Academic literature on the topic 'Ca2+-Dependent Lipid-Binding Domain'
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Journal articles on the topic "Ca2+-Dependent Lipid-Binding Domain"
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Davis, Alison J., Jonathan T. Butt, John H. Walker, Stephen E. Moss, and Debra J. Gawler. "The Ca2+-dependent Lipid Binding Domain of P120GAPMediates Protein-Protein Interactions with Ca2+-dependent Membrane-binding Proteinss." Journal of Biological Chemistry 271, no. 40 (1996): 24333–36. http://dx.doi.org/10.1074/jbc.271.40.24333.
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Yu, Haijia, Yinghui Liu, Daniel R. Gulbranson, Alex Paine, Shailendra S. Rathore, and Jingshi Shen. "Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites." Proceedings of the National Academy of Sciences 113, no. 16 (2016): 4362–67. http://dx.doi.org/10.1073/pnas.1517259113.
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Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca2+, allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca2+. In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum–plasma membrane contact sites, are Ca2+-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca2+. E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca2+-bound C2 domains. Strikingly, the Ca2+-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca2+ sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca2+ regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca2+-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca2+ regulation.
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FUKUDA, Mitsunori. "The C2A domain of synaptotagmin-like protein 3 (Slp3) is an atypical calcium-dependent phospholipid-binding machine: comparison with the C2A domain of synaptotagmin I." Biochemical Journal 366, no. 2 (2002): 681–87. http://dx.doi.org/10.1042/bj20020484.
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The synaptotagmin-like protein (Slp) family consists of an N-terminal Rab27-binding domain and C-terminal tandem C2 motifs, and although it has been suggested to regulate Rab27-dependent membrane trafficking, such as Ca2+-regulated granule exocytosis in T-lymphocytes [Kuroda, Fukuda, Ariga and Mikoshiba (2002) J. Biol. Chem. 277, 9212–9218], little is known about the Ca2+-binding property of the Slp family. In this study, I demonstrated that the C2A domain of Slp3 exhibits Ca2+-dependent phospholipid-binding activity similar to that of the C2A domain of synaptotagmin I (Syt I) with regard to phospholipid selectivity, bivalent cation selectivity and effect of ionic strength. This finding was surprising because the C2A domains of other C-terminal-type (C-type) tandem C2 proteins require five conserved acidic residues in the putative Ca2+-binding loops 1 and 3 on the top of the β-sandwich structure for their Ca2+-/phospholipid-binding activity, whereas the C2A domain of Slp3 contains only one conserved acidic residue in the putative Ca2+-binding loop 1. Site-directed mutagenesis and chimaeric analysis of the C2A domains of Syt I and Slp3 showed that Glu-336 and Glu-337 in the putative Ca2+-binding loop 1 and polybasic sequence (Lys-359, Lys-360 and Lys-361) in the β-4 strand of the C2 structure are crucial for Ca2+-dependent phospholipid-binding activity of the Slp3 C2A domain, whereas the similar polybasic sequence in the C2A domain of Syt I is dispensable for Ca2+-dependent phospholipid-binding activity. These results indicate that the C2A domain of Slp3 is an atypical Ca2+-/phospholipid-binding machine, compared with other C-type tandem C2 proteins.
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Brohus, Malene, Mads T. Søndergaard, Sui Rong Wayne Chen, Filip van Petegem, and Michael T. Overgaard. "Ca2+-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains." Biochemical Journal 476, no. 2 (2019): 193–209. http://dx.doi.org/10.1042/bcj20180545.
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Abstract The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM–RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2 µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.
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STAHELIN, Robert V., and Wonhwa CHO. "Roles of calcium ions in the membrane binding of C2 domains." Biochemical Journal 359, no. 3 (2001): 679–85. http://dx.doi.org/10.1042/bj3590679.
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The C2 domain is a membrane-targeting domain found in many cellular proteins involved in signal transduction or membrane trafficking. The majority of C2 domains co-ordinate multiple Ca2+ ions and bind the membrane in a Ca2+-dependent manner. To understand the mechanisms by which Ca2+ mediates the membrane binding of C2 domains, we measured the membrane binding of the C2 domains of group IV cytosolic phospholipase A2 (cPLA2) and protein kinase C-α (PKC-α) by surface plasmon resonance and lipid monolayer analyses. Ca2+ ions mainly slow the membrane dissociation of cPLA2-C2, while modulating both membrane association and dissociation rates for PKC-α-C2. Further studies with selected mutants showed that for cPLA2 a Ca2+ ion bound to the C2 domain of cPLA2 induces the intra-domain conformational change that leads to the membrane penetration of the C2 domain whereas the other Ca2+ is not directly involved in membrane binding. For PKC-α, a Ca2+ ion induces the inter-domain conformational changes of the protein and the membrane penetration of non-C2 residues. The other Ca2+ ion of PKC-α-C2 is involved in more complex interactions with the membrane, including both non-specific and specific electrostatic interactions. Together, these studies of isolated C2 domains and their parent proteins allow for the determination of the distinct and specific roles of each Ca2+ ion bound to different C2 domains.
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Boswell, Kristin L., Declan J. James, Joseph M. Esquibel, et al. "Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion." Journal of Cell Biology 197, no. 2 (2012): 301–12. http://dx.doi.org/10.1083/jcb.201109132.
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Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca2+, but Ca2+-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis.
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Rahier, Renaud, Alexandre Noiriel та Abdelkarim Abousalham. "Functional Characterization of the N-Terminal C2 Domain fromArabidopsis thalianaPhospholipase Dαand Dβ". BioMed Research International 2016 (2016): 1–15. http://dx.doi.org/10.1155/2016/2721719.
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Most of plant phospholipases D (PLD) exhibit a C2-lipid binding domain of around 130 amino acid residues at their N-terminal region, involved in their Ca2+-dependent membrane binding. In this study, we expressed and partially purified catalytically active PLDαfromArabidopsis thaliana(AtPLDα) in the yeastPichia pastoris. The N-terminal amino acid sequence of the recombinant AtPLDαwas found to be NVEETIGV and thus to lack the first 35 amino acid belonging to the C2 domain, as found in other recombinant or plant purified PLDs. To investigate the impact of such a cleavage on the functionality of C2 domains, we expressed, inE. coli, purified, and refolded the mature-like form of the C2 domain of the AtPLDαalong with its equivalent C2 domain of the AtPLDβ, for the sake of comparison. Using Förster Resonance Energy Transfer and dot-blot assays, both C2 domains were shown to bind phosphatidylglycerol in a Ca2+-independent manner while phosphatidic acid and phosphatidylserine binding were found to be enhanced in the presence of Ca2+. Amino acid sequence alignment and molecular modeling of both C2 domains with known C2 domain structures revealed the presence of a novel Ca2+-binding site within the C2 domain of AtPLDα.
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Xue, Renhao, Jon D. Gaffaney та Edwin R. Chapman. "Structural elements that underlie Doc2β function during asynchronous synaptic transmission". Proceedings of the National Academy of Sciences 112, № 31 (2015): E4316—E4325. http://dx.doi.org/10.1073/pnas.1502288112.
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Double C2-like domain-containing proteins alpha and beta (Doc2α and Doc2β) are tandem C2-domain proteins proposed to function as Ca2+ sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca2+ to the β isoform. The Ca2+ ligands in the C2A domain were dispensable for Ca2+-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca2+ ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca2+/lipid-binding loops of synaptotagmin 1, a Ca2+ sensor for synchronous release, with corresponding loops from Doc2β, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2β that underlie its function during the slow phase of synaptic transmission.
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Thomas, Jessica R., Jussara Hagen, Daniel Soh, and Amy Lee. "Molecular moieties masking Ca2+-dependent facilitation of voltage-gated Cav2.2 Ca2+ channels." Journal of General Physiology 150, no. 1 (2017): 83–94. http://dx.doi.org/10.1085/jgp.201711841.
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Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 α1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand–like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.
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HAYEK, Salim M., Xinsheng ZHU, Manjunatha B. BHAT, et al. "Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor." Biochemical Journal 351, no. 1 (2000): 57–65. http://dx.doi.org/10.1042/bj3510057.
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A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872–1923, is involved in Ca 2+-dependent regulation of the Ca2+-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca2+ exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca2+ concentrations respectively. To characterize the role of D3 in the Ca2+-dependent regulation of the Ca2+-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (∆D3-RyR1) using a heterologous expression system, [3H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca2+-dependent regulation of RyR1, but did not alter [3H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca2+-dependence curve of ∆D3-RyR1 is broader, reflecting increased sensitivity to Ca2+ activation and decreased sensitivity to Ca2+ inactivation. In addition, ∆D3-RyR1 was more resistant to inhibition by Mg2+. Comparison of the effect of caffeine on wt-RyR1 and ∆D3-RyR1 suggested that D3 is an important region of RyR that participates in Ca2+-dependent activation and inactivation of the Ca2+-release channel.
Dissertations / Theses on the topic "Ca2+-Dependent Lipid-Binding Domain"
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Rodríguez, Solovey Leisa Natacha. "IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs)." Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/58862.
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[EN] ABSTRACT
Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches.
Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.<br>[ES] RESUMEN
La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas.
La transducción de la señal del ABA localizada en la membrana plasmática celular
juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA.<br>[CAT] RESUM
La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses.
Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA.<br>Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862<br>TESIS
Books on the topic "Ca2+-Dependent Lipid-Binding Domain"
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Stamenova, M., and S. Sanvito. Atomistic spin-dynamics. Edited by A. V. Narlikar and Y. Y. Fu. Oxford University Press, 2017. http://dx.doi.org/10.1093/oxfordhb/9780199533046.013.7.
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This article reviews recent advances towards the development of a truly atomistic time-dependent theory for spin-dynamics. The focus is on the s-d tight-binding model [where conduction electrons (s) are exchange-coupled to a number of classical spins (d)], including electrostatic corrections at the Hartree level, as the underlying electronic structure theory. In particular, the article considers one-dimensional (1D) magnetic atomic wires and their electronic structure, described by means of the s-d model. The discussion begins with an overview of the model spin Hamiltonian, followed by molecular-dynamics simulations of spin-wave dispersion in a s-d monoatomic chain and spin impurities in a non-magnetic chain. The current-induced motion in a magnetic domain wall (DW) is also explored, along with how an electric current can affect the magnetization landscape of a magnetic nano-object. The article concludes with an assessment of spin-motive force, and especially whether a driven magnetization dynamics can generate an electrical signal.
Book chapters on the topic "Ca2+-Dependent Lipid-Binding Domain"
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Brisson, A., and O. Lambert. "Two-Dimensional Crystallization of Soluble Proteins on Planar Lipid Films." In Crystallization of Nucleic Acids and Proteins. Oxford University Press, 1999. http://dx.doi.org/10.1093/oso/9780199636792.003.0016.
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Electron crystallography of protein two-dimensional (2D) crystals constitutes a fast-expanding method for determining the structure of macromolecules at near-atomic resolution (1, 2). The main limitation in the application and generalization of this approach remains in obtaining highly ordered 2D crystals, as is the case of 3D crystals in X-ray crystallography. Several methods of 2D crystallization are available which can be classified into two families, depending on the type of proteins under investigation, either membrane proteins (3, 4) or soluble proteins (5, 6). In both cases, 2D crystallization is a self-organization process which spontaneously occurs between macromolecules which are restricted to diffusing by translation and rotation in a 2D space, with a fixed orientation along the normal to this plane. The scope of this chapter is restricted to the 2D crystallization of soluble proteins on planar lipid films, by the so-called ‘lipid monlayer crystallization method’ (5). Our aim is to present a step-by-step description of the experimental procedures involved in the application of this method. The method of protein 2D crystallization on planar lipid films was introduced about 15 years ago (5) and has since been successfully applied to about 30 proteins. Its principle is based on the specific interaction between soluble proteins and lipid ligands inserted in a lipid monolayer, at an air-water interface. In practice, a lipid monolayer is formed by spreading lipids dissolved in an organic solvent on a water surface. Proteins present in the aqueous subphase bind to their ligand of lipidic nature and spontaneously form 2D domains and, in favourable cases, 2D crystals. The process of 2D crystal formation relies on three successive steps: (a) Molecular recognition between a protein and its ligand. (b) Diffusion and concentration of the protein-lipid complexes in the plane of the lipid film. (c) Self-organization of the proteins into 2D crystals. As indicated in Table 1, three different types of systems can be distinguished, depending on the nature of the lipid ligand: • natural lipids • synthetic lipids made of a protein ligand coupled to a lipid molecule • charged lipids.
Conference papers on the topic "Ca2+-Dependent Lipid-Binding Domain"
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Sugo, T., S. Tanabe, K. Shinoda, and M. Matsuda. "MONOCLONAL ANTIBODIES THAT RECOGNIZE Ca2+-INDUCED CONFORMER OF PROTEIN C, INDEPENDENT OF GLA RESIDUES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643644.
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Monoclonal antibodies (MCA’s) were prepared against human protein C (PC) according to Köhler & Milstein, and those that recognize the Ca2+-dependent PC conformers were screened by direct ELISA in the presence of 2 mM either CaCl2 or EDTA. Out of nine MCAߣs thus screened, five MCA's designated as HPC-1˜5, respectively, were found to react with PC in the presence of Ca2+ but not EDTA. By SDS-PAGE coupled with Western Blotting performed in the presence of 2 mM CaCl2, we found that two MCA’s HPC-1 and 2, recognized the light chain, and two others, HPC-3 and 4, recognized the heavy chain of PC. But another MCA, HPC-5 was found to react with only non-reduced antigens. Further study showed that HPC-1 and 5 failed to react with the Gla-domainless PC, i.e. PC from which the N-terminal Gla-domain of the light chain had been cleaved off by α-chymotrypsin. However, all the other three MCA's retained the reactivity with the antigen in the presence of Ca2+ even after the Gla-domain had been removed. The binding of these MCA’s to PC in the presence of Ca2+ was found to be saturable with respect to the Ca2+ concentration and the half maximal binding for each MCA was calculated to be about 0.5+mM. Moreover, many other divalent cations such as Mg2+, Mn2+ , Ba2+, Zn2+, Co2+, Sr2+, were found to substitute for Ca2+ in inducing the metal ion-dependent but Gla-domain-independent conformer of PC.Cross-reactivity to other vitamin K-aependent plasma proteins was examined by direct ELISA; HPC-2 and 3 reacted solely to PC, but HPC-1 and 4 also reacted with prothrombin and HPC-5 with both prothrombin and factor X.These findings indicated that there are two or more metal binding sites besides the Gla-domain, possibly one in the light chain and the other(s) in the heavy chain. The presence of these metal binding sites may contribute to the unique conformer of vitamin K-dependent plasma proteins including protein C.
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Stenflo, J., A.-K. öhlin, Å. Lundvall та B. Dahlback. "β-HYDROXY ASPARTIC ACID AND ft-HYDROXYASPARAGINE IN THEEGF-HOMOLOGY REGIONS OF PROTEIN C AND PROTEINS". У XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643995.
Full textAbstract:
The amino acid sequence has been determined for all of the vitamin K-dependent proteins and the gene structure is known for some of them. These findings have shown the proteins to consist of four clearly discernible domains, except protein S which has six domains. The protein domains seem to be coded on separate exons (Foster, D. C. et. al. 1985 Proc. Natl. Acad. Sci. USA 82,4673). The vitamin K-dependent γ-carboxyglutamic acid (Gla) containing domain isthe common structural denominator of the members of this protein family. In addition, all of these proteins except prothrombin contain domains that are homologous to the precursor of the epidermal growth factor (EGF). Such domains arealso found in proteins that are not vitamin K-dependent, such as the low density lipoprotein receptor, thrombomodulin, factor XII, plasminogen, the tissue type plasminogen activator, urokinase and the complement protein Clr. The vitamin K-dependent proteins can be dividedinto three groups. Factors VII, IX, X, protein C and protein Z form one group, which in addition to the Gla-region have two EGF-homology regions and one domain that is homologous to the serine proteases. Prothrombin has two 'kringle' structures and a serine protease domain and constitutes a group of its own. Protein S is also unique in that it has four EGF-homology regions and a COOH-terminal region that is homologous to the sexual hormone binding globulin (see poster by Edenbrand et. al.).Recently a posttranslationally modified amino acid, B-hydroxyaspatic acid (Hya), was identified in position 71 in the NH2-terminal EGF-homology region ofbovine protein C. The amino acid is formed by hydroxylation of aspartic acid. It has also been identified in the corresponding positions in factors VII, IX,X and protein Z (i. e. proteins which like protein C have two EGF-homology regions each). In protein S the N2-terminal of four EGF-homology regions has hydroxy lated aspartic acid .whereas the following three EGF-like domains have B-hydroxyasparagine. The nucleotide sequence codes for asparagine in the three latter positions. Neither vitamin K nor vitamin C seem to be involvedin the formation of the two hydroxylated amino acids. Recently, Hya was identified in acid hydrolysates of the complement protein Clr. Hya and Hyn have onlybeen found in domains that are homologous to the EGF precursor. In an attempt to identify the structural requirement of the hydroxylating enzyme, we have compared the sequences of EGF-homology regions that contain Hya or Hyn with the corresponding sequences that have been shown not to contain the modified amino acids. The domains that have Hya or Hyn have the consensus sequence Cx xxxx xCxC. This sequence has been found in three EGF-like domains in the EGF-precursor, in two in the LDL-receptor and in two in thrombomodulin. Furthermore, the neurogenic Notch locus in Drosophila melanogaster codes for 36 EGF-homolgy regions, 22 of which contain the consensussequence, whereas the Lin-12 locus in Caenorhabditis elegans codes for at least 11 EGF-like repeats, two of which comply with the consensus sequence. Whether any of these proteins contain Hya orHyn is not yet known with certainty.It has been hypothesized that Hya isinvolved in the Gla independent Ca2+binding of factors IX, X and protein C. In an attempt to resolve this issue, we have isolated the EGF-homology region from human protein C and been able to demonstrate that it binds Ca2+ (see poster by öhlin and Stenflo). However, we do not yet know whether Hya is directly involved in the Ca2+binding.
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KÖhlin, A., and J. Stenflo. "HIGH AFFINITY CALCIUM BINDING TO DOMAINES OF PROTEIN C THAT ARE HOMOLOGUS TO THE EPIDERMAL GROWTH FACTOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643645.
Full textAbstract:
In addition to γ-carboxyglutamic acid (Gla)-dependent calcium binding all of the vitamin K-dependent plasma proteins, except prothrombin, have one or two high affinity calcium binding sites that do not require the Gla residues. A common denominator among these proteins (factors IX, X, protein C, protein Z and protein S) is that they have domaines that are homologus to the epidermal growth factor (EGF) precursor. In factors VII,IX,X, protein C and in protein Z the aminoterminal of two EGF homology regions contain one residue of β-hydroxyaspartic acid (Hya) whereas in protein S the aminoterminal EGF homology region contains Hya and the three following contain one β-hydroxyasparagine residue each.In an attempt to elucidate the role of the EGF homology regions in the Gla independent calcium binding we have isolated a tryptic fragment (residue 44-138) from the light chain of human protein C. The fragment was isolated using a monoclonal antibody that recognizes a calcium ion stabilized epitope that is expressed both in intact protein C and in protein C lacking the Gla domaine.The antibody bound the isolated EGF homology region in the presence of calcium ions but not in EDTA containing buffer. A calcium ion titration showed half maximal binding at approximately 200 μM Ca2+. The metal ion induced conformational change in the isolated fragment was also studied with affinity purified rabbit antibodies against Gla domainless protein C. Antibodies that bound in the presence of calcium ions and that could be eluted with EDTA recognized the metal ion induced conformational change in the isolated EGF homology domain. Our results suggest that one or both of the EGF homology regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.
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"Impact of Heparan Sulphate Binding Domain of Chemokine CCL21 to Migration of Breast Cancer Cells." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0132.
Full textAbstract:
Lymph node metastasis constitutes a key event in breast cancer progression. Chemokines are small proteins, which can promote metastatic spread by inducing cancer cell migration and invasion. Chemokine function is dependant upon their binding to both cell surface heparan sulphate (HS) molecules and to their specific receptor. Our group has demonstrated a significant increase in chemokine receptor CCR7 expression in cancerous breast epithelia compared to healthy controls. This study is designed to test the hypothesis that a non-HS binding forms of chemokine CCL21 can disrupt the normal response to CCL21, therefore reducing the metastasis of CCR7-expressing cancer cells. Truncated CCL21 chemokine (Δ98- 134 c-terminal basic extension), was synthesised to investigate a possible linkage between chemokine binding capacity and cell activation. Wild type (WT) and mutant-CCL21 were tested for their ability to stimulate a dose-dependent increase in intracellular-free calcium in peripheral blood mononuclear cell (PBMC) and breast cancer epithelial cells MDA-MB-231. Mutant-CCL21 at concentrations 5 and 10nM showed potential to mobilise Ca2+ at levels similar to that produced by WT-CCl21. A series of experiments was performed to determine how deletion of the HS-binding site altered the ability of CCL21 to stimulate chemotaxis within a concentration gradient generated by free solute diffusion. PBMC stimulated to migrate by wild-type CCL21 was not significantly different from that stimulated by mutant (P> 0.05). Similar results were observed in assays using MDA-MB-231 cells. A further series of experiments was performed to compare the potential of WT and mutant-CCL21 to stimulate the migration of cells across endothelium. In contrast to results for trans-filter migration, it was found that the non HSbinding mutant stimulated no increased in transendothelial cell migration above the background at each of the tested concentrations, 10, 30 and 50 nM respectively (P>0.05). However, WT-CCL21 stimulated significant increased PBMC migration at each of the tested concentration (all P <0.001). Furthermore, the effect of heparin on chemotactic properties of WT and mutant- CCL21 was examined. Interestingly, heparin (250 µg/ml) completely inhibit the chemotaxis mediated by WT-CCL21 (5nM) (P < 0.001), whereas it did not inhibit the chemotaxis at concentrations 100, 250 & 500 µg/ml in response to mutant CCL21 (5nM) (P > 0.05). Similar assay will be performed using MDA-MB-231 cells. Work is ongoing to characterise the biophysical properties of mutant-CCL21 and determine its potential role for a therapeutic blockade of the migration of breast cancer cells in-vivo. Our primarily data showed that mutant CCL21 in xenograft brain tumor models showed substantial inhibition of tumour growth. Our results indicate that truncated CCL21 chemokine might be a potential preventive biofactor for human breast cancer metastasis by targeting chemokine receptor genes.
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Church, F. C., R. E. Treanor, and H. C. Whinna. "ACTIVATION OF HEPARIN COFACTOR II BY PHOSVITIN, A PHOSPHOGLYCO-PROTEIN, AND OTHER PHOSPHATE-CONTAINING POLYANIONS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643867.
Full textAbstract:
We are characterizing the specificity of the polyanion-binding domain of the heparin/dermatan sulfate-dependent plasma protease inhibitor, heparin cofactor II (HCII). Various phosphate-containing polyanions accelerate the HCII-catalyzed inhibition of thrombin (T). Phosvitin, a phosphoprotein, enhances the HCII/T reaction at 25°C and pH 8.0 with the apparent second-order rate constant value (K2) increasing from 5 × 104 M−1 min−1 (in the absence of phosvitin) to 8 x 10' M”1 min as phosvitin increased from 0.05 to 30 ug/ml and then decreases as phosvitin is increased above 30 ug/ml. Apparent dissociation constant values for phosvitin-HCII and phosvitin-T are 450 nM and 10 nM, respectively. Polynucleotides accelerate the HCII/T reaction and have the following specificity (concentrations examined from 1-200 ug/ml): poly(guanylic acid) >> poly(adeny-lic acid, guanylic acid) > poly(inosinic acid) > poly(guanylic acid, uridylic acid) > poly(uridylic acid) = poly(adenylic acid) > poly(cytidylic acid). Polyphosphate anions (phosphate chain length, n, ranging from 5-100) enhance the HCII/T reaction. When compared at an equimolar phosphate concentration (1 mM), the rate was saturated at n = 50 with a maximum V.2 of about 5 × 107 M−1 min−1. Ca2+ (or Mg2+)-phosvitin/polyphosphate anion complexes and salmon protamine-polynucleotide complexes have lost the ability to enhance the HCII/T reaction. Phospho-pyridoxylated-HCII (lysine modified), with greatly reduced heparin cofactor activity, has lost its accelerating effect with phosvitin, polynucleotides and the polyphosphate anions. None of the above mentioned polyphosphate-containing compounds are effective at accelerating either the HCII-catalyzed inhibition of chymotrypsin or the antithrombin Ill-catalyzed T reaction. Our results suggest that (i) HCII is activated by the multiple negative charges of phosphate polyanions but they alone are not sufficient; (ii) the effective phosphate polyanions must also possess a specific conformation for maximum activity; and (iii) the phosphoserine-containing protein, phosvitin, can serve as a "template" for HCII/T.
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Shattil, S. J., J. A. Hoxie, M. Cunningham, C. S. Abrahms, J. O’Brien, and Z. Budzynski. "DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643830.
Full textAbstract:
Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.
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DePoli, Patricia, Theresa Bacon-Baquley, and Daniel A. Walz. "THROMBOSPONDIN INTERACTION WITH PLASMINOGEN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643824.
Full textAbstract:
Platelet thrombospondin (TSP) interacts with plasminogen in a specific and saturable manner. TSP can form a trimolecular complex with histidine-rich glycoprotein and plasminogen and the plasminogen within such complexes can reportedly be activated by tissue plasminogen activator. We have studied the interaction of TSP with plasminogen using Western blotting of plasminogen, reduced plasmin and the elastase-generated fragments of plasminogen and their binding of iodinated TSP. TSP was found to specifically bind to plasminogen and the heavy (non-enzyme) chain of plasmin in a calcium-independent manner. Binding could be blocked by preincubation of the immobilized plasminogen or plasmin with an excess of unlabeled TSP. Plasminogen domains (kringles) were generated by limited eTastase proteolysis. TSP bound specifically to a single 51 kDa plasminogen fragment. The elastase-generated fragments were separated by lysine-Sepharose chromatography and their identities established by amino acid composition and amino-terminal sequence analysis. The 51 kDa plasminogen fragment bound to lysine-Sepharose and had an amino-terminal sequence corresponding to kringle 4 (K4) and a composition consistent with that of K4-K5-plasmin. TSP binding to this fragment was not blocked by the presence of an excess of the fragment K1-K2-K3, K4, nor miniplasminogen (K5-plasmin). Binding does not appear to be directly dependent upon the specific high-affinity lysine binding site of the 51 kDa fragment. Our data suggests that thrombospondin interacts with plasminogen at a single distinct site, and that this recognition site is at or near the K4-K5 contiguous region of plasminogen.
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Stern, David M., Sara Rimon, Todd Scott, and Peter P. Nawroth. "MODULATION OF ENDOTHELIAL CELL COAGULANT PROPERTIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642946.
Full textAbstract:
As the cells forming the luminal vascular surface, endothelium is strategically located to play a role in the regulation of coagulation. Participation of endothelium in coagulation involves specific receptors on the cell surface functioning at the level of initiation and propagation of hemostatic reactions. In the anticoagulant protein C pathway, for example, the receptor thrombomodulin initiates thrombin-mediated activation of protein C and a binding site for protein S on bovine endothelium promotes assembly of the functional activated protein C/protein S complex. Endothelium also synthesizes, stores and releases functional protein S constitutively and in response to specific stimuli such as norepinephrine.Since activation of protein C requires thrombin formation in proximity to the vessel wall, we have examined procoagulant reactions on the endothelial cell surface. Endothelium provides a receptor for Factor IX/IXa which is relatively selective for the enzyme form and facilitates Factor IXa-VIII-mediated activation of Factor X. Half-maximal Factor Xa formation occurs at a Factor IXa concentration of 0.4nM on endothelium, whereas lOnM is required on liposomes. This concentration of Factor IXa corresponds to that which results in half-maximal occupancy of endothelial cell Factor IXa binding sites in the presence of Factors VIII and X, thus correlating kinetics and binding measurements. Crosslinking and ligand blotting studies have shown that the receptor is a protein with a molecular weight of ∼160,000. The clinical significance of this receptor is suggested by the moderately severe bleeding disorder observed in a patient with hemophilia B due to an abnormal Factor IX molecule, Factor IXalabama (Factor IXala). Although the coagulant activity of Factor IXala is only mildly decreased on phospholipids, it is severely impaired on endothelium. The affinity of Factor IXala for the endothelial cell Factor X activation complex is decreased by 20-fold compared with the normal enzyme and the binding affinity is similarly decreased. Since the molecular defect in Factor IXala has been previously shown to consist of a single point mutation in the growth factor domain, this indicates a role for the growth factor domain in receptor, recognition.The picture of endothelial cell coagulant properties which emerges from these and other studies is one in which endothelium has either an anticoagulant or procoagulant potential, depending on modulation of receptor expression and release of secreted products. In the quiescent state, anticoagulant mechanisms predominate with only limited amounts of procoagulant activity: there is little tissue factor activity and only a basal level of receptors for Factor IX/lXa. Activation of endothelium by Tumor Necrosis Factor (TNF) or Interleukin 1 can shift this balance. Tissue factor synthesis and expression occurs in a dose-dependent manner, being half-maximal at a TNF concentration of about 150pM. TNF also increases the number of Factor IX/lXa binding sites. Concomitant with enhancement of endothelial cell procoagulant properties is a suppression of cell surface cofactor activity for the anticoagulant protein C pathway. Endothelial cell-dependent, thrombin-mediated activated protein C formation is decreased by 70-80% and activated protein C-protein S-mediated Factor Va inactivation decreases by over 90%. Following the in vivo infusion of Interleukin 1, similar changes in endothelial cell coagulant properties were observed on aortic segments with fibrin deposition occurring on the functionally altered, but morphologically intact endothelium. This modulation of endothelial cell coagulant properties could underlie the prothrombotic state associated with inflammatory disorders and could also explain the recently observed selective intravascular thrombosis of tumor vasculature seen in vivo in meth A sarcomas after administration of TNF.Thus, although endothelium was initially felt to be hemostatically inert, this apparent lack of activity actually masks a delicate balance of procoagulant and anticoagulant mechanisms. The balance can be effectively shifted by physiologic mediators, such as monokines, which alter receptor expression on the endothelial cell surface. Changes in endothelial cell hemostatic properties may be an early indicator of vessel wall disease and underlie the pathogenesis of localized thrombotic processes.
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Berndt, M. "STRUCTURE AND FUNCTION OF THE GLYCOPROTEIN Ib-IX COMPLEX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643729.
Full textAbstract:
At high shear flow, the adhesion of platelets to the exposed vascular subendothelium requires von Willebrand factor (vWF) and is dependent upon a specific platelet membrane adhesion receptor, the human platelet membrane glycoprotein (GP) Ib-IX complex. Recent evidence suggests that vWFbinding to the GP Ib-IX complex plays an important role in other key aspects of hemostasis and thrombosis such as shear-induced platelet aggregation and the interaction of platelets with fibrin.Studies in our laboratory with a seriesof murine monoclonal antibodies directed against epitopes on GP lb, GP IX, or against complex-specificepitopes indicate that GP lb and GP IX exist in the intact platelet membrane as a native heterodimer complex(-25,000 copies/platelet). By analysis onSDS-polyacrylamide gels, GP lb has an apparent molecular weight of 170,000 and cnsists of two disulfide-linked subunits, GP Iba (Mr = 135,000) and GP Ibβ (Mr = 25,000),whilst GP IX has an equivalent molecularweight under both nonreducing and reducing conditions (Mr = 22,000).The ±-chain ofGP lb has a central macroglycopeptide core (Mr =90,000) which is highly glycosylated. At each end of themacroglycopeptide region is a domainsensitive to proteolytic cleavage. Cleavage at the end proximal to the platelet membrane, e.g. by calpain, Serratia marcescens metalloprotease and trypsin, generates two fragments :a Mr =130,000 highly glycosylated fragment termed glycocalicin anda membrane-associated region consisting ofa Mr -25,000 fragment that remains disulfide-linkedto GP Ibβ and associated with GP IX. In resting platelets, the membrane-associated region spans the lipid bilayer linking the GP Ib-IX complex to the platelet endoskeleton via actin-binding protein. This membrane-associated region also contains the domain(s) recognized by quinine/quinidine drug-dependent antibodies. Cleavage at the plasma end of the macroglycopeptide, e.g. by human leukocyte elastase, generates a poorly glycosylated Mr = 45,000 fragment of GP Ibα (peptide tail region) and a heavily glycosylated Mr = 100,000 fragment that remains disulfide-linked to GP Ibg and associated with GP IX. Platelets lacking the N-terminal peptide tail region of GP Iba fail to agglutinate with ristocetin and vWF and show a delayed response to a-thrombin.Polyclonal and monoclonal antibodies against this region also inhibit both these platelet responses suggesting that the peptide tail region contains the binding sites for both α-thrombinand vWF. Rotary shadowingelectron microscopy of purified GP Ib-IX complex shows the structure to be highlyasymmetric with each complex existing asa flexible rod with a globular domain at each end. The overall length of the complexwas =60 nm.The smaller globular domain (peptidetail region) has a diameter of =9nm; the larger globular domain (membrane-associated region), a diameter of =16 nmWe have recently examined whetherthe human platelet GP Ib-IX complex is the receptor for the ristocetin-dependent binding of vWF by reconstitution with the purified components using a solid-phasebead assay. Our approach was to indirectlybind and orientate the GP Ib-IX complex onthe beads via a monoclonal antibody directed against the membrane-associated region of the complex (FMC 25, epitope on GP IX).Immunobeads were chosen as the insoluble matrix because they are uniform in size (=10μm in diameter), impermeable,specifically designed for the coupling of IgG, and because, like platelets, the beads have a net negative charge atneutral pH.Specific binding of 125I-labelled human vWF tothe GP Ib-IX complex-coated immunobeads was strictly ristocetin-dependent with maximal binding occurring atristocetin concentrations >1 mg/ml. Ristocetin-dependent specificbinding of 125I-labelled vWF was saturable.Scatchardanalysis revealed a single classof binding sites for vWF with purified GP Ib-IX complex.Monoclonal antibodies against the Mr = 45,000 peptide tail region ofGP lb which stronglyinhibitthe ristocetin-dependent binding ofvWF toplatelets also strongly inhibited the ristocetin-dependent binding of vWFto the GP Ib-IX coated beads. Monoclonalantibody against either themacroglycopeptide or membrane-associated regions of the GPIb-IX complex did not inhibit the ristocetin-dependent binding of vWF to platelets or to the GP Ib-IX complex-coated beads. Similar functional correlations were obtained with anti-vWFmonoclonal antibodies. The reconstitutiondata therefore confirm the functional roleof the GP Ib-IX complex as a major plateletvWF receptor. The region ofthe vWF molecule involved in binding to the GP Ib-IX complex has been localized toa Mr =50,000 domain towards theN-terminal end of the vWF subunit. The reconstitution assay should prove useful in the further definition of active peptides of vWF that bind tothe human platelet GP Ib-IX complex.
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Farahat, Waleed A., and H. Harry Asada. "Control of Eukaryotic Cell Migration Through Modulation of Extracellular Chemoattractant Gradients." In ASME 2010 Dynamic Systems and Control Conference. ASMEDC, 2010. http://dx.doi.org/10.1115/dscc2010-4190.
Full textAbstract:
Cell migration is fundamental to a wide range of biological and physiological functions including: wound healing, immune defense, cancer metastasis, as well as the formation and development of biological structures such as vascular and neural networks. In these diverse processes, cell migration is influenced by a broad set of external mechanical and biochemical cues, particularly the presence of (time dependent) spatial gradients of soluble chemoattractants in the extracellular domain. Many biological models have been proposed to explain the mechanisms leading to the migratory response of cells as a function of these external cues. Based on such models, here we propose approaches to controlling the chemotactic response of eukaryotic cells by modulating their micro-environments in vitro (for example, using a microfluidic chemotaxis chamber). By explicitly modeling i) chemoattractant-receptor binding kinetics, ii) diffusion dynamics in the extracellular domain, and iii) the chemotactic response of cells, models for the migration processes arise. Based on those models, optimal control formulations are derived. We present simulation results, and suggest experimental approaches to controlling cellular motility in vitro, which can be used as a basis for cellular manipulation and control.