E.S. Mullins; M.J. Flick; K.C.S. Queiroz; K. Shi; C.A. Spek; E.M. Conway; B.P. Monia; H. Weiler; J.L. Degen; J.S. Palumbo

    2011-01-01

    Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of

  14. Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor*

    OpenAIRE

    Hamouda, Ayman K.; Stewart, Deirdre S.; Husain, S. Shaukat; Cohen, Jonathan B.

    2011-01-01

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our unders...

  15. Domain Engineering

    Science.gov (United States)

    Bjørner, Dines

    Before software can be designed we must know its requirements. Before requirements can be expressed we must understand the domain. So it follows, from our dogma, that we must first establish precise descriptions of domains; then, from such descriptions, “derive” at least domain and interface requirements; and from those and machine requirements design the software, or, more generally, the computing systems.

  16. Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children

    DEFF Research Database (Denmark)

    Lavstsen, Thomas; Turner, Louise; Saguti, Fredy;

    2012-01-01

    The clinical outcome of Plasmodium falciparum infections ranges from asymptomatic parasitemia to severe malaria syndromes associated with high mortality. The virulence of P. falciparum infections is associated with the type of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the...

  17. G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

    Science.gov (United States)

    Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

    2011-01-01

    G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.

  18. Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity

    DEFF Research Database (Denmark)

    Daniel, Philip B; Morgan, Tim; Alanay, Yasemin;

    2012-01-01

    Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin rep...... actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism....... repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin...... exception disease-associated substitutions surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced...

  19. Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

    Science.gov (United States)

    Mulepati, Sabin; Bailey, Scott

    2011-09-01

    RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

  20. Effect of Cocaine on Fas-Associated Protein with Death Domain in the Rat Brain: Individual Differences in a Model of Differential Vulnerability to Drug Abuse

    OpenAIRE

    García-Fuster, María-Julia; Clinton, Sarah M; Watson, Stanley J.; Akil, Huda

    2008-01-01

    This study was designed to (1) assess the effects of cocaine on Fas-associated protein with death domain (FADD) system and its role in the activation of apoptotic vs nonapoptotic events and (2) ascertain whether animals selectively bred for their differential propensity to drug-seeking show differences in FADD levels or response to cocaine. Acute cocaine, through D2 dopamine receptors, induced a dose–response increase in FADD protein in the cortex, with opposite effects over pFADD (Ser191/194...

  1. Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

    OpenAIRE

    Arora, Shikha; Lapinski, Philip Edward; Raghavan, Malini

    2001-01-01

    The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT...

  2. Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains*

    OpenAIRE

    Jackson, Heather M.; Kawahara, Tsukasa; Nisimoto, Yukio; Smith, Susan M. E.; Lambeth, J. David

    2010-01-01

    By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1–4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-l...

  3. The transmembrane domain of acid trehalase mediates ubiquitin-independent multivesicular body pathway sorting

    NARCIS (Netherlands)

    Huang, Ju; Reggiori, Fulvio; Klionsky, Daniel J; Reggiori, Fulvio

    2007-01-01

    Trehalose serves as a storage source of carbon and plays important roles under various stress conditions. For example, in many organisms trehalose has a critical function in preserving membrane structure and fluidity during dehydration/rehydration. In the yeast Saccharomyces cerevisiae, trehalose ac

  4. Polar transmembrane domains target proteins to the interior of the yeast vacuole

    NARCIS (Netherlands)

    Reggiori, F; Black, M W; Pelham, H R; Reggiori, Fulvio

    2000-01-01

    Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, w

  5. Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2014-01-01

    A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight. T....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance.......A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight...

  6. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking*

    Science.gov (United States)

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B.; Holding, Andrew N.; Montgomery, Martin G.; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. PMID:25851905

  7. A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides.

    Science.gov (United States)

    Venegas, Leon A; Pershad, Kritika; Bankole, Oluwadamilola; Shah, Noman; Kay, Brian K

    2016-09-25

    Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such post-translational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases, including human Myc, calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2). The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). The FHA domains binding Myc, CaMKII, and ERK1/2 were characterized and compared against commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains. PMID:26772725

  8. Grafting PNIPAAm from β-barrel shaped transmembrane nanopores.

    Science.gov (United States)

    Charan, Himanshu; Kinzel, Julia; Glebe, Ulrich; Anand, Deepak; Garakani, Tayebeh Mirzaei; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich; Böker, Alexander

    2016-11-01

    The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a β-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the β-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N-isopropylacrylamide (NIPAAm) polymerized under copper-mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors. PMID:27614163

  9. Surfactantlipid biosynthesis: Regulation of transmembrane transport of palmitate

    OpenAIRE

    Guthmann, Florian

    2010-01-01

    Considering the mechanisms by which antenatal maturation of lung can be induced, the role of long chain fatty acids as precursors of surfactant lipid synthesis has not been thoroughly investigated. To specifically increase surfactant synthesis during the fetal and/or neonatal period we studied the regulation of de novo phosphatidyl synthesis in type II pneumocytes. First, we characterised the transmembrane transport of palmitate, a long chain fatty acid prevalent in surfactant lipids, with...

  10. Cache Domains That are Homologous to, but Different from PAS Domains Comprise the Largest Superfamily of Extracellular Sensors in Prokaryotes.

    Science.gov (United States)

    Upadhyay, Amit A; Fleetwood, Aaron D; Adebali, Ogun; Finn, Robert D; Zhulin, Igor B

    2016-04-01

    Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms. Furthermore, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes. PMID:27049771

  11. Partial association of restriction polymorphism of the ligand binding domain of human androgen receptor in prostate cancer

    Directory of Open Access Journals (Sweden)

    Mohamed Hessien

    2016-04-01

    Conclusion: Our results indicate that the loss of the restriction integrity in the C-terminal part (exons: 7 and 8 of the LBD is associated with the progression of benign prostatic hyperplasia to prostate cancer.

  12. Free energetics of rigid body association of ubiquitin binding domains: a biochemical model for binding mediated by hydrophobic interaction.

    Science.gov (United States)

    Cui, Di; Ou, Shuching; Patel, Sandeep

    2014-07-01

    Weak intermolecular interactions, such as hydrophobic associations, underlie numerous biomolecular recognition processes. Ubiquitin is a small protein that represents a biochemical model for exploring thermodynamic signatures of hydrophobic association as it is widely held that a major component of ubiquitin's binding to numerous partners is mediated by hydrophobic regions on both partners. Here, we use atomistic molecular dynamics simulations in conjunction with the Adaptive Biasing Force sampling method to compute potentials of mean force (the reversible work, or free energy, associated with the binding process) to investigate the thermodynamic signature of complexation in this well-studied biochemical model of hydrophobic association. We observe that much like in the case of a purely hydrophobic solute (i.e., graphene, carbon nanotubes), association is favored by entropic contributions from release of water from the interprotein regions. Moreover, association is disfavored by loss of enthalpic interactions, but unlike in the case of purely hydrophobic solutes, in this case protein-water interactions are lost and not compensated for by additional water-water interactions generated upon release of interprotein and moreso, hydration, water. We further find that relative orientations of the proteins that mutually present hydrophobic regions of each protein to its partner are favored over those that do not. In fact, the free energy minimum as predicted by a force field based method recapitulates the experimental NMR solution structure of the complex.

  13. Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors.

    Science.gov (United States)

    Bye, Chris R; Jönsson, Marie E; Björklund, Anders; Parish, Clare L; Thompson, Lachlan H

    2015-04-14

    An important challenge for the continued development of cell therapy for Parkinson's disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a "FACS-array" approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations.

  14. Resolving the biophysics of axon transmembrane polarization in a single closed-form description

    International Nuclear Information System (INIS)

    When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis

  15. Resolving the biophysics of axon transmembrane polarization in a single closed-form description

    Science.gov (United States)

    Melendy, Robert F.

    2015-12-01

    When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

  16. Resolving the biophysics of axon transmembrane polarization in a single closed-form description

    Energy Technology Data Exchange (ETDEWEB)

    Melendy, Robert F., E-mail: rfmelendy@liberty.edu [School of Engineering and Computational Sciences, Liberty University, Lynchburg, Virginia 24515 (United States)

    2015-12-28

    When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

  17. The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement

    Science.gov (United States)

    Mitra, Ruchira; Krishnamurthy, Konduru; Blancaflor, Elison; Payton, Mark; Nelson, Richard S.; Verchot-Lubicz, Jeanmarie

    2003-01-01

    Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association.

  18. Is the LIM-domain protein HaWLIM1 associated with cortical microtubules in sunflower protoplasts?

    Science.gov (United States)

    Brière, Christian; Bordel, Anne-Claire; Barthou, Henri; Jauneau, Alain; Steinmetz, André; Alibert, Gilbert; Petitprez, Michel

    2003-10-01

    Flowering plants express several LIM-domain proteins related to the animal cystein-rich proteins. The expression of sunflower LIM genes was followed by RT-PCR in cultured sunflower protoplasts. A transcript was detected only for HaWLIM1, but not for the other two genes HaPLIM1 and HaPLIM2. Polyclonal antibodies raised against either full length recombinant HaWLIM1 protein or peptides recognized a 27 kDa polypeptide on Western blots. Immunocytolocalization studies showed that HaWLIM1 is located in the cytoplasm and in the nucleus. In the cytoplasm, HaWLIM1 is localized in punctate structures, distributed along microtubule bundles. Depolymerizing microtubules with oryzalin resulted in a strong modification of the HaWLIM1 cortical pattern. In contrast, treatment of protoplasts with latrunculin B, which disrupts actin filaments, had no effect on HaWLIM1 localization. HaWLIM1 was also located within the nucleus of interphase protoplasts. During mitosis, nuclear labelling was observed in prophase, which decreased in metaphase, disappeared in anaphase, and recovered in telophase. These results suggest a dual role for HaWLIM1: in the cytoplasm, as a component of molecular complexes which may interact with microtubules, and in the nucleus, as a partner of transcription factors during interphase. PMID:14581630

  19. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

    Science.gov (United States)

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

    2016-08-01

    The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

  20. Multi-objective Numeric Association Rules Mining via Ant Colony Optimization for Continuous Domains without Specifying Minimum Support and Minimum Confidence

    Directory of Open Access Journals (Sweden)

    Parisa Moslehi

    2011-09-01

    Full Text Available Currently, all search algorithms which use discretization of numeric attributes for numeric association rule mining, work in the way that the original distribution of the numeric attributes will be lost. This issue leads to loss of information, so that the association rules which are generated through this process are not precise and accurate. Based on this fact, algorithms which can natively handle numeric attributes would be interesting. Since association rule mining can be considered as a multi-objective problem, rather than a single objective one, a new multi-objective algorithm for numeric association rule mining is presented in this paper, using Ant Colony Optimization for Continuous domains (ACOR. This algorithm mines numeric association rules without any need to specify minimum support and minimum confidence, in one step. In order to do this we modified ACOR for generating rules. The results show that we have more precise and accurate rules after applying this algorithm and the number of rules is more than the ones resulted from previous works.

  1. The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity

    NARCIS (Netherlands)

    P.W.J. de Groot; F.M. Klis

    2008-01-01

    Yeast cell wall-associated, lectin-like adhesins form large families that mediate flocculation and host cell recognition. The glycan specificity of individual adhesins is largely unknown. Zupancic et al. (this issue of Molecular Microbiology) used glycan microarrays to compare the glycan-binding cha

  2. Functional Diversity of Tandem Substrate-Binding Domains in ABC Transporters from Pathogenic Bacteria

    NARCIS (Netherlands)

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Vujicic - Zagar, Andreja; Guskov, Albert; Slotboom, Dirk-Jan; Poolman, Bert

    2013-01-01

    The ATP-binding cassette (ABC) transporter GInPQ is an essential uptake system for amino acids in gram-positive pathogens and related nonpathogenic bacteria. The transporter has tandem substrate-binding domains (SBDs) fused to each transmembrane domain, giving rise to four SBDs per functional transp

  3. Association of different cognitive domains with lifetime history of psychosis and reported antipsychotic-treatment adverse events in bipolar disorders

    OpenAIRE

    Kałwa, Agnieszka; Piróg-Balcerzak, Agnieszka Magdalena; Święcicki, Łukasz

    2013-01-01

    Aim of the study. The present work aimed to assess the association between cognitive functions, the lifetime occurrence of psychotic symptoms, and reported adverse effects of antipsychotic treatments inpatients with bipolar disorders.Methods. In the present work, 44 bipolar disorder inpatients hospitalized in the Affective Disorders Unit of the Institute of Psychiatry and Neurology in Warsaw, were investigated. All of them met the criteria of remission and were prepared for release from the h...

  4. An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application.

    Science.gov (United States)

    Kratochwil, Nicole A; Malherbe, Pari; Lindemann, Lothar; Ebeling, Martin; Hoener, Marius C; Mühlemann, Andreas; Porter, Richard H P; Stahl, Martin; Gerber, Paul R

    2005-01-01

    G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.

  5. Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC regulates inflammasomes

    Directory of Open Access Journals (Sweden)

    Rojanasakul Yon

    2010-05-01

    Full Text Available Abstract Background The apoptotic speck-like protein containing a caspase recruitment domain (ASC is the essential adaptor protein for caspase 1 mediated interleukin (IL-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs, which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an

  6. Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, L.; Morgan, D.O.; Jong, S.M.; Wang, L.H.; Roth, R.A.; Rutter, W.J.

    1987-08-01

    A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68/sup gag-ros/ has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into ..cap alpha.. and hybrid ..beta.. subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine resides of the hybrid ..beta..-subunit in vivo and the phosphorylation of an exogeneous substrate (poly(Glu,Tyr)) in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of (/sup 3/H)thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling

  7. Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68/sup gag-ros/ has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into α and hybrid β subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine resides of the hybrid β-subunit in vivo and the phosphorylation of an exogeneous substrate [poly(Glu,Tyr)] in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of [3H]thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling

  8. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  9. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor.

    Science.gov (United States)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-20

    Water molecules inside a G-protein coupled receptor (GPCR) have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal water molecules in GPCR activity, we studied the A2A adenosine receptor using microsecond molecular-dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times more slowly than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from ∼O(10(2)) ps to ∼O(10(2)) ns. Especially, water molecules, exhibiting ultraslow relaxation (∼O(10(2)) ns) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow water molecules in the activation of GPCRs. PMID:27653477

  10. High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Kledal, Thomas N; Schwartz, Thue W

    2005-01-01

    The highly conserved Arg in the so-called DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3 is in general considered as an essential residue for G protein coupling in rhodopsin-like seven transmembrane (7TM) receptors. In the open reading frame 74 (ORF74) receptor encoded by...

  11. Mutational Analysis of Region-cytotoxicity Relationship in Human Transmembrane Tumor Necrosis Factor-alpha

    Institute of Scientific and Technical Information of China (English)

    ZHENGFang; GONGFeili; LIZhuoya; JIANGXiaodan; XIONGPing; FENGWei; XUYong

    2002-01-01

    Objective:To determine the region of human transmembrane tumor necrosis factor-alpha (TM-TNFa), essential for cytotoxic activity a-gainst human breast cancer cell line MCF-7. Methods:Single amino-acid-substituted TM-TNFα mutant proteins (muteins) were produced by in vitro transcription linked translation techniques. The cDNA of TM-TNFα was site-directed mutagenized by recombinant PCR. Results:13 single amino-acid substituted TM-TNFα muteins were generated and assayed for cytotoxic activity. The cytotoxic activities of TM-TNFα muteins, eg, TM-TNFα-71/Lys, -28/Phe and 117/Leu were significantly decreased (P<0.01) compared to that of parent TM-TNFα, 143/Tyr decreased 4-folds, and-17/Thr,-39/Ser,ll9/His,35/Gly,95/Cys and 147/Phe decreased 1.5-2.5-folds, respectively. However, the cytotoxic activities of TM-TNFα-8/Arg, 31/Gly and 87/Phe showed no significant change. Conclusion:These results indicate that the regions associated with cytotoxic-activity of TM-TNFα are different with that of secretory TNF-lpha (S-TNFα). The inner cell region and transmembrane region of TM-TNFα are related to the cytotoxic activity of TM-TNFα.

  12. Transmembrane and truncated (SEC isoforms of MUC1 in the human endometrium and Fallopian tube

    Directory of Open Access Journals (Sweden)

    Wreschner Daniel H

    2003-01-01

    Full Text Available Abstract The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.

  13. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    Energy Technology Data Exchange (ETDEWEB)

    Schoenitzer, Veronika [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Eichner, Norbert [Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Clausen-Schaumann, Hauke [Munich University of Applied Sciences, Lothstrasse 34, D-80335 Muenchen, Germany, and Center for NanoScience (CeNS), Geschwister-Scholl-Platz 1, D-80539 Muenchen (Germany); Weiss, Ingrid M., E-mail: ingrid.weiss@inm-gmbh.de [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  14. HMM_RA: An Improved Method for Alpha-Helical Transmembrane Protein Topology Prediction

    Directory of Open Access Journals (Sweden)

    Changhui Yan

    2008-01-01

    Full Text Available α-helical transmembrane (TM proteins play important and diverse functional roles in cells. The ability to predict the topology of these proteins is important for identifying functional sites and inferring function of membrane proteins. This paper presents a Hidden Markov Model (referred to as HMM_RA that can predict the topology of α-helical transmembrane proteins with improved performance. HMM_RA adopts the same structure as the HMMTOP method, which has five modules: inside loop, inside helix tail, membrane helix, outside helix tail and outside loop. Each module consists of one or multiple states. HMM_RA allows using reduced alphabets to encode protein sequences. Thus, each state of HMM_RA is associated with n emission probabilities, where n is the size of the reduced alphabet set. Direct comparisons using two standard data sets show that HMM_RA consistently outperforms HMMTOP and TMHMM in topology prediction. Specifically, on a high-quality data set of 83 proteins, HMM_RA outperforms HMMTOP by up to 7.6% in topology accuracy and 6.4% in α-helices location accuracy. On the same data set, HMM_RA outperforms TMHMM by up to 6.4% in topology accuracy and 2.9% in location accuracy. Comparison also shows that HMM_RA achieves comparable performance as Phobius, a recently published method.

  15. Electric fingerprint of voltage sensor domains

    Science.gov (United States)

    Souza, Caio S.; Amaral, Cristiano; Treptow, Werner

    2014-01-01

    A dynamic transmembrane voltage field has been suggested as an intrinsic element in voltage sensor (VS) domains. Here, the dynamic field contribution to the VS energetics was analyzed via electrostatic calculations applied to a number of atomistic structures made available recently. We find that the field is largely static along with the molecular motions of the domain, and more importantly, it is minimally modified across VS variants. This finding implies that sensor domains transfer approximately the same amount of gating charges when moving the electrically charged S4 helix between fixed microscopic configurations. Remarkably, the result means that the observed operational diversity of the domain, including the extension, rate, and voltage dependence of the S4 motion, as dictated by the free energy landscape theory, must be rationalized in terms of dominant variations of its chemical free energy. PMID:25422443

  16. Analysis of Medical Domain Using CMARM: Confabulation Mapreduce Association Rule Mining Algorithm for Frequent and Rare Itemsets

    Directory of Open Access Journals (Sweden)

    Dr. Jyoti Gautam

    2015-11-01

    Full Text Available In Human Life span, disease is a major cause of illness and death in the modern society. There are various factors that are responsible for diseases like work environment, living and working conditions, agriculture and food production, housing, unemployment, individual life style etc. The early diagnosis of any disease that frequently and rarely occurs with the growing age can be helpful in curing the disease completely or to some extent. The long-term prognosis of patient records might be useful to find out the causes that are responsible for particular diseases. Therefore, human being can take early preventive measures to minimize the risk of diseases that may supervene with the growing age and hence increase the life expectancy chances. In this paper, a new CMARM: Confabulation-MapReduce based association rule mining algorithm is proposed for the analysis of medical data repository for both rare and frequent itemsets using an iterative MapReduce based framework inspired by cogency. Cogency is the probability of the assumed facts being true if the conclusion is true, means it is based on pairwise item conditional probability, so the proposed algorithm mine association rules by only one pass through the file. The proposed algorithm is also valuable for dealing with infrequent items due to its cogency inspired approach.

  17. SGTA Recognizes a Noncanonical Ubiquitin-like Domain in the Bag6-Ubl4A-Trc35 Complex to Promote Endoplasmic Reticulum-Associated Degradation

    Directory of Open Access Journals (Sweden)

    Yue Xu

    2012-12-01

    Full Text Available Elimination of aberrantly folded polypeptides from the endoplasmic reticulum (ER by the ER-associated degradation (ERAD system promotes cell survival under stress conditions. This quality control mechanism requires movement of misfolded proteins across the ER membrane for targeting to the cytosolic proteasome, a process facilitated by a “holdase” complex, consisting of Bag6 and the cofactors Ubl4A and Trc35. This multiprotein complex also participates in several other protein quality control processes. Here, we report SGTA as a component of the Bag6 system, which cooperates with Bag6 to channel dislocated ERAD substrates that are prone to aggregation. Using nuclear magnetic resonance spectroscopy and biochemical assays, we demonstrate that SGTA contains a noncanonical ubiquitin-like-binding domain that interacts specifically with an unconventional ubiquitin-like protein/domain in Ubl4A at least in part via electrostatics. This interaction helps recruit SGTA to Bag6, enhances substrate loading to Bag6, and thus prevents the formation of nondegradable protein aggregates in ERAD.

  18. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis.

    Science.gov (United States)

    Xiao, Dong; Cui, Yanjiao; Xu, Fan; Xu, Xinxin; Gao, Guanxiao; Wang, Yaxin; Guo, Zhaoxia; Wang, Dan; Wang, Ning Ning

    2015-10-01

    Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an important role in the regulation of leaf senescence. We previously reported that the leucine-rich repeat receptor-like kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (AtSARK) positively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). Here, we report the involvement of a protein serine/threonine phosphatase 2C-type protein phosphatase, SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence. SSPP transcript levels decreased greatly during both natural senescence and SARK-induced precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf senescence and changes in hormonal responses. All our results suggested that SSPP functions in sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing SARK-mediated senescence signal transduction.

  19. Human LINE1 endonuclease domain as a putative target of SARS-associated autoantibodies involved in the pathogenesis of severe acute respiratory syndrome

    Institute of Scientific and Technical Information of China (English)

    HE Wei-ping; SHU Cui-li; LI Bo-an; ZHAO Jun; CHENG Yun

    2008-01-01

    Background Severe acute respiratory syndrome(SARS)is a disease with a mortality of 9.56%.Although SARS is etiologically linked to a new coronavirus(SARS-CoV)and functional cell receptor has been identified,the pathogenesis of the virus infection is largely unclear.Methods The clinical specimens were processed and analyzed using an indirect enzyme-linked immunosorbent assay (ELISA) in-house.Further investigations of target antigen included reviews of phage display technique,rapid amplification of cDNA ends(RACE)technique,protein expression and purification,Western blotting validation,serological and immunohistochemical staining in postmortem tissue.Results A type of medium or low titer anti-lung tissue antibodies were found in the sera of SARS patients at the early stage of the disease.Human long interspersed nuclear element 1(LINE1)gene endonuclease(EN)domain protein was one of the target autoantigens and it was aberrantly expressed in the lung tissue of SARS patients.Anti-EN antibody was positive in the sera of 40.9% of SARS patients.Conclusions Human LINE1 endonuclease domain was identified as a putative target of SARS-associated autoantibodies,which were presented in the serum of SARS patients and may be involved in the pathogenesis of SARS.

  20. Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax.

    Directory of Open Access Journals (Sweden)

    Eldin Talundzic

    Full Text Available Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

  1. The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.

    Science.gov (United States)

    Yokoyama, A; Kawaguchi, Y; Kitabayashi, I; Ohki, M; Hirai, K

    2001-01-20

    There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation. PMID:11162796

  2. The matrix-binding domain of microfibril-associated glycoprotein-1 targets active connective tissue growth factor to a fibroblast-produced extracellular matrix.

    Science.gov (United States)

    Weinbaum, Justin S; Tranquillo, Robert T; Mecham, Robert P

    2010-11-10

    It is advantageous to use biomaterials in tissue engineering that stimulate extracellular matrix (ECM) production by the cellular component. Connective tissue growth factor (CTGF) stimulates type I collagen (COL1A1) transcription, but is functionally limited as a free molecule. Using a matrix-binding domain (MBD) from microfibril-associated glycoprotein-1, the fusion protein MBD-CTGF was targeted to the ECM and tested for COL1A1 transcriptional activation. MBD-CTGF produced by the ECM-synthesizing fibroblasts, or provided exogenously, localized to the elastic fiber ECM. MBD-CTGF, but not CTGF alone, led to a two-fold enhancement of COL1A1 expression. This study introduces a targeting technology that can be used to elevate collagen transcription in engineered tissues and thereby improve tissue mechanics.

  3. Positional cloning of zinc finger domain transcription factor Zfp69, a candidate gene for obesity-associated diabetes contributed by mouse locus Nidd/SJL.

    Directory of Open Access Journals (Sweden)

    Stephan Scherneck

    2009-07-01

    Full Text Available Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1 contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO mice. A critical interval of distal chromosome 4 (2.1 Mbp conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT-PCR, and RACE-PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lep(ob background, the diabetogenic Zfp69(SJL allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes.

  4. JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis.

    Science.gov (United States)

    He, Zhimin; Wu, Junyu; Su, Xiaonan; Zhang, Ye; Pan, Lixia; Wei, Huimin; Fang, Qiang; Li, Haitao; Wang, Da-Liang; Sun, Fang-Lin

    2016-02-26

    Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.

  5. Annexin A5 anticoagulant activity in children with systemic lupus erythematosus and the association with antibodies to domain I of β2-glycoprotein I.

    Science.gov (United States)

    Wahezi, D M; Ilowite, N T; Wu, X X; Pelkmans, L; Laat, B; Schanberg, L E; Rand, J H

    2013-06-01

    Children with systemic lupus erythematosus (SLE) have a high prevalence of antiphospholipid (aPL) antibodies and are at increased risk for aPL-related thrombosis. We investigated the association between annexin A5 anticoagulant activity and antibodies to the domain I portion of β2-glycoprotein I (anti-DI antibodies), and propose a potential mechanism for the pathogenesis of aPL-related thrombosis. Using samples from 183 children with SLE collected during the Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) trial, we examined resistance to the anticoagulant effects of annexin A5, using the annexin A5 resistance (A5R) assay, and evaluated for anti-DI IgG antibodies. Children with SLE had higher frequency of anti-D1 antibodies (p = 0.014) and significantly reduced A5R compared to pediatric controls: mean A5R = 172 ± 30% versus 242 ± 32% (p antibodies had significantly lower mean A5R levels compared to those with negative anti-DI antibodies: mean A5R = 155 ± 24% versus 177 ± 30% (p antibodies (p = 0.013) and lupus anticoagulant (LA) (p = 0.036) were both independently associated with reduced A5R. Children with SLE have significantly reduced annexin A5 anticoagulant activity that is associated with the presence of LA and anti-DI antibodies.

  6. Sialidase NEU3 dynamically associates to different membrane domains specifically modifying their ganglioside pattern and triggering Akt phosphorylation.

    Directory of Open Access Journals (Sweden)

    Dario Bonardi

    Full Text Available Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM. Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i newly synthesized NEU3 is initially associated to non-DRM; ii at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii NEU3 is degraded via the proteasomal pathway; iv the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.

  7. Association of different cognitive domains with lifetime history of psychosis and reported antipsychotic-treatment adverse events in bipolar disorders

    Directory of Open Access Journals (Sweden)

    Kałwa, Agnieszka

    2013-06-01

    Full Text Available Aim of the study. The present work aimed to assess the association between cognitive functions, the lifetime occurrence of psychotic symptoms, and reported adverse effects of antipsychotic treatments inpatients with bipolar disorders.Methods. In the present work, 44 bipolar disorder inpatients hospitalized in the Affective Disorders Unit of the Institute of Psychiatry and Neurology in Warsaw, were investigated. All of them met the criteria of remission and were prepared for release from the hospital unit. Twenty-two patients were hospitalized in the manic stage of the illness, and 22 were in the depressive stage of illness. Both groups were assessed using adequate psychiatric rating scales (HDRS or YMRS and CAMRS and neuropsychological tests (WCST, TMT, Stroop Test and Verbal Fluency Test.Results. Patients who had a prior history of psychotic symptoms had poorer verbal functioning in comparison to subjects without such a history. However, individuals hospitalized in the manic state of disease, and who reported more adverse events after antipsychotic medication during the whole course of illness, had worse results in some parameters of executive function measurements in the WCST test, namely occurring in a greater percentage of nonperseverative errors and a lower number of completed categories.Discussion. Generally the results confirm findings according to which, patients with the history of psychosis performe worse on neurocognitive tasks. However, the nature of dysfunctions found, generates questions about its relations with the experience of psychosis and antipsychotic treatment. Conclusion: Different aspects of cognitive dysfunctions may be related to the experience of psychosis and antipsychotic treatment in patients with bipolar disorders.

  8. Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

    Directory of Open Access Journals (Sweden)

    Fumio Yoshikawa

    Full Text Available BACKGROUND: Phospholipase D (PLD catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x(4-Asp (HKD motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non

  9. Transmembrane transport of peptidoglycan precursors across model and bacterial membranes.

    Science.gov (United States)

    van Dam, Vincent; Sijbrandi, Robert; Kol, Matthijs; Swiezewska, Ewa; de Kruijff, Ben; Breukink, Eefjan

    2007-05-01

    Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane. PMID:17501931

  10. Stability analysis of the inverse transmembrane potential problem in electrocardiography

    Science.gov (United States)

    Burger, Martin; Mardal, Kent-André; Nielsen, Bjørn Fredrik

    2010-10-01

    In this paper we study some mathematical properties of an inverse problem arising in connection with electrocardiograms (ECGs). More specifically, we analyze the possibility for recovering the transmembrane potential in the heart from ECG recordings, a challenge currently investigated by a growing number of groups. Our approach is based on the bidomain model for the electrical activity in the myocardium, and leads to a parameter identification problem for elliptic partial differential equations (PDEs). It turns out that this challenge can be split into two subproblems: the task of recovering the potential at the heart surface from body surface recordings; the problem of computing the transmembrane potential inside the heart from the potential determined at the heart surface. Problem (1), which can be formulated as the Cauchy problem for an elliptic PDE, has been extensively studied and is well known to be severely ill-posed. The main purpose of this paper is to prove that problem (2) is stable and well posed if a suitable prior is available. Moreover, our theoretical findings are illuminated by a series of numerical experiments. Finally, we discuss some aspects of uniqueness related to the anisotropy in the heart.

  11. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    Science.gov (United States)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  12. Transmembrane proteins--Mining the cattle tick transcriptome.

    Science.gov (United States)

    Richards, Sabine A; Stutzer, Christian; Bosman, Anna-Mari; Maritz-Olivier, Christine

    2015-09-01

    Managing the spread and load of pathogen-transmitting ticks is an important task worldwide. The cattle tick, Rhipicephalus microplus, not only impacts the economy through losses in dairy and meat production, but also raises concerns for human health in regards to the potential of certain transmitted pathogens becoming zoonotic. However, novel strategies to control R. microplus are hindered by lack of understanding tick biology and the discovery of suitable vaccine or acaricide targets. The importance of transmembrane proteins as vaccine targets are well known, as is the case in tick vaccines with Bm86 as antigen. In this study, we describe the localization and functional annotation of 878 putative transmembrane proteins. Thirty proteins could be confirmed in the R. microplus gut using LC-MS/MS analysis and their roles in tick biology are discussed. To the best of our knowledge, 19 targets have not been reported before in any proteomics study in various tick species and the possibility of using the identified proteins as targets for tick control are discussed. Although tissue expression of identified putative proteins through expansive proteomics is necessary, this study demonstrates the possibility of using bioinformatics for the identification of targets for further evaluation in tick control strategies. PMID:26096851

  13. Structure and dynamics of nano-sized raft-like domains on the plasma membrane

    Science.gov (United States)

    Herrera, Fernando E.; Pantano, Sergio

    2012-01-01

    Cell membranes are constitutively composed of thousands of different lipidic species, whose specific organization leads to functional heterogeneities. In particular, sphingolipids, cholesterol and some proteins associate among them to form stable nanoscale domains involved in recognition, signaling, membrane trafficking, etc. Atomic-detail information in the nanometer/second scale is still elusive to experimental techniques. In this context, molecular simulations on membrane systems have provided useful insights contributing to bridge this gap. Here we present the results of a series of simulations of biomembranes representing non-raft and raft-like nano-sized domains in order to analyze the particular structural and dynamical properties of these domains. Our results indicate that the smallest (5 nm) raft domains are able to preserve their distinctive structural and dynamical features, such as an increased thickness, higher ordering, lower lateral diffusion, and specific lipid-ion interactions. The insertion of a transmembrane protein helix into non-raft, extended raft-like, and raft-like nanodomain environments result in markedly different protein orientations, highlighting the interplay between the lipid-lipid and lipid-protein interactions.

  14. Association of genetic variations of genes encoding thrombospondin, type 1, domain-containing 4 and 7A with low bone mineral density in Japanese women with osteoporosis.

    Science.gov (United States)

    Mori, Seijiro; Kou, Ikuyo; Sato, Hidenori; Emi, Mitsuru; Ito, Hideki; Hosoi, Takayuki; Ikegawa, Shiro

    2008-01-01

    Twins and family studies have shown that genetic factors are important determinants of bone mass. Important aspects of bone mineral density (BMD) regulation are endocrine systems, notably hormonal regulation of adrenal corticoids, as indicated by clinical knowledge of glucocorticoid-induced osteoporosis. Glucocorticoid is known to negatively regulate bone mass in vivo, and glucocorticoid increases thrombospondin messenger ribonucleic acid (mRNA) levels. We studied single nucleotide polymorphisms (SNPs) in genes encoding thrombospondin, type 1, domain-containing 4 and 7A (THSD4 and THSD7A) for possible association with lumbar and femoral BMD among 337 Japanese women with osteoporosis who participated in the BioBank Japan project. Genetic variations of THSD4 and THSD7A loci displayed significant association with lumbar and femoral BMD. Most significant correlation was observed for THSD7A SNP rs12673692 with lumbar BMD (P = 0.00017). Homozygous carriers of the major (G) allele had the highest BMD [0.886 +/- 0.011 g/cm2, mean +/- standard deviation (SD)], whereas heterozygous carriers were intermediate (0.872 +/- 0.013 g/cm2) and homozygous A-allele carriers had the lowest (0.753 +/- 0.023 g/cm2). THSD4 SNP rs10851839 also displayed strong association with lumbar BMD (P = 0.0092). In addition, both THSD7A and THSD4 displayed significant association with femoral BMD in a recessive model (P = 0.036 and P = 0.0046, respectively). Results suggest that variations of THSD7A and THSD4 loci may be important determinants of osteoporosis in Japanese women.

  15. Effects of La3+ on H+ Transmembrane Gradient and Membrane Potential in Rice Seedling Roots

    Institute of Scientific and Technical Information of China (English)

    郑海雷; 张春光; 赵中秋; 马建华; 李利

    2002-01-01

    The effects of LaCl3 on membrane potential and transmembrane proton gradient for rice (Oryza sativa) seedling roots were studied. Highly purified plasma membrane was isolated by aqueous two-phase partitioning method. Both the gradient of transmembrane proton and membrane potential were stimulated by certain low concentration of LaCl3 and depressed by high concentration of LaCl3. The optimal concentration of La3+ is around 40~60 μmolL-1 for transmembrane proton gradient and membrane potential. It shows that La3+ can influence the generations and maintenances of membrane potential and transmembrane proton gradient in rice seedling roots.

  16. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers.

    Science.gov (United States)

    Hou, Yuning; Guan, Xiaoqing; Yang, Zhe; Li, Chunying

    2016-03-15

    Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers. PMID:26989463

  17. Rapid Evolution to Blast Crisis Associated with a Q252H ABL1 Kinase Domain Mutation in e19a2 BCR-ABL1 Chronic Myeloid Leukaemia

    Directory of Open Access Journals (Sweden)

    Sarah L. McCarron

    2013-01-01

    Full Text Available A minority of chronic myeloid leukaemia (CML patients express variant transcripts of which the e19a2 BCR-ABL1 fusion is the most common. Instances of tyrosine kinase inhibitor (TKI resistance in e19a2 BCR-ABL1 CML patients have rarely been reported. A case of e19a2 BCR-ABL1 CML is described in whom imatinib resistance, associated with a Q252H ABL1 kinase domain mutation, became apparent soon after initiation of TKI therapy. The patient rapidly transformed to myeloid blast crisis (BC with considerable bone marrow fibrosis and no significant molecular response to a second generation TKI. The clinical course was complicated by comorbidities with the patient rapidly succumbing to advanced disease. This scenario of Q252H-associated TKI resistance with rapid BC transformation has not been previously documented in e19a2 BCR-ABL1 CML. This case highlights the considerable challenges remaining in the management of TKI-resistant BC CML, particularly in the elderly patient.

  18. Polymorphisms of clip domain serine proteinase and serine proteinase homolog in the swimming crab Portunus trituberculatus and their association with Vibrio alginolyticus

    Science.gov (United States)

    Liu, Meng; Liu, Yuan; Hui, Min; Song, Chengwen; Cui, Zhaoxia

    2016-05-01

    Clip domain serine proteases (cSPs) and their homologs (SPHs) play an important role in various biological processes that are essential components of extracellular signaling cascades, especially in the innate immune responses of invertebrates. Here, polymorphisms of PtcSP and PtSPH from the swimming crab Portunus trituberculatus were investigated to explore their association with resistance/ susceptibility to Vibrio alginolyticus. Polymorphic loci were identified using Clustal X, and characterized with SPSS 16.0 software, and then the significance of genotype and allele frequencies between resistant and susceptible stocks was determined by a χ2 test. A total of 109 and 77 single nucleotide polymorphisms (SNPs) were identified in the genomic fragments of PtcSP and PtSPH, respectively. Notably, nearly half of PtSPH polymorphisms were found in the non-coding exon 1. Fourteen SNPs investigated were significantly associated with susceptibility/resistance to V. alginolyticus (P<0.05). Among them, eight SNPs were observed in introns, and one synonymous, four non-synonymous SNPs and one ins-del were found in coding exons. In addition, five simple sequence repeats (SSRs) were detected in intron 3 of PtcSP. Although there was no statistically significant difference of allele frequencies, the SSRs showed different polymorphic alleles on the basis of the repeat number between resistant and susceptible stocks. After further validation, polymorphisms investigated here might be applied to select potential molecular markers of P. trituberculatus with resistance to V. alginolyticus.

  19. The Action of Discoidin Domain Receptor 2 in Basal Tumor Cells and Stromal Cancer-Associated Fibroblasts Is Critical for Breast Cancer Metastasis

    Directory of Open Access Journals (Sweden)

    Callie A.S. Corsa

    2016-06-01

    Full Text Available High levels of collagen deposition in human and mouse breast tumors are associated with poor outcome due to increased local invasion and distant metastases. Using a genetic approach, we show that, in mice, the action of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2 in both tumor and tumor-stromal cells is critical for breast cancer metastasis yet does not affect primary tumor growth. In tumor cells, DDR2 in basal epithelial cells regulates the collective invasion of tumor organoids. In stromal cancer-associated fibroblasts (CAFs, DDR2 is critical for extracellular matrix production and the organization of collagen fibers. The action of DDR2 in CAFs also enhances tumor cell collective invasion through a pathway distinct from the tumor-cell-intrinsic function of DDR2. This work identifies DDR2 as a potential therapeutic target that controls breast cancer metastases through its action in both tumor cells and tumor-stromal cells at the primary tumor site.

  20. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains

    Energy Technology Data Exchange (ETDEWEB)

    Wun, T.C.; Kretzmer, K.K.; Girard, T.J.; Miletich, J.P.; Broze, G.J. Jr.

    1988-05-05

    Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor X/sub a/ directly, and in a X/sub a/-dependent fashion also inhibits the VII/sub a/-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI antiserum was used to screen human placental and fetal liver lambdagt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind /sup 125/I-factor X/sub a/. Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (lambdaP9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that lambdaP9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH/sub 2/ terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH/sub 2/ terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA which hybridize with LACI cDNA.

  1. Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8

    DEFF Research Database (Denmark)

    Kniazeff, Julie; Loland, Claus Juul; Goldberg, Naomi;

    2005-01-01

    The extracellular concentration of the neurotransmitters dopamine, serotonin, norepinephrine, GABA and glycine is tightly controlled by plasma membrane transporters belonging to the SLC6 gene family. A very large number of putative transport proteins with a remarkable homology to the SLC6...... transporters has recently been identified in prokaryotes. Here we have probed structural relationships in a 'microdoman' corresponding to the extracellular ends of transmembrane segments (TM) 7 and 8 in one of these homologs, the tryptophan transporter TnaT from Symbiobacterium thermophilum. We found...... proximity between TM 7 and 8 in the tertiary structure of TnaT as previously suggested for the mammalian counterparts. Furthermore, the inhibition of uptake upon cross-linking the two cysteines provides indirect support for a conserved conformational role of these transmembrane domains in the transport...

  2. Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

    Energy Technology Data Exchange (ETDEWEB)

    Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N. (INSERM U 28, Hopital Broussais, Paris (France))

    1990-12-01

    Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes.

  3. 白氏文昌鱼FADD的克隆及功能研究%MOLECULAR CLONING AND CHARACTERIZATION OF FAS-ASSOCIATED DEATH DOMAIN (FADD) FROM BRANCHIOSTOMA BELCHERI

    Institute of Scientific and Technical Information of China (English)

    张晶; 黄贝; 高谦; 聂品

    2009-01-01

    FADD (Fas-associated death domain protein) , also termed MORT1, plays a critical role in the apoptotic signaling pathways of both CD95 ( Fas/AP0-l ) and certain members of the tumor necrosis factor receptor (TNFR) superfami-ly, and is well conserved in vertebrates, especially in mammals. To reveal the features of FADD in lower animals, we have characterized FADD from amphioxus, Branchiostoma belcheri (Chordata, Cephalochordata) , which is a model animal, and have an insight into the origin and evolution of apoptotic signaling system of the vertebrate. Branchiostoma belcheri FADD (bbFADD) cDNA and genomic DNA sequences were obtained by using RACE-PCR and Genomic Walking, respectively. The full-length cDNA of bbFADD consisted of 1239 base pairs (bp) encoding 217 amino acid residues (aa) with a death effector domain (DED) (12-95~(th); 84 aa) near the N-terminal and a death domain (DD) (129-211(st); 83 aa) by the C-terminal. bbFADD genomic sequence, the length of which was 2840 bp, consisted of three exons and two introns in the gene coding region. The structure of bbFADD gene was different from that of its vertebrate counterparts, with two exons. The result of multiple alignments among FADDs from various species supported that DED and DD regions were more conservative than other regions. Furthermore, the 33~(rd) phenylalanine residue ( F33) of bbFADD amino acid sequence, which was equivalent to the 25~(th) phenylalanine residue ( F25) in human FADD. This site, which was crucial to mediate FADD self-association, was well conserved through sea urchin and human. Homology analysis showed that the percent identity and the percent similarity between bbFADD and vertebrate FADDs were 27.5%-30.0% and 44.7%-55.3%, while 35.1%-36.9% and 51.1%-52.0% between bbFADD and sea urchin FADD, respectively. In NJ phylogenetic tree based on amino acid sequences of FADDs, B. belcheri was grouped with sea urchin with the suggestion that comparing with lower vertebrate, fish, amphioxus was

  4. Team work at its best – TAPL and its two domains.

    Science.gov (United States)

    Zollmann, Tina; Bock, Christoph; Graab, Philipp; Abele, Rupert

    2015-09-01

    The transporter associated with antigen processing (TAPL, ABCB9) is a homodimeric ABC transporter, shuttling cytosolic polypeptides into the lumen of lysosomes energized by ATP hydrolysis. Here we give a short overview of the superfamily of ABC transporters and summarize the current state of knowledge on TAPL in detail. The architecture of TAPL and its substrate specificity are described and we discuss the function of an extra N-terminal transmembrane domain, called TMD0, in respect of subcellular targeting and interaction with proteins, contributing to long-term stability. As TAPL shows – besides a ubiquitous basal expression – an elevated expression in antigen presenting cells, we present models of TAPL function in adaptive immunity.

  5. Large-Conductance Transmembrane Porin Made from DNA Origami.

    Science.gov (United States)

    Göpfrich, Kerstin; Li, Chen-Yu; Ricci, Maria; Bhamidimarri, Satya Prathyusha; Yoo, Jejoong; Gyenes, Bertalan; Ohmann, Alexander; Winterhalter, Mathias; Aksimentiev, Aleksei; Keyser, Ulrich F

    2016-09-27

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance. PMID:27504755

  6. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-01-01

    in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue......A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed....... The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium...

  7. Glycosylation and the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    Glick Mary Catherine

    2001-08-01

    Full Text Available Abstract The cystic fibrosis transmembrane conductance regulator (CFTR has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wtCFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF, and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

  8. Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin.

    Science.gov (United States)

    Tremouilhac, Pierre; Strandberg, Erik; Wadhwani, Parvesh; Ulrich, Anne S

    2006-10-27

    The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure. PMID:16877761

  9. Transmembrane protein topology prediction using support vector machines

    Directory of Open Access Journals (Sweden)

    Nugent Timothy

    2009-05-01

    Full Text Available Abstract Background Alpha-helical transmembrane (TM proteins are involved in a wide range of important biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, cell recognition and cell adhesion. Many are also prime drug targets, and it has been estimated that more than half of all drugs currently on the market target membrane proteins. However, due to the experimental difficulties involved in obtaining high quality crystals, this class of protein is severely under-represented in structural databases. In the absence of structural data, sequence-based prediction methods allow TM protein topology to be investigated. Results We present a support vector machine-based (SVM TM protein topology predictor that integrates both signal peptide and re-entrant helix prediction, benchmarked with full cross-validation on a novel data set of 131 sequences with known crystal structures. The method achieves topology prediction accuracy of 89%, while signal peptides and re-entrant helices are predicted with 93% and 44% accuracy respectively. An additional SVM trained to discriminate between globular and TM proteins detected zero false positives, with a low false negative rate of 0.4%. We present the results of applying these tools to a number of complete genomes. Source code, data sets and a web server are freely available from http://bioinf.cs.ucl.ac.uk/psipred/. Conclusion The high accuracy of TM topology prediction which includes detection of both signal peptides and re-entrant helices, combined with the ability to effectively discriminate between TM and globular proteins, make this method ideally suited to whole genome annotation of alpha-helical transmembrane proteins.

  10. Photometric recording of transmembrane potential in outer hair cells

    Science.gov (United States)

    Nakagawa, Takashi; Oghalai, John S.; Saggau, Peter; Rabbitt, Richard D.; Brownell, William E.

    2006-06-01

    Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 µm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

  11. Magnetic resonance study of the transmembrane nitrite diffusion.

    Science.gov (United States)

    Samouilov, A; Woldman, Ya Yu; Zweier, J L; Khramtsov, V V

    2007-05-01

    Nitrite (NO(2)-), being a product of metabolism of both nitric oxide (NO(*)) and nitrate (NO(3)-), can accumulate in tissues and regenerate NO() by several mechanisms. The effect of NO(2)- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO(2)- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO(2)). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO(2) transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO(2)(-)-mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by (31)P NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO(2)- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 microM NO(2)- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NO(x) in myocardium increased from initial 7.8 to 24.7 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.

  12. TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins

    Directory of Open Access Journals (Sweden)

    Mutai Hideki

    2003-06-01

    Full Text Available Abstract Background Mutations in the transmembrane cochlear expressed gene 1 (TMC1 cause deafness in human and mouse. Mutations in two homologous genes, EVER1 and EVER2 increase the susceptibility to infection with certain human papillomaviruses resulting in high risk of skin carcinoma. Here we report that TMC1, EVER1 and EVER2 (now TMC6 and TMC8 belong to a larger novel gene family, which is named TMC for trans membrane channel-like gene family. Results Using a combination of iterative database searches and reverse transcriptase-polymerase chain reaction (RT-PCR experiments we assembled contigs for cDNA encoding human, murine, puffer fish, and invertebrate TMC proteins. TMC proteins of individual species can be grouped into three subfamilies A, B, and C. Vertebrates have eight TMC genes. The majority of murine TMC transcripts are expressed in most organs; some transcripts, however, in particular the three subfamily A members are rare and more restrictively expressed. Conclusion The eight vertebrate TMC genes are evolutionary conserved and encode proteins that form three subfamilies. Invertebrate TMC proteins can also be categorized into these three subfamilies. All TMC genes encode transmembrane proteins with intracellular amino- and carboxyl-termini and at least eight membrane-spanning domains. We speculate that the TMC proteins constitute a novel group of ion channels, transporters, or modifiers of such.

  13. Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.

    Directory of Open Access Journals (Sweden)

    Tina L Gumienny

    2010-05-01

    Full Text Available Bone morphogenetic protein (BMP pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

  14. Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

    Science.gov (United States)

    Gao, Quan Q; Wyatt, Eugene; Goldstein, Jeff A; LoPresti, Peter; Castillo, Lisa M; Gazda, Alec; Petrossian, Natalie; Earley, Judy U; Hadhazy, Michele; Barefield, David Y; Demonbreun, Alexis R; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M

    2015-11-01

    Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations. PMID:26457733

  15. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    Science.gov (United States)

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  16. WW domains of the yes-kinase-associated-protein (YAP) transcriptional regulator behave as independent units with different binding preferences for PPxY motif-containing ligands.

    Science.gov (United States)

    Iglesias-Bexiga, Manuel; Castillo, Francisco; Cobos, Eva S; Oka, Tsutomu; Sudol, Marius; Luque, Irene

    2015-01-01

    YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling. PMID:25607641

  17. WW domains of the yes-kinase-associated-protein (YAP transcriptional regulator behave as independent units with different binding preferences for PPxY motif-containing ligands.

    Directory of Open Access Journals (Sweden)

    Manuel Iglesias-Bexiga

    Full Text Available YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.

  18. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cystic fibrosis transmembrane conductance... DEVICES Immunological Test Systems § 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR... intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis...

  19. Understanding Function of Transmembrane Proteins by Single-Molecule Experiments on Native-like Environments

    DEFF Research Database (Denmark)

    Jørgensen, Sune Klamer

    Transmembrane proteins are vital for a range of biological processes. Here we investigate three representatives from three different classes of transmembrane proteins; an active ion transporter, a photosynthetic electron transporter, and a signaling protein. To isolate them from other cellular in...

  20. The histone acetyltransferase domains of CREB-binding protein (CBP) and p300/CBP-associated factor are not necessary for cooperativity with the class II transactivator.

    Science.gov (United States)

    Harton, J A; Zika, E; Ting, J P

    2001-10-19

    The class II transactivator (CIITA) is a transcriptional co-activator regulating the constitutive and interferon-gamma-inducible expression of class II major histocompatibility complex (MHC) and related genes. Promoter remodeling occurs following CIITA induction, suggesting the involvement of chromatin remodeling factors. Transcription of numerous genes requires the histone acetyltransferase (HAT) activities of CREB-binding protein (CBP), p300, and/or p300/CBP-associated factor (pCAF). These co-activators cooperate with CIITA and are hypothesized to promote class II major histocompatibility complex transcription through their HAT activity. To directly test this, we used HAT-defective CBP and pCAF. We demonstrate that cooperation between CIITA and CBP is independent of CBP HAT activity. Further, although pCAF enhances CIITA-mediated transcription, pCAF HAT domain dependence appears contingent upon the concentration of available CIITA. When HAT-defective CBP and pCAF are both present, cooperativity with CIITA is maintained. Consistent with a recent report, we show that nuclear localization of CIITA is enhanced by lysine 144, an in vitro target of pCAF-mediated HAT. Yet we find that neither mutation of lysine 144 nor deletion of residues 132-209 affects transcriptional cooperation with CBP or pCAF. Thus, acetylation of this residue may not be the primary mechanism for pCAF/CBP cooperation with CIITA. In conclusion, the HAT activities of the co-activators are not necessary for cooperation with CIITA.

  1. Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Khaiboullina, Svetlana F., E-mail: sv.khaiboullina@gmail.com [Whittemore Peterson Institute, University of Nevada-Reno, Reno (United States); Morzunov, Sergey P. [Department of Pathology and Nevada State Health Laboratory, University of Nevada-Reno, Reno (United States); Boichuk, Sergei V. [Kazan State Medical University, Kazan (Russian Federation); Palotás, András [Asklepios-Med (private medical practice and research center), Szeged (Hungary); Jeor, Stephen St. [Department of Microbiology and Immunology, University of Nevada-Reno, Reno (United States); Lombardi, Vincent C. [Whittemore Peterson Institute, University of Nevada-Reno, Reno (United States); Rizvanov, Albert A. [Department of Genetics, Kazan (Volga Region) Federal University, Kazan (Russian Federation)

    2013-09-01

    Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.

  2. A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma.

    Science.gov (United States)

    Liu, Chunqiao; Widen, Sonya A; Williamson, Kathleen A; Ratnapriya, Rinki; Gerth-Kahlert, Christina; Rainger, Joe; Alur, Ramakrishna P; Strachan, Erin; Manjunath, Souparnika H; Balakrishnan, Archana; Floyd, James A; Li, Tiansen; Waskiewicz, Andrew; Brooks, Brian P; Lehmann, Ordan J; FitzPatrick, David R; Swaroop, Anand

    2016-04-01

    Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

  3. Electrostatics analysis of the mutational and pH effects of the N-terminal domain self-association of the major ampullate spidroin.

    Science.gov (United States)

    Barroso da Silva, Fernando Luís; Pasquali, Samuela; Derreumaux, Philippe; Dias, Luis Gustavo

    2016-07-01

    Spider silk is a fascinating material combining mechanical properties such as maximum strength and high toughness comparable or better than man-made materials, with biocompatible degradability characteristics. Experimental measurements have shown that pH triggers the dimer formation of the N-terminal domain (NTD) of the major ampullate spidroin 1 (MaSp 1). A coarse-grained model accounting for electrostatics, van der Waals and pH-dependent charge-fluctuation interactions, by means of Monte Carlo simulations, gave us a more comprehensive view of the NTD dimerization process. A detailed analysis of the electrostatic properties and free energy derivatives for the NTD homoassociation was carried out at different pH values and salt concentrations for the protein wild type and for several mutants. We observed an enhancement of dipole-dipole interactions at pH 6 due to the ionization of key amino acids, a process identified as the main driving force for dimerization. Analytical estimates based on the DVLO theory framework corroborate our findings. Molecular dynamics simulations using the OPEP coarse-grained force field for proteins show that the mutant E17Q is subject to larger structural fluctuations when compared to the wild type. Estimates of the association rate constants for this mutant were evaluated by the Debye-Smoluchowski theory and are in agreement with the experimental data when thermally relaxed structures are used instead of the crystallographic data. Our results can contribute to the design of new mutants with specific association properties. PMID:27250106

  4. THE COMBINATION PREDICTION OF TRANSMEMBRANE REGIONS BASED ON DEMPSTER-SHAFER THEORY OF EVIDENCE

    Institute of Scientific and Technical Information of China (English)

    Deng Xinyang; Xu Peida; Deng Yong

    2012-01-01

    Transmembrane proteins are some special and important proteins in cells.Because of their importance and specificity,the prediction of the transmembrane regions has very important theoretical and practical significance.At present,the prediction methods are mainly based on the physicochemical property and statistic analysis of amino acids.However,these methods are suitable for some environments but inapplicable for other environments.In this paper,the multi-sources information fusion theory has been introduced to predict the transmembrane regions.The proposed method is test on a data set of transmembrane proteins.The results show that the proposed method has the ability of predicting the transmembrane regions as a good performance and powerful tool.

  5. Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

    Science.gov (United States)

    Chouhan, Bhanupratap; Denesyuk, Alexander; Heino, Jyrki; Johnson, Mark S.; Denessiouk, Konstantin

    2011-01-01

    Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem

  6. Conservation of the human integrin-type beta-propeller domain in bacteria.

    Directory of Open Access Journals (Sweden)

    Bhanupratap Chouhan

    Full Text Available Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca(2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca(2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and

  7. Cystic Fibrosis Transmembrane Conductance Regulator is an Epithelial Cell Receptor for Clearance of Pseudomonas aeruginosa from the Lung

    Science.gov (United States)

    Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.

    1997-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant Δ F508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

  8. Trusted Domain

    DEFF Research Database (Denmark)

    Hjorth, Theis Solberg; Torbensen, Rune

    2012-01-01

    In the digital age of home automation and with the proliferation of mobile Internet access, the intelligent home and its devices should be accessible at any time from anywhere. There are many challenges such as security, privacy, ease of configuration, incompatible legacy devices, a wealth...... of wireless standards, limited resources of embedded systems, etc. Taking these challenges into account, we present a Trusted Domain home automation platform, which dynamically and securely connects heterogeneous networks of Short-Range Wireless devices via simple non-expert user. interactions, and allows...... that enables secure end-to-end communication with home automation devices, and it supports device revocations as well as a structure of intersecting sets of nodes for scalability. Devices in the Trusted Domain are registered in a list that is distributed using a robust epidemic protocol optimized...

  9. Exosome-related multi-pass transmembrane protein TSAP6 is a target of rhomboid protease RHBDD1-induced proteolysis.

    Directory of Open Access Journals (Sweden)

    Chunhua Wan

    Full Text Available We have previously reported that rhomboid domain containing 1 (RHBDD1, a mammalian rhomboid protease highly expressed in the testis, can cleave the Bcl-2 protein Bik. In this study, we identified a multi-pass transmembrane protein, tumor suppressor activated pathway-6 (TSAP6 as a potential substrate of RHBDD1. RHBDD1 was found to induce the proteolysis of TSAP6 in a dose- and activity-dependent manner. The cleavage of TSAP6 was not restricted to its glycosylated form and occurred in three different regions. In addition, mass spectrometry and mutagenesis analyses both indicated that the major cleavage site laid in the C-terminal of the third transmembrane domain of TSAP6. A somatic cell knock-in approach was used to genetically inactivate the endogenous RHBDD1 in HCT116 and RKO colon cancer cells. Exosome secretion was significantly elevated when RHBDD1 was inactivated in the two cells lines. The increased exosome secretion was verfied through the detection of certain exosomal components, including Tsg101, Tf-R, FasL and Trail. In addition, the elevation of exosome secretion by RHBDD1 inactivation was reduced when TSAP6 was knocked down, indicating that the role of RHBDD1 in regulating exosomal trafficking is very likely to be TSAP6-dependent. We found that the increase in FasL and Trail increased exosome-induced apoptosis in Jurkat cells. Taken together, our findings suggest that RHBDD1 is involved in the regulation of a nonclassical exosomal secretion pathway through the restriction of TSAP6.

  10. 调控黑色素瘤转移的新信号蛋白: Syntenin%Syntenin: a novel PDZ domain-containing scaffolding protein associated with human melanoma metastasis

    Institute of Scientific and Technical Information of China (English)

    Jian-bo Yang; James B. McCarthy

    2007-01-01

    Syntenin蛋白在多种肿瘤中表达增强,最近被认为是一个新的黑色素瘤转移调节因子.作为一类支架信号蛋白,Syntenin通过它的两个PDZ 功能基团可与许多细胞膜受体胞内末端或细胞内的信号分子结合,调控多种重要的细胞生理过程和信号传导途径,包括细胞膜受体的聚集,细胞内蛋白质的转运,细胞骨架的重建,转录因子的激活,以增强肿瘤细胞的生长、黏附以及肿瘤的血管生成、侵袭和转移能力.本文简要综述了syntenin的结构和功能,相关的信号途径,及其在黑色素瘤研究领域的最新进展.%Syntenin is overexpressed in multiple human cancers and is newly recognized as a novel regulator in melanoma metastasis. It functions as a scaffolding protein, via its two PDZ domains interacting with multiple transmembrane and cytoplasmic partners to regulate many of the major signaling pathways involved in various cellular processes, such as cell surface receptor clustering, protein trafficking, cytoskeleton remodeling, and activation of transcription factor, and results in the increased abilities for tumor cell growth, adhesion, angiogenesis, invasion and metastasis. The present article attempts to review the structure and functions of syntenin by summarizing our current knowledge on the interacting partners and diverse signaling pathways related to syntenin, and highlight the importance of syntenin as a new potential therapeutic target for the aggressive human melanoma.

  11. The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes.

    Science.gov (United States)

    Wernig, Gerlinde; Gonneville, Jeffrey R; Crowley, Brian J; Rodrigues, Margret S; Reddy, Mamatha M; Hudon, Heidi E; Walz, Christoph; Reiter, Andreas; Podar, Klaus; Royer, Yohan; Constantinescu, Stefan N; Tomasson, Michael H; Griffin, James D; Gilliland, D Gary; Sattler, Martin

    2008-04-01

    The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.

  12. Control of Transmembrane Protein Diffusion within the Postsynaptic Density Assessed by Simultaneous Single-Molecule Tracking and Localization Microscopy.

    Science.gov (United States)

    Li, Tuo P; Blanpied, Thomas A

    2016-01-01

    Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using universal point accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). The results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins. A protein with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize

  13. Control of Transmembrane Protein Diffusion within the Postsynaptic Density Assessed by Simultaneous Single-Molecule Tracking and Localization Microscopy

    Science.gov (United States)

    Li, Tuo P.; Blanpied, Thomas A.

    2016-01-01

    Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using universal point accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). The results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins. A protein with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize

  14. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    Science.gov (United States)

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

  15. [Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies].

    Science.gov (United States)

    Bocharova, O V; Kuzmichev, P K; Urban, A S; Goncharuk, S A; Bocharov, E V; Arsenyev, A S

    2015-01-01

    Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy. PMID:27125024

  16. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    Science.gov (United States)

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

  17. "Affective contingencies in the affiliative domain: Physiological assessment, associations with the affiliation motive, and prediction of behavior": Correction to Dufner et al. (2015).

    Science.gov (United States)

    2016-06-01

    Reports an error in "Affective contingencies in the affiliative domain: Physiological assessment, associations with the affiliation motive, and prediction of behavior" by Michael Dufner, Ruben C. Arslan, Birk Hagemeyer, Felix D. Schönbrodt and Jaap J. A. Denissen (Journal of Personality and Social Psychology, 2015[Oct], Vol 109[4], 662-676). In this article an erroneous statement was made regarding the high cutoff filter for the EMG raw signal. The high cutoff filter reported in Appendix B in the Technical Details of the EMG Recording Procedure section should be 300 Hz. (The following abstract of the original article appeared in record 2015-37761-001.) According to classical motive disposition theory, individuals differ in their propensity to derive pleasure from affiliative experiences. This propensity is considered a core process underlying the affiliation motive and a pervasive cause of motivated behavior. In this study, we tested these assumptions. We presented participants with positive affiliative stimuli and used electromyography to record changes in facial muscular activity that are indicative of subtle smiling. We were thus able to physiologically measure positive affect following affiliative cues. Individual differences in these affective contingencies were internally consistent and temporally stable. They converged with affiliation motive self- and informant reports and picture story exercise scores, indicating that they are partly accessible to the self, observable to outsiders, and overlap with implicit systems. Finally, they predicted affiliative behavior in terms of situation selection and modification across a wide variety of contexts (i.e., in daily life, the laboratory, and an online social network). These findings corroborate the long-held assumption that affective contingencies represent a motivational core aspect of affiliation. (PsycINFO Database Record PMID:27281355

  18. Effect of cocaine on Fas-associated protein with death domain in the rat brain: individual differences in a model of differential vulnerability to drug abuse.

    Science.gov (United States)

    García-Fuster, María-Julia; Clinton, Sarah M; Watson, Stanley J; Akil, Huda

    2009-04-01

    This study was designed to (1) assess the effects of cocaine on Fas-associated protein with death domain (FADD) system and its role in the activation of apoptotic vs nonapoptotic events and (2) ascertain whether animals selectively bred for their differential propensity to drug-seeking show differences in FADD levels or response to cocaine. Acute cocaine, through D(2) dopamine receptors, induced a dose-response increase in FADD protein in the cortex, with opposite effects over pFADD (Ser191/194), and no induction of apoptotic cell death (poly-(ADP-ribose) polymerase cleavage). FADD was increased by cocaine in cytosol (approximately 142%), membranes (approximately 23%) and nucleus (approximately 54%). The modulation of the FADD system showed tolerance of the acute effect over time, as well as a compensatory response on withdrawal that mirrored the acute effect--ie a transient FADD decrease on day 3 of withdrawal, both at mRNA and protein levels. In a second experiment, possible FADD differences were investigated in rats selectively bred for differential responsiveness to novelty, propensity for drug-seeking and cocaine sensitization. High-responders (HR), who were more prone to drug abuse, exhibited higher FADD and lower pFADD levels than low-responder (LR) rats. However, HR and LR rats showed similar rates of cocaine-induced apoptosis, and exhibited a parallel impact of cocaine over FADD within each phenotype. Thus, FADD is a signaling protein modulated by cocaine, regulating apoptosis/proliferative mechanisms in relation to its FADD/pFADD content. Interestingly, animals selectively bred for differential propensity to substance abuse show basal differences in the expression of this protein, suggesting FADD may also be a molecular correlate for the HR/LR phenotype. PMID:18580876

  19. Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Rat Ovary

    Institute of Scientific and Technical Information of China (English)

    Lei JIN; Ruiling TANG

    2008-01-01

    Summary: The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10U hCG 48h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72h after hCG treatment. The immnnohistochemistry revealed that administration of PMSG stimulated the CFTR expression in theeal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granuiose lutein cell layer and theeal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170kD. It is concluded that cAMP-dependent Cl- channels are involved in regulation of follicle development and luteum formation.

  20. The Cystic Fibrosis Transmembrane Regulator (CFTR in the kidney

    Directory of Open Access Journals (Sweden)

    MORALES MARCELO M.

    2000-01-01

    Full Text Available The cystic fibrosis transmembrane regulator (CFTR is a Cl- channel. Mutations of this transporter lead to a defect of chloride secretion by epithelial cells causing the Cystic Fibrosis disease (CF. In spite of the high expression of CFTR in the kidney, patients with CF do not show major renal dysfunction, but it is known that both the urinary excretion of drugs and the renal capacity to concentrate and dilute urine is deficient. CFTR mRNA is expressed in all nephron segments and its protein is involved with chloride secretion in the distal tubule, and the principal cells of the cortical (CCD and medullary (IMCD collecting ducts. Several studies have demonstrated that CFTR does not only transport Cl- but also secretes ATP and, thus, controls other conductances such as Na+ (ENaC and K+ (ROMK2 channels, especially in CCD. In the polycystic kidney the secretion of chloride through CFTR contributes to the cyst enlargement. This review is focused on the role of CFTR in the kidney and the implications of extracellular volume regulators, such as hormones, on its function and expression.

  1. Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion.

    Science.gov (United States)

    Guo, Yanmeng; Wang, Yuping; Zhang, Wei; Meltzer, Shan; Zanini, Damiano; Yu, Yue; Li, Jiefu; Cheng, Tong; Guo, Zhenhao; Wang, Qingxiu; Jacobs, Julie S; Sharma, Yashoda; Eberl, Daniel F; Göpfert, Martin C; Jan, Lily Yeh; Jan, Yuh Nung; Wang, Zuoren

    2016-06-28

    Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs. PMID:27298354

  2. TOPDOM: database of conservatively located domains and motifs in proteins

    Science.gov (United States)

    Varga, Julia; Dobson, László; Tusnády, Gábor E.

    2016-01-01

    Summary: The TOPDOM database—originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins—has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. Availability and implementation: TOPDOM database is available at http://topdom.enzim.hu. The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. Contact: tusnady.gabor@ttk.mta.hu. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153630

  3. Domains in Ferroelectric Nanostructures

    Science.gov (United States)

    Gregg, Marty

    2010-03-01

    Ferroelectric materials have great potential in influencing the future of small scale electronics. At a basic level, this is because ferroelectric surfaces are charged, and so interact strongly with charge-carrying metals and semiconductors - the building blocks for all electronic systems. Since the electrical polarity of the ferroelectric can be reversed, surfaces can both attract and repel charges in nearby materials, and can thereby exert complete control over both charge distribution and movement. It should be no surprise, therefore, that microelectronics industries have already looked very seriously at harnessing ferroelectric materials in a variety of applications, from solid state memory chips (FeRAMs) to field effect transistors (FeFETs). In all such applications, switching the direction of the polarity of the ferroelectric is a key aspect of functional behavior. The mechanism for switching involves the field-induced nucleation and growth of domains. Domain coarsening, through domain wall propagation, eventually causes the entire ferroelectric to switch its polar direction. It is thus the existence and behavior of domains that determine the switching response, and ultimately the performance of the ferroelectric device. A major issue, associated with the integration of ferroelectrics into microelectronic devices, has been that the fundamental properties associated with ferroelectrics, when in bulk form, appear to change quite dramatically and unpredictably when at the nanoscale: new modes of behaviour, and different functional characteristics from those seen in bulk appear. For domains, in particular, the proximity of surfaces and boundaries have a dramatic effect: surface tension and depolarizing fields both serve to increase the equilibrium density of domains, such that minor changes in scale or morphology can have major ramifications for domain redistribution. Given the importance of domains in dictating the overall switching characteristics of a device

  4. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  5. Structural basis of Smoothened regulation by its extracellular domains

    Science.gov (United States)

    Byrne, Eamon F. X.; Sircar, Ria; Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Newstead, Simon; Rohatgi, Rajat; Siebold, Christian

    2016-07-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

  6. Synergistic Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Gating by Two Chemically Distinct Potentiators, Ivacaftor (VX-770) and 5-Nitro-2-(3-Phenylpropylamino) Benzoate.

    Science.gov (United States)

    Lin, Wen-Ying; Sohma, Yoshiro; Hwang, Tzyh-Chang

    2016-09-01

    Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated but ATP-gated chloride channel. Previous studies suggested that VX-770 [ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide], a CFTR potentiator now used in clinics, increases the open probability of CFTR by shifting the gating conformational changes to favor the open channel configuration. Recently the chloride channel blocker and CFTR potentiator 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) has been reported to enhance CFTR activity by a mechanism that exploits the ATP hydrolysis-driven, nonequilibrium gating mechanism unique to CFTR. Surprisingly however, NPPB increased the activity of nonhydrolytic G551D-CFTR, the third most common disease-associated mutation. Here, we further investigated the mechanism of NPPB's effects on CFTR gating by assessing its interaction with well-studied VX-770. Interestingly, once G551D-CFTR was maximally potentiated by VX-770, NPPB further increased its activity. However, quantitative analysis of this drug-drug interaction suggests that this pharmacologic synergism is not due to independent actions of NPPB and VX-770 on CFTR gating; instead, our data support a dependent mechanism involving two distinct binding sites. This latter idea is further supported by the observation that the locked-open time of a hydrolysis-deficient mutant K1250A was shortened by NPPB but prolonged by VX-770. In addition, the effectiveness of NPPB, but not of VX-770, was greatly diminished in a mutant whose second nucleotide-binding domain was completely removed. Interpreting these results under the framework of current understanding of CFTR gating not only reveals insights into the mechanism of action for different CFTR potentiators but also brings us one step forward to a more complete schematic for CFTR gating. PMID:27413118

  7. Chemical synthesis of transmembrane peptide and its application for research on the transmembrane-juxtamembrane region of membrane protein.

    Science.gov (United States)

    Sato, Takeshi

    2016-11-01

    Membrane proteins possess one or more hydrophobic regions that span the membrane and interact with the lipids that constitute the membrane. The interactions between the transmembrane (TM) region and lipids affect the structure and function of these membrane proteins. Molecular characterization of synthetic TM peptides in lipid bilayers helps to understand how the TM region participates in the formation of the structure and in the function of membrane proteins. The use of synthetic peptides enables site-specific labeling and modification and allows for designing of an artificial TM sequence. Research involving such samples has resulted in significant increase in the knowledge of the mechanisms that govern membrane biology. In this review, the chemical synthesis of TM peptides has been discussed. The preparation of synthetic TM peptides is still not trivial; however, the accumulated knowledge summarized here should provide a basis for preparing samples for spectroscopic analyses. The application of synthetic TM peptides for gaining insights into the mechanism of signal transduction by receptor tyrosine kinase (RTK) has also been discussed. RTK is a single TM protein and is one of the difficult targets in structural biology as crystallization of the full-length receptor has not been successful. This review describes the structural characterization of the synthetic TM-juxtamembrane sequence and proposes a possible scheme for the structural changes in this region for the activation of ErbBs, the epidermal growth factor receptor family. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 613-621, 2016. PMID:26573237

  8. EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system

    Science.gov (United States)

    Zoltner, Martin; Ng, Wui M.A.V.; Money, Jillian J.; Fyfe, Paul K.; Kneuper, Holger; Palmer, Tracy; Hunter, William N.

    2016-01-01

    The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein. PMID:27130157

  9. EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system.

    Science.gov (United States)

    Zoltner, Martin; Ng, Wui M A V; Money, Jillian J; Fyfe, Paul K; Kneuper, Holger; Palmer, Tracy; Hunter, William N

    2016-07-01

    The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1-D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.

  10. Transmembrane electric potential difference in the protein-pigment complex of photosystem 2.

    Science.gov (United States)

    Mamedov, M D; Kurashov, V N; Petrova, I O; Semenov, A Yu

    2012-09-01

    The protein-pigment complex of photosystem 2 (PS2) localized in the thylakoid membranes of higher plants, algae, and cyanobacteria is the main source of oxygen on Earth. The light-induced functioning of PS2 is directly linked to electron and proton transfer across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). The major contribution to ΔΨ of the PS2 reaction center is due to charge separation between the primary chlorophyll donor P(680) and the quinone acceptor Q(A), accompanied by re-reduction of P(680)(+) by the redox-active tyrosine residue Y(Z). The processes associated with the uptake and release of protons on the acceptor and donor sides of the enzyme, respectively, are also coupled with ΔΨ generation. The objective of this work was to describe the mechanisms of ΔΨ generation associated with the S-state transitions of the water-oxidizing complex in intact PS2 complex and in PS2 preparation depleted of Mn(4)Ca cluster in the presence of artificial electron donors. The findings elucidate the mechanisms of electrogenic reactions on the PS2 donor side and may be a basis for development of an effective solar energy conversion system. PMID:23157254

  11. [Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel].

    Science.gov (United States)

    Cao, Huan-Huan; Fang, Fang; Yu, Bo; Luan, Jian; Jiang, Yu; Yang, Hong

    2015-04-25

    Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation. PMID:25896054

  12. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

    Directory of Open Access Journals (Sweden)

    Thomas Hoffmann

    2015-07-01

    Full Text Available T-cell receptors (TCR play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR, which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

  13. Conformational dynamics of cancer-associated MyD88-TIR domain mutant L252P (L265P) allosterically tilts the landscape toward homo-dimerization.

    Science.gov (United States)

    Zhan, Chendi; Qi, Ruxi; Wei, Guanghong; Guven-Maiorov, Emine; Nussinov, Ruth; Ma, Buyong

    2016-09-01

    MyD88 is an essential adaptor protein, which mediates the signaling of the toll-like and interleukin-1 receptors' superfamily. The MyD88 L252P (L265P) mutation has been identified in diffuse large B-cell lymphoma. The identification of this mutation has been a major advance in the diagnosis of patients with aldenstrom macroglobulinemia and related lymphoid neoplasms. Here we used computational methods to characterize the conformational effects of the mutation. Our molecular dynamics simulations revealed that the mutation allosterically quenched the global conformational dynamics of the toll/IL-1R (TIR) domain, and readjusted its salt bridges and dynamic community network. Specifically, the mutation changed the orientation and reduced the fluctuation of α-helix 3, possibly through eliminating/weakening ~8 salt bridges and enhancing the salt bridge D225-K258. Using the energy landscape of the TIR domains of MyD88, we identified two dynamic conformational basins, which correspond to the binding sites used in homo- and hetero-oligomerization, respectively. Our results indicate that the mutation stabilizes the core of the homo-dimer interface of the MyD88-TIR domain, and increases the population of homo-dimer-compatible conformational states in MyD88 family proteins. However, the dampened motion restricts its ability to heterodimerize with other TIR domains, thereby curtailing physiological signaling. In conclusion, the L252P both shifts the landscape toward homo-dimerization and restrains the dynamics of the MyD88-TIR domain, which disfavors its hetero-dimerization with other TIR domains. We further put these observations within the framework of MyD88-mediated cell signaling. PMID:27503954

  14. Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

    Science.gov (United States)

    Barber-Zucker, Shiran; Uebe, René; Davidov, Geula; Navon, Yotam; Sherf, Dror; Chill, Jordan H; Kass, Itamar; Bitton, Ronit; Schüler, Dirk; Zarivach, Raz

    2016-08-23

    Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins' proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.

  15. Free energy barrier for melittin reorientation from a membrane-bound state to a transmembrane state

    OpenAIRE

    Irudayam, Sheeba J.; Pobandt, Tobias; Berkowitz, Max L.

    2013-01-01

    An important step in a phospholipid membrane pore formation by melittin antimicrobial peptide is a reorientation of the peptide from a surface into a transmembrane conformation. In this work we perform umbrella sampling simulations to calculate the potential of mean force (PMF) for the reorientation of melittin from a surface-bound state to a transmembrane state and provide a molecular level insight into understanding peptide and lipid properties that influence the existence of the free energ...

  16. Transmembrane Protein Diffusion in Gel-Supported Dual-Leaflet Membranes

    OpenAIRE

    Wang, Chih-Ying; Hill, Reghan J.

    2014-01-01

    Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed per...

  17. TMRPres2D: high quality visual representation of transmembrane protein models.

    Science.gov (United States)

    Spyropoulos, Ioannis C; Liakopoulos, Theodore D; Bagos, Pantelis G; Hamodrakas, Stavros J

    2004-11-22

    The 'TransMembrane protein Re-Presentation in 2-Dimensions' (TMRPres2D) tool, automates the creation of uniform, two-dimensional, high analysis graphical images/models of alpha-helical or beta-barrel transmembrane proteins. Protein sequence data and structural information may be acquired from public protein knowledge bases, emanate from prediction algorithms, or even be defined by the user. Several important biological and physical sequence attributes can be embedded in the graphical representation. PMID:15201184

  18. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

    OpenAIRE

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A.; Kappes, John C.; DeLucas, Lawrence J.; Riordan, John R.; Urbatsch, Ina L.; Hunt, John F.; Brouillette, Christie G.

    2014-01-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Not...

  19. Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia

    DEFF Research Database (Denmark)

    Mitchelmore, Cathy; Kjaerulff, Karen M; Pedersen, Hans C;

    2002-01-01

    BTB/POZ (broad complex tramtrack bric-a-brac/poxvirus and zinc finger) zinc finger factors are a class of nuclear DNA-binding proteins involved in development, chromatin remodeling, and cancer. However, BTB/POZ domain zinc finger factors linked to development of the mammalian cerebral cortex......, cerebellum, and macroglia have not been described previously. We report here the isolation and characterization of two novel nuclear BTB/POZ domain zinc finger isoforms, designated HOF(L) and HOF(S), that are specifically expressed in early hippocampal neurons, cerebellar granule cells, and gliogenic...

  20. Protein domain organisation: adding order

    Directory of Open Access Journals (Sweden)

    Kummerfeld Sarah K

    2009-01-01

    Full Text Available Abstract Background Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. Results We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Conclusion Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected

  1. Natural variation of the amino-terminal glutamine-rich domain in Drosophila argonaute2 is not associated with developmental defects.

    Directory of Open Access Journals (Sweden)

    Daniel Hain

    Full Text Available The Drosophila argonaute2 (ago2 gene plays a major role in siRNA mediated RNA silencing pathways. Unlike mammalian Argonaute proteins, the Drosophila protein has an unusual amino-terminal domain made up largely of multiple copies of glutamine-rich repeats (GRRs. We report here that the ago2 locus produces an alternative transcript that encodes a putative short isoform without this amino-terminal domain. Several ago2 mutations previously reported to be null alleles only abolish expression of the long, GRR-containing isoform. Analysis of drop out (dop mutations had previously suggested that variations in GRR copy number result in defects in RNAi and embryonic development. However, we find that dop mutations genetically complement transcript-null alleles of ago2 and that ago2 alleles with variant GRR copy numbers support normal development. In addition, we show that the assembly of the central RNAi machinery, the RISC (RNA induced silencing complex, is unimpaired in embryos when GRR copy number is altered. In fact, we find that GRR copy number is highly variable in natural D. melanogaster populations as well as in laboratory strains. Finally, while many other insects share an extensive, glutamine-rich Ago2 amino-terminal domain, its primary sequence varies drastically between species. Our data indicate that GRR variation does not modulate an essential function of Ago2 and that the amino-terminal domain of Ago2 is subject to rapid evolution.

  2. BCR-ABL isoforms associated with intrinsic or acquired resistance to imatinib : more heterogeneous than just ABL kinase domain point mutations?

    NARCIS (Netherlands)

    Gruber, Franz X.; Lundan, Tuija; Goll, Rasmus; Silye, Aleksandra; Mikkola, Ingvild; Rekvig, Ole Petter; Knuutila, Sakari; Remes, Kari; Gedde-Dahl, Tobias; Porkka, Kimmo; Hjorth-Hansen, Henrik

    2012-01-01

    Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mu

  3. The Chinese Version of the Revised Creativity Domain Questionnaire (CDQ-R): First Evidence for Its Factorial Validity and Systematic Association with the Big Five

    Science.gov (United States)

    Werner, Christian H.; Tang, Min; Kruse, Joachim; Kaufman, James C.; Spörrle, Matthias

    2014-01-01

    The present study examines the factor structure of a Chinese version of the Revised Creativity Domain Questionnaire (CDQ-R; Kaufman, Waterstreet, Ailaouni, Whitcomb, Roe, & Riggs, 2009) as well as its relation to Big Five personality traits within a Chinese sample (N = 787). Analyses indicate the appropriateness of the Chinese version of the…

  4. Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization.

    Directory of Open Access Journals (Sweden)

    Céline Lafourcade

    Full Text Available The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase, a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0 and the cytoplasmic V(1. Here we found that the ratio of membrane associated V(1/Vo varies along the endocytic pathway, the relative abundance of V(1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.

  5. Towards the rational design of a candidate vaccine against pregnancy associated malaria: conserved sequences of the DBL6epsilon domain of VAR2CSA.

    Directory of Open Access Journals (Sweden)

    Cyril Badaut

    Full Text Available BACKGROUND: Placental malaria is a disease linked to the sequestration of Plasmodium falciparum infected red blood cells (IRBC in the placenta, leading to reduced materno-fetal exchanges and to local inflammation. One of the virulence factors of P. falciparum involved in cytoadherence to chondroitin sulfate A, its placental receptor, is the adhesive protein VAR2CSA. Its localisation on the surface of IRBC makes it accessible to the immune system. VAR2CSA contains six DBL domains. The DBL6epsilon domain is the most variable. High variability constitutes a means for the parasite to evade the host immune response. The DBL6epsilon domain could constitute a very attractive basis for a vaccine candidate but its reported variability necessitates, for antigenic characterisations, identifying and classifying commonalities across isolates. METHODOLOGY/PRINCIPAL FINDINGS: Local alignment analysis of the DBL6epsilon domain had revealed that it is not as variable as previously described. Variability is concentrated in seven regions present on the surface of the DBL6epsilon domain. The main goal of our work is to classify and group variable sequences that will simplify further research to determine dominant epitopes. Firstly, variable sequences were grouped following their average percent pairwise identity (APPI. Groups comprising many variable sequences sharing low variability were found. Secondly, ELISA experiments following the IgG recognition of a recombinant DBL6epsilon domain, and of peptides mimicking its seven variable blocks, allowed to determine an APPI cut-off and to isolate groups represented by a single consensus sequence. CONCLUSIONS/SIGNIFICANCE: A new sequence approach is used to compare variable regions in sequences that have extensive segmental gene relationship. Using this approach, the VAR2CSA DBL6 domain is composed of 7 variable blocks with limited polymorphism. Each variable block is composed of a limited number of consensus types

  6. Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove

    Energy Technology Data Exchange (ETDEWEB)

    Ivanova, Marina E.; Fletcher, Georgina C.; O’Reilly, Nicola; Purkiss, Andrew G.; Thompson, Barry J. [Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY (United Kingdom); McDonald, Neil Q., E-mail: neil.mcdonald@cancer.org.uk [Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY (United Kingdom); Birkbeck College, University of London, Malet Street, London WC1E 7HX (United Kingdom)

    2015-03-01

    This study characterizes the interaction between the carboxy-terminal (ERLI) motif of the essential polarity protein Crb and the Pals1/Stardust PDZ-domain protein. Structures of human Pals1 PDZ with and without a Crb peptide are described, explaining the highly conserved nature of the ERLI motif and revealing a sterically blocked peptide-binding groove in the absence of ligand. Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein–protein interaction.

  7. Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle.

    Science.gov (United States)

    Joseph, Benesh; Jeschke, Gunnar; Goetz, Birke A; Locher, Kaspar P; Bordignon, Enrica

    2011-11-25

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

  8. Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus.

    Science.gov (United States)

    Acharya, Rudresh; Carnevale, Vincenzo; Fiorin, Giacomo; Levine, Benjamin G; Polishchuk, Alexei L; Balannik, Victoria; Samish, Ilan; Lamb, Robert A; Pinto, Lawrence H; DeGrado, William F; Klein, Michael L

    2010-08-24

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2(+) and 3(+) with a pK(a) near 6. A 1.65 A resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  9. Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

    Directory of Open Access Journals (Sweden)

    Heidi O Nousiainen

    Full Text Available Prostatic acid phosphatase (PAP, the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG, but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP in the brain by utilizing mice deficient in TMPAP (PAP-/- mice. Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

  10. Transmembrane prostatic acid phosphatase (TMPAP interacts with snapin and deficient mice develop prostate adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ileana B Quintero

    Full Text Available The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP, a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP and a transmembrane type-I (TMPAP. The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/- with C57BL/6J background. The PAP(-/- mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

  11. Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

    Directory of Open Access Journals (Sweden)

    Stefanie Löffek

    Full Text Available The hemidesmosomal transmembrane component collagen XVII (ColXVII plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

  12. A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

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    Christine Dorothee Schmeitz

    2013-09-01

    Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

  13. Functional Interactions of HCO3- with Cystic Fibrosis Transmembrane Conductance Regulator

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    Gray MA

    2001-07-01

    Full Text Available Disruption of normal cystic fibrosis transmembrane conductance regulator- (CFTR-mediated Cl(- transport is associated with cystic fibrosis (CF. CFTR is also required for HCO(3(- transport in many tissues such as the lungs, gastro-intestinal tract, and pancreas, although the exact role CFTR plays is uncertain. Given the importance of CFTR in HCO(3(- transport by so many CF-affected organ systems, it is perhaps surprising that relatively little is known about the interactions of HCO(3(- ions with CFTR. We have used patch clamp recordings from native pancreatic duct cells to study HCO(3(- permeation and interaction with CFTR. Ion selectivity studies shows that CFTR is between 3-5 times more selective for Cl(- over HCO(3(-. In addition, extracellular HCO(3(- has a novel inhibitory effect on cAMP-stimulated CFTR currents carried by Cl(-. The block by HCO(3(- was rapid, relatively independent of voltage and occurred over the physiological range of HCO(3(- concentrations. These data show that luminal HCO(3(- acts as a potent regulator of CFTR, and suggests that inhibition involves an external anion-binding site on the channel. This work has implications not only for elucidating mechanisms of HCO(3(- transport in epithelia, but also for approaches used to treat CF.

  14. MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes.

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    Helena Bujalka

    Full Text Available The myelination of axons is a crucial step during vertebrate central nervous system (CNS development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf, as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

  15. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    Science.gov (United States)

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

  16. Somatic mutations, allele loss, and DNA methylation of the Cub and Sushi Multiple Domains 1 (CSMD1 gene reveals association with early age of diagnosis in colorectal cancer patients.

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    Austin Y Shull

    Full Text Available BACKGROUND: The Cub and Sushi Multiple Domains 1 (CSMD1 gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients. METHODS: We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A. RESULTS: Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%. The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15:1, a ratio significantly higher than the expected 2:1 ratio (p = 0.014. This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%. Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years than those without somatic mutations (average age, 68 years. The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%, suggesting extensive cytosine methylation predisposing to somatic mutations. CONCLUSIONS: Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1

  17. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Knaap, E. van der; Sauter, M.; Kende, H. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. (Univ. of California, Davis, CA (United States). Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  18. CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain

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    Mariano A. Ostuni

    2014-11-01

    Full Text Available The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain, and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.

  19. Origin of the Diversity in DNA Recognition Domains in Phasevarion Associated modA Genes of Pathogenic Neisseria and Haemophilus influenzae

    OpenAIRE

    Gawthorne, Jayde A.; Beatson, Scott A.; Srikhanta, Yogitha N.; Fox, Kate L.; Jennings, Michael P.

    2012-01-01

    Phase variable restriction-modification (R-M) systems have been identified in a range of pathogenic bacteria. In some it has been demonstrated that the random switching of the mod (DNA methyltransferase) gene mediates the coordinated expression of multiple genes and constitutes a phasevarion (phase variable regulon). ModA of Neisseria and Haemophilus influenzae contain a highly variable, DNA recognition domain (DRD) that defines the target sequence that is modified by methylation and is used ...

  20. Association of Academic Performance with Outcome Expectations and Its Domains in Nursing and Midwifery Students at Kermanshah University of Medical Sciences

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    Sepideh Bakhtiari

    2014-09-01

    Full Text Available Introduction: Outcome expectation is considered as a basic and significant variable in education. It is a cognitive-motivational component that takes the individual into account as an active and sensible decision-maker. The present study was conducted to investigate the correlation of outcome expectations with academic performance of students of nursing and midwifery in Kermanshah University of Medical Sciences. Methods: In this descriptive cross-sectional study, the sample size included 218 nursing and midwifery students selected through convenient random sampling method. The instrument for data collection was the questionnaire of “outcome expectations of career decision-making and discovery targets”, which comprised of 13 questions in three domains of future orientation, job satisfaction and personal expectations. The questionnaires were coded after being completed and the obtained data were fed into SPSS-16 software and analyzed by descriptive statistics, t-test, Kolmogrov-Smirnov, ANOVA and Mann-Whitney tests. Results: The findings indicated no statistically significant difference between place of living (dormitory or home and outcome expectations along with its domains (39.4% and 60-6%. However, a significant correlation was reported between discipline, gender, admittance year and academic performance of the students (p0.05. Conclusion: The findings of this study indicated a positively positive significant relationship between students’ academic performance and outcome expectations along with its domains.

  1. Bisociative Discovery of Interesting Relations between Domains

    OpenAIRE

    Nagel, Uwe; Thiel, Kilian; Kötter, Tobias; Piatek, Dawid; Berthold, Michael

    2011-01-01

    The discovery of surprising relations in large, heterogeneous information repositories is gaining increasing importance in real world data analysis. If these repositories come from diverse origins, forming different domains, domain bridging associations between otherwise weakly connected domains can provide insights into the data that can otherwise not be accomplished. In this paper, we propose a first formalization for the detection of such potentially interesting, domain-crossing relations ...

  2. Hydrophobic compounds reshape membrane domains.

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    Jonathan Barnoud

    2014-10-01

    Full Text Available Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo.

  3. The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance.

    Science.gov (United States)

    Kwon, Byungsu; Hong, Mei

    2016-09-27

    The influenza M2 protein is the target of the amantadine family of antiviral drugs, and its transmembrane (TM) domain structure and dynamics have been extensively studied. However, little is known about the structure of the highly conserved N-terminal ectodomain, which contains epitopes targeted by influenza vaccines. In this study, we synthesized an M2 construct containing the N-terminal ectodomain and the TM domain, to understand the site-specific conformation and dynamics of the ectodomain and to investigate the effect of the ectodomain on the TM structure. We incorporated (13)C- and (15)N-labeled residues into both domains and measured their chemical shifts and line widths using solid-state nuclear magnetic resonance. The data indicate that the entire ectodomain is unstructured and dynamic, but the motion is slower for residues closer to the TM domain. (13)C line shapes indicate that this ecto-TM construct undergoes fast uniaxial rotational diffusion, like the isolated TM peptide, but drug binding increases the motional rates of the TM helix while slowing the local motion of the ectodomain residues that are close to the TM domain. Moreover, (13)C and (15)N chemical shifts indicate that the ectodomain shifts the conformational equilibria of the TM residues toward the drug-bound state even in the absence of amantadine, thus providing a molecular structural basis for the lower inhibitory concentration of full-length M2 compared to that of the ectodomain-truncated M2. We propose that this conformational selection may result from electrostatic repulsion between negatively charged ectodomain residues in the tetrameric protein. Together with the recent study of the M2 cytoplasmic domain, these results show that intrinsically disordered extramembrane domains in membrane proteins can regulate the functionally relevant conformation and dynamics of the structurally ordered TM domains.

  4. Substrate-induced changes in domain interaction of vacuolar H⁺-pyrophosphatase.

    Science.gov (United States)

    Hsu, Shen-Hsing; Lo, Yueh-Yu; Liu, Tseng-Huang; Pan, Yih-Jiuan; Huang, Yun-Tzu; Sun, Yuh-Ju; Hung, Cheng-Chieh; Tseng, Fan-Gang; Yang, Chih-Wei; Pan, Rong-Long

    2015-01-01

    Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog. PMID:25451931

  5. Thermal unfolding simulations of NBD1 domain variants reveal structural motifs associated with the impaired folding of F508del-CFTR.

    Science.gov (United States)

    Estácio, Sílvia G; Martiniano, Hugo F M C; Faísca, Patrícia F N

    2016-08-16

    We employed high-temperature classical molecular dynamics (MD) simulations to investigate the unfolding process of the wild-type (WT) and F508del-NBD1 domains of CFTR protein, with and without second-site mutations. To rationalize the in vitro behavior of F508del-NBD1, namely its lower folding yield and higher aggregation propensity, we focused our analysis of the MD data on the existence of intermediate states with aggregation potential and/or stabilized by a significant number of non-native interactions (i.e. misfolded states). We find that the deletion of phenylalanine 508 is able to forcefully reshape the conformational space of the NBD1 domain to the extent that it uniquely populates intermediate states whose structural traits provide important insights into the molecular events that underlie the impaired folding of F508del-NBD1. In particular, our simulations predict the formation of a misfolded intermediate whose population is highly enhanced by deletion of residue 508. The stabilization of this intermediate is a direct consequence of the enhanced non-native coupling between various key regions of the α-helical subdomain and ATP-binding subdomain; it is singularly characterized by a set of non-native interactions within the ATP-binding subdomain and between that domain and the α-helical subdomain region. The formation of this intermediate is not blocked by second-site suppressor mutations which indicates a limited role of the latter in correcting the rather complex folding process of the CFTR protein missing residue 508. PMID:27354240

  6. Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Antoinette M Dummer

    2016-06-01

    Full Text Available The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s, but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and

  7. Molecular Characterization of an Ice Nucleation Protein Variant (InaQ from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli

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    Qianqian Li, Qi Yan, Jinsi Chen, Yan He, Jing Wang, Hongxing Zhang, Ziniu Yu, Lin Li

    2012-01-01

    Full Text Available The ice nucleation protein (INP of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l but not by culture temperatures (15 ºC to 37 ºC. Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated

  8. Docking ellipticine to the V-VI transmembrane domain of the Kv11.1 potassium channel

    Science.gov (United States)

    Lipscomb, Dawn; Brancaleon, Lorenzo; Gentile, S.

    2011-03-01

    Ellipticines such as 9-methoxy-N-2-methylellipticinium acetate (MMEA) and 9-hydroxy-N-2-methylellipticinium acetate (NMEA, Celiptium ) are antineoplastic drugs exerting their selective cytotoxicity against leukemia and endometrial carcinoma. Ellipticine's action is also related to severe physical side effects, but the link between undesired effects and pharmacological application is not well understood. We investigated the binding of Ellipticine derivatives with the Kv11.1 potassium ion channel using Autodock and revealed that hydroxyellipticinium derivatives provide binding configurations with Kv11.1, but the energy, location and estimated dissociation constant varied. The binding energy is as follows: Chloroceliptium (-6.60 kcal/mol) Celiptium (- 6.37 kcal / mol) > Methoxyceliptium (- 6.20 kcal / mol) Datelliptium (-6.08 kcal/mol). The data shows that some configurations enable these molecules to bridge among channel subunits, thus potentially inhibiting the flow of ions.

  9. Vaccination with Recombinant Non-transmembrane Domain of Protein Mannosyltransferase 4 Improves Survival during Murine Disseminated Candidiasis.

    Science.gov (United States)

    Wang, Li; Yan, Lan; Li, Xing Xing; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2015-01-01

    Candida albicans is the most common cause of invasive fungal infections in humans. The C. albicans cell wall proteins play an important role in crucial host-fungus interactions and might be ideal vaccine targets to induce protective immune response in host. Meanwhile, protein that is specific to C. albicans is also an ideal target of vaccine. In this study, 11 proteins involving cell wall biosynthesis, yeast-to-hypha formation, or specific to C. albicans were chosen and were successfully cloned, purified and verified. The immune protection of vaccination with each recombinant protein respectively in preventing systemic candidiasis in BALB/c mice was assessed. The injection of rPmt4p vaccination significantly increased survival rate, decreased fungal burdens in the heart, liver, brain, and kidneys, and increased serum levels of both immunoglobulin G (IgG) and IgM against rPmt4p in the immunized mice. Histopathological assessment demonstrated that rPmt4p vaccination protected the tissue structure, and decreased the infiltration of inflammatory cells. Passive transfer of the rPmt4p immunized serum increased survival rate against murine systemic candidiasis and significantly reduced organ fungal burden. The immune serum enhanced mouse neutrophil killing activity by directly neutralizing rPmt4p effects in vitro. Levels of interleukin (IL)-4, IL-10, IL-12p70, IL-17A and tumor necrosis factor (TNF)-α in serum were higher in the immunized mice compared to those in the adjuvant control group. In conclusion, our results suggested that rPmt4p vaccination may be considered as a potential vaccine candidate against systemic candidiasis.

  10. Novel mutation in the ligand-binding domain of the androgen receptor gene (1790p) associated with complete androgen insensitivity syndrome

    Institute of Scientific and Technical Information of China (English)

    Florina Raicu; Rossella Giuliani; Valentina Gatta; Chiara Palka; Paolo Guanciali Franchi; Pierluigi Lelli-Chiesa; Stefano Tumini; Liborio Stuppia

    2008-01-01

    Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators. (Asian J Androl 2008 Jul; 10: 687-691)

  11. Loss of Expression of Human Spectrin Src Homology Domain Binding Protein 1 is Associated with 10p Loss in Human Prostatic Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Jill A. Macoska

    2001-01-01

    Full Text Available The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bpl, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bpl. These experiments demonstrated that 4/6 tumors (67% with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46% tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

  12. TMM@: a web application for the analysis of transmembrane helix mobility

    Directory of Open Access Journals (Sweden)

    Jonassen Inge

    2007-07-01

    Full Text Available Abstract Background To understand the mechanism by which a protein transmits a signal through the cell membrane, an understanding of the flexibility of its transmembrane (TM region is essential. Normal Mode Analysis (NMA has become the method of choice to investigate the slowest motions in macromolecular systems. It has been widely used to study transmembrane channels and pumps. It relies on the hypothesis that the vibrational normal modes having the lowest frequencies (also named soft modes describe the largest movements in a protein and are the ones that are functionally relevant. In particular NMA can be used to study dynamics of TM regions, but no tool making this approach available for non-experts, has been available so far. Results We developed the web-application TMM@ (TransMembrane α-helical Mobility analyzer. It uses NMA to characterize the propensity of transmembrane α-helices to be displaced. Starting from a structure file at the PDB format, the server computes the normal modes of the protein and identifies which helices in the bundle are the most mobile. Each analysis is performed independently from the others and results can be visualized using only a web browser. No additional plug-in or software is required. For users who would like to further analyze the output data with their favourite software, raw results can also be downloaded. Conclusion We built a novel and unique tool, TMM@, to study the mobility of transmembrane α-helices. The tool can be applied to for example membrane transporters and provides biologists studying transmembrane proteins with an approach to investigate which α-helices are likely to undergo the largest displacements, and hence which helices are most likely to be involved in the transportation of molecules in and out of the cell.

  13. Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

    Science.gov (United States)

    Jeřábek, Petr; Florián, Jan; Martínek, Václav

    2016-04-18

    Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex. PMID:26918755

  14. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Silvennoinen, O;

    1994-01-01

    Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not...

  15. Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

    DEFF Research Database (Denmark)

    Woods, A; Longley, R L; Tumova, S;

    2000-01-01

    fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component...

  16. The N-terminal extension domain of the C. elegans half-molecule ABC transporter, HMT-1, is required for protein-protein interactions and function.

    Directory of Open Access Journals (Sweden)

    Sungjin Kim

    Full Text Available BACKGROUND: Members of the HMT-1 (heavy metal tolerance factor 1 subfamily of the ATP-binding cassette (ABC transporter superfamily detoxify heavy metals and have unique topology: they are half-molecule ABC transporters that, in addition to a single transmembrane domain (TMD1 and a single nucleotide-binding domain (NBD1, possess a hydrophobic NH2-terminal extension (NTE. These structural features distinguish HMTs from other ABC transporters in different species including Drosophila and humans. Functional ABC transporters, however, are comprised of at least four-domains (two TMDs and two NDBs formed from either a single polypeptide or by the association of two or four separate subunits. Whether HMTs act as oligomers and what role the NTE domain plays in their function have not been determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the oligomeric status of Caenorhabditis elegans HMT-1 and the functional significance of its NTE using gel-filtration chromatography in combination with the mating-based split-ubiquitin yeast two-hybrid system (mbSUS and functional in vivo assays. We found that HMT-1 exists in a protein complex in C. elegans. Studies in S. cerevisiae showed that HMT-1 at a minimum homodimerizes and that oligomerization is essential for HMT-1 to confer cadmium tolerance. We also established that the NTE domain plays an important structural and functional role: it is essential for HMT-1 oligomerization and Cd-detoxification function. However, the NTE itself was not sufficient for oligomerization suggesting that multiple structural features of HMT-1 must associate to form a functional transporter. CONCLUSIONS: The prominence of heavy metals as environmental toxins and the remarkable conservation of HMT-1 structural architecture and function in different species reinforce the value of continued studies of HMT-1 in model systems for identifying functional domains in HMT1 of humans.

  17. Predicting domain-domain interaction based on domain profiles with feature selection and support vector machines

    Directory of Open Access Journals (Sweden)

    Liao Li

    2010-10-01

    Full Text Available Abstract Background Protein-protein interaction (PPI plays essential roles in cellular functions. The cost, time and other limitations associated with the current experimental methods have motivated the development of computational methods for predicting PPIs. As protein interactions generally occur via domains instead of the whole molecules, predicting domain-domain interaction (DDI is an important step toward PPI prediction. Computational methods developed so far have utilized information from various sources at different levels, from primary sequences, to molecular structures, to evolutionary profiles. Results In this paper, we propose a computational method to predict DDI using support vector machines (SVMs, based on domains represented as interaction profile hidden Markov models (ipHMM where interacting residues in domains are explicitly modeled according to the three dimensional structural information available at the Protein Data Bank (PDB. Features about the domains are extracted first as the Fisher scores derived from the ipHMM and then selected using singular value decomposition (SVD. Domain pairs are represented by concatenating their selected feature vectors, and classified by a support vector machine trained on these feature vectors. The method is tested by leave-one-out cross validation experiments with a set of interacting protein pairs adopted from the 3DID database. The prediction accuracy has shown significant improvement as compared to InterPreTS (Interaction Prediction through Tertiary Structure, an existing method for PPI prediction that also uses the sequences and complexes of known 3D structure. Conclusions We show that domain-domain interaction prediction can be significantly enhanced by exploiting information inherent in the domain profiles via feature selection based on Fisher scores, singular value decomposition and supervised learning based on support vector machines. Datasets and source code are freely available on

  18. The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

    Science.gov (United States)

    Knight, Jonathan D.; Li, Rong; Botchan, Michael

    1991-04-01

    The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

  19. Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae.

    Science.gov (United States)

    Yamamoto, Shouji; Mitobe, Jiro; Ishikawa, Takahiko; Wai, Sun Nyunt; Ohnishi, Makoto; Watanabe, Haruo; Izumiya, Hidemasa

    2014-01-01

    In Vibrio cholerae, 41 chitin-inducible genes, including the genes involved in natural competence for DNA uptake, are governed by the orphan two-component system (TCS) sensor kinase ChiS. However, the mechanism by which ChiS controls the expression of these genes is currently unknown. Here, we report the involvement of a novel transcription factor termed 'TfoS' in this process. TfoS is a transmembrane protein that contains a large periplasmic domain and a cytoplasmic AraC-type DNA-binding domain, but lacks TCS signature domains. Inactivation of tfoS abolished natural competence as well as transcription of the tfoR gene encoding a chitin-induced small RNA essential for competence gene expression. A TfoS fragment containing the DNA-binding domain specifically bound to and activated transcription from the tfoR promoter. Intracellular TfoS levels were unaffected by disruption of chiS and coexpression of TfoS and ChiS in Escherichia coli recovered transcription of the chromosomally integrated tfoR::lacZ gene, suggesting that TfoS is post-translationally modulated by ChiS during transcriptional activation; however, this regulation persisted when the canonical phosphorelay residues of ChiS were mutated. The results presented here suggest that ChiS operates a chitin-induced non-canonical signal transduction cascade through TfoS, leading to transcriptional activation of tfoR. PMID:24236404

  20. High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

    Science.gov (United States)

    Prasad, Joni M; Young, Patricia A; Strickland, Dudley K

    2016-08-26

    The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor. PMID:27402839

  1. Regulation of cytoskeletal organization by syndecan transmembrane proteoglycans

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Couchman, John R

    2003-01-01

    have recently suggested that signaling through core protein of syndecans can regulate cytoskeletal organization through their clustering, association with cytoskeletal structures, binding to cytoplasmic binding proteins, and intracellular phosphorylation. Here we will review current understanding...... of signaling through syndecans in cytoskeletal organization....

  2. Structural basis of typhoid: Salmonella typhi type IVb pilin (PiLS) and cystic fibrosis transmembrane conductance regulator interaction

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishna, A.M.; Saxena, A.; Mok, H. Y.-K.; Swaminathan, K.

    2009-11-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein ({Delta}PilS), which makes the pilus, was determined at 1.9 {angstrom} resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of {Delta}PilS and a target CFTR peptide, determined at 1.8 {angstrom}, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  3. Structural Basis of Typhoid: Salmonella typhi Type IVb pilin (PilS) and Cystic Fibrosis Transmembrane Conductance Regulatory Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

    2009-01-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  4. Structural basis of typhod: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

    2009-01-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  5. Modeling a dehalogenase fold into the 8-A density map for Ca(2+)-ATPase defines a new domain structure.

    OpenAIRE

    Stokes, D.L.; Green, N M

    2000-01-01

    Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extende...

  6. Mutational Analysis of Intracellular Loops Identify Cross Talk with Nucleotide Binding Domains of Yeast ABC Transporter Cdr1p

    OpenAIRE

    Shah, Abdul Haseeb; Rawal, Manpreet Kaur; Dhamgaye, Sanjiveeni; Komath, Sneha Sudha; Saxena, Ajay Kumar; Prasad, Rajendra

    2015-01-01

    The ABC transporter Cdr1 protein (Cdr1p) of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that are interconnected by extracellular (ECLs) and intracellular (ICLs) loops. To examine the communication interface between the NBDs and ICLs of Cdr1p, we subjected all four ICLs to alanine scanning mutagenesis, replacing each of the 85 residues with an alanine. The resulting ICL mutant library was an...

  7. Lack of gender-specific antibody recognition of products from domains of a var gene implicated in pregnancy-associated Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Jensen, Anja T R; Zornig, Hanne D; Buhmann, Caecilie;

    2003-01-01

    Gender-specific and parity-dependent acquired antibody recognition is characteristic of variant surface antigens (VSA) expressed by chondroitin sulfate A (CSA)-adherent Plasmodium falciparum involved in pregnancy-associated malaria (PAM). However, antibody recognition of recombinant products...

  8. COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation.

    Science.gov (United States)

    Kang, Minho; Ross, Gracious R; Akbarali, Hamid I

    2007-12-01

    The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

  9. Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System.

    Science.gov (United States)

    Ishida, Hiroaki; Nguyen, Leonard T; Gopal, Ramamourthy; Aizawa, Tomoyasu; Vogel, Hans J

    2016-09-01

    In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells. PMID:27502305

  10. Structure of the cystic fibrosis transmembrane conductance regulator in the inward-facing conformation revealed by single particle electron microscopy

    Directory of Open Access Journals (Sweden)

    Ateeq Al-Zahrani

    2015-05-01

    Full Text Available The most common inherited disease in European populations is cystic fibrosis. Mutations in the gene lead to loss of function of the cystic fibrosis transmembrane conductance regulator protein (CFTR. CFTR is a member of the ATP-binding cassette family of membrane proteins that mostly act as active transporters using ATP to move substances across membranes. These proteins undergo large conformational changes during the transport cycle, consistent with an inward-facing to outward-facing translocation mechanism that was originally proposed by Jardetzky. CFTR is the only member of this family of proteins that functions as an ion channel, and in this case ATP and phosphorylation of a regulatory domain controls the opening of the channel. In this article we describe the inward-facing conformation of the protein and show it can be modulated by the presence of a purified recombinant NHERF1-PDZ1 domain that binds with high affinity to the CFTR C-terminal PDZ motif (-QDTRL. ATP hydrolysis activity of CFTR can also be modulated by glutathione, which we postulate may bind to the inward-facing conformation of the protein. A homology model for CFTR, based on a mitochondrial ABC transporter of glutathione in the inward-facing configuration has been generated. The map and the model are discussed with respect to the biology of the channel and the specific relationship between glutathione levels in the cell and CFTR. Finally, disease-causing mutations are mapped within the model and discussed in terms of their likely physiological effects.

  11. Critical Role of Cystic Fibrosis Transmembrane Conductance Regulation(CFTR)in Female Reproduction

    Institute of Scientific and Technical Information of China (English)

    Hsiao Chang CHAN

    2003-01-01

    @@ Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- channel, mutations of which are responsible for defective Cl- and/or HCO-3 secretions seen in cystic fibrosis (CF), a common lethal genetic disease affecting most exocrine glands/organs, including the lungs, intestine, pancreas and reproductive tracts of both sexes.

  12. Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum Ca2+-ATPase

    Institute of Scientific and Technical Information of China (English)

    屠亚平; 徐红; 杨福愉

    1995-01-01

    The sarcoplasmic reticulum (SR) Ca2+-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role ofphospholipids, especially phosphatidylcholine(PC), in the modulation of Ca2+-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows, (i) Incubated with phospholiplds, the enzyme activity of the delipidated Ca2+-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1 000μmol/L:50μmol/L, similar to the physiological condition), the inhibition of Ca2+-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50.50 molar ratio of PC:PE. (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of Ca2+-ATPase is observed in vesicles containin

  13. Trans-membrane area asymmetry controls the shape of cellular organelles

    NARCIS (Netherlands)

    Beznoussenko, Galina V; Pilyugin, Sergei S; Geerts, Willie J C; Kozlov, Michael M; Burger, Koert N J; Luini, Alberto; Derganc, Jure; Mironov, Alexander A

    2015-01-01

    Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle.