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1.
Blood ; 135(17): 1484-1496, 2020 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-32078672

RESUMEN

Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-D'D3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.


Asunto(s)
Factor VIII/metabolismo , Hemofilia A/terapia , Hemorragia/prevención & control , Proteínas Recombinantes de Fusión/administración & dosificación , Factor de von Willebrand/metabolismo , Animales , Factor VIII/genética , Hemofilia A/metabolismo , Hemofilia A/patología , Hemostasis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Primates , Factor de von Willebrand/genética
2.
Mol Ther ; 29(5): 1729-1743, 2021 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-33484965

RESUMEN

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.


Asunto(s)
Vesículas Extracelulares/trasplante , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas/administración & dosificación , Proteínas Represoras/metabolismo , Animales , Comunicación Celular , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Femenino , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/genética
3.
Blood ; 125(3): 562-9, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25359992

RESUMEN

How glycoprotein (GP)Ib-IX complex on the platelet surface senses the blood flow through its binding to the plasma protein von Willebrand factor (VWF) and transmits a signal into the platelet remains unclear. Here we show that optical tweezer-controlled pulling of the A1 domain of VWF (VWF-A1) on GPIb-IX captured by its cytoplasmic domain induced unfolding of a hitherto unidentified structural domain before the dissociation of VWF-A1 from GPIb-IX. Additional studies using recombinant proteins and mutant complexes confirmed its existence in GPIb-IX and enabled localization of this quasi-stable mechanosensitive domain of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIbα subunit. These results suggest that VWF-mediated pulling under fluid shear induces unfolding of the mechanosensitive domain in GPIb-IX, which may possibly contribute to platelet mechanosensing and/or shear resistance of VWF-platelet interaction. The identification of the mechanosensitive domain in GPIb-IX has significant implications for the pathogenesis and treatment of related blood diseases.


Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Adhesividad Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Estrés Mecánico , Factor de von Willebrand/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dicroismo Circular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/genética
4.
Blood ; 126(8): 935-8, 2015 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-26065652

RESUMEN

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.


Asunto(s)
Factor VIII/química , Factor VIII/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Sitios de Unión , Factor VIII/ultraestructura , Células HEK293 , Humanos , Espectrometría de Masas , Microscopía Electrónica , Estructura Terciaria de Proteína , Factor de von Willebrand/ultraestructura
5.
Biophys J ; 111(3): 601-608, 2016 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-27508443

RESUMEN

Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.


Asunto(s)
Citoesqueleto/metabolismo , Fenómenos Mecánicos , Adhesividad Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Animales , Fenómenos Biomecánicos , Células CHO , Cricetinae , Cricetulus , Filaminas/metabolismo , Humanos
6.
Blood ; 123(4): 582-9, 2014 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-24297869

RESUMEN

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo.


Asunto(s)
Vitamina K/antagonistas & inhibidores , Vitamina K/metabolismo , Warfarina/química , Alcoholes/química , Anticoagulantes/química , Doxiciclina/química , Factor IX/química , Citometría de Flujo , Células HEK293 , Humanos , Concentración 50 Inhibidora , Hígado/metabolismo , Fenol/química , Unión Proteica , Estructura Terciaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , Vitamina K/química , Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Vitamina K Epóxido Reductasas/metabolismo , Warfarina/análogos & derivados
7.
Blood ; 115(3): 706-12, 2010 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-19812385

RESUMEN

Elevated plasma von Willebrand factor (VWF) and low ADAMTS13 activity have been reported in several inflammatory states, including sepsis and acute respiratory distress syndrome. One hallmark of inflammation is neutrophil activation and production of reactive oxygen species, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). HOCl is produced from hydrogen peroxide and chloride ions through the action of myeloperoxidase. HOCl can oxidize methionine to methionine sulfoxide and tyrosine to chlorotyrosine. This is of interest because the ADAMTS13 cleavage site in VWF, the Tyr(1605)-Met(1606) peptide bond, contains both oxidation-prone residues. We hypothesized that HOCl would oxidize either or both of these residues and possibly inhibit ADAMTS13-mediated cleavage. We therefore treated ADAMTS13 substrates with HOCl and examined their oxidative modification by mass spectrometry. Met(1606) was oxidized to the sulfoxide in a concentration-dependent manner, with complete oxidation at 75muM HOCl, whereas only a miniscule percentage of Tyr(1605) was converted to chlorotyrosine. The oxidized substrates were cleaved much more slowly by ADAMTS13 than the nonoxidized substrates. A similar result was obtained with multimeric VWF. Taken together, these findings indicate that reactive oxygen species released by activated neutrophils have a prothrombotic effect, mediated in part by inhibition of VWF cleavage by ADAMTS13.


Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Neutrófilos/metabolismo , Oxidantes/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factor de von Willebrand/metabolismo , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Secuencia de Aminoácidos , Dominio Catalítico , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Ácido Hipocloroso/farmacología , Metionina/metabolismo , Metionina/fisiología , Modelos Biológicos , Neutrófilos/efectos de los fármacos , Oxidantes/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Multimerización de Proteína/fisiología , Especies Reactivas de Oxígeno/farmacología , Tirosina/metabolismo , Tirosina/fisiología , Factor de von Willebrand/química
8.
J Theor Biol ; 279(1): 143-9, 2011 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-21453708

RESUMEN

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO(2) and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.


Asunto(s)
Ligasas de Carbono-Carbono/metabolismo , Modelos Biológicos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Multimerización de Proteína , Vitamina K/metabolismo , Ligasas de Carbono-Carbono/química , Ligasas de Carbono-Carbono/genética , Dominio Catalítico , Mutación/genética , NAD(P)H Deshidrogenasa (Quinona)/química , NAD(P)H Deshidrogenasa (Quinona)/genética , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Teoría Cuántica , Warfarina/farmacología
9.
CRISPR J ; 3(6): 440-453, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33346710

RESUMEN

The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.


Asunto(s)
Edición Génica/ética , Edición Génica/métodos , Edición Génica/tendencias , Artefactos , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma Humano/genética , Inestabilidad Genómica/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , ARN Guía de Kinetoplastida/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad
10.
Thromb Res ; 135(5): 970-6, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25721936

RESUMEN

Recombinant Factor VIIa (rFVIIa) is utilized for on-demand treatment of bleeding episodes in hemophilia patients with neutralizing antibodies (inhibitors) against Factor VIII or Factor IX, but a short half-life in the circulation (~2.5hrs) limits its use in a prophylactic setting. Recombinant FVIIa variants with improved pharmacokinetic properties may enable improved treatment and prevention of bleeding episodes in the inhibitor population. In this study we describe recombinant FVIIaFc (rFVIIaFc), a recombinant Fc-fusion protein generated to utilize the neonatal Fc receptor (FcRn)-mediated recycling pathway that protects immunoglobulin G from catabolism. On the basis of activity, rFVIIaFc exhibited a 5.5-fold extension in terminal half-life in hemophilia A mice compared to rFVIIa. The potency of rFVIIaFc was comparable to that of rFVIIa in thrombin generation assay and ROTEM. In agreement with these data, rFVIIaFc and rFVIIa showed similar acute efficacy at comparable molar doses in the tail clip bleeding model in hemophilia A mice. Taken together, these studies demonstrate enhanced pharmacokinetics and similar hemostatic properties for rFVIIaFc compared to rFVIIa.


Asunto(s)
Factor VIIa/farmacocinética , Animales , Modelos Animales de Enfermedad , Factor VIIa/genética , Factor VIIa/uso terapéutico , Semivida , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Hemofilia A/genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tiempo de Protrombina , Receptores Fc/deficiencia , Receptores Fc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéutico
11.
PLoS One ; 9(11): e113600, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25415306

RESUMEN

Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.


Asunto(s)
Factor VIII/genética , Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Clonación Molecular/métodos , Modelos Animales de Enfermedad , Factor VIII/química , Factor VIII/uso terapéutico , Semivida , Hemofilia A/sangre , Hemorragia , Humanos , Técnicas In Vitro , Masculino , Ratones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Trombina/metabolismo , Factor de von Willebrand/metabolismo
12.
Protein Expr Purif ; 57(2): 280-9, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18042400

RESUMEN

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.


Asunto(s)
Biotina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animales , Biotinilación , Células CHO , Línea Celular , Cricetinae , Cricetulus , Doxiciclina/farmacología , Vectores Genéticos , Glicosilación/efectos de los fármacos , Humanos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores Acoplados a Proteínas G/biosíntesis , Tetraciclina/farmacología
13.
Protein Expr Purif ; 52(2): 320-8, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17084093

RESUMEN

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.


Asunto(s)
Biotina/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Avidina/metabolismo , Biotinilación , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Recombinantes/genética , Proteínas Represoras/genética , Transfección , Ubiquitina-Proteína Ligasas/genética
14.
Proc Natl Acad Sci U S A ; 104(21): 8767-72, 2007 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-17502622

RESUMEN

Proline-rich Gla protein 2 (PRGP2) is one of four known vertebrate transmembrane gamma-carboxyglutamic acid (Gla) proteins. Members of this protein family are broadly expressed in fetal and adult human tissues and share a common architecture consisting of a predicted propeptide and Gla domain, a single-pass transmembrane segment, and tandem Pro/Leu-Pro-Xaa-Tyr (PY) motifs near their C termini. Using a methodology developed for the regulated expression of enzymatically biotinylated proteins in mammalian cells, we demonstrate that PRGP2 undergoes gamma-glutamyl carboxylation in a manner that is both dependent upon the presence of a proteolytically cleavable propeptide and sensitive to warfarin, a vitamin K antagonist that is widely used as an antithrombotic agent. When expressed at physiologically relevant levels, the majority of PRGP2 is present in the gamma-glutamyl carboxylated, propeptide-cleaved (mature) form. We additionally demonstrate, by Western blotting and flow cytometry, that mature PRGP2 is predominantly located on the cell surface with the Gla domain exposed extracellularly. In a yeast two-hybrid screen that used the C-terminal cytoplasmic region of PRGP2 as bait, we identified the WW domain-containing transcriptional coactivator Yes-associated protein (YAP) as a binding partner for PRGP2. In GST pull-down experiments, both PRGP2 PY motifs and both YAP WW domains were essential for complex formation, as were residues proximal to the core sequence of the first PY motif. These findings suggest that PRGP2 may be involved in a signal transduction pathway, the impairment of which may be an unintended consequence of warfarin therapy.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Vitamina K/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Portadoras/química , Proteínas Portadoras/genética , Cricetinae , Cricetulus , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Procesamiento Proteico-Postraduccional , Transactivadores/genética , Factores de Transcripción , Proteínas Señalizadoras YAP
15.
Semin Thromb Hemost ; 32 Suppl 1: 3-15, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16673262

RESUMEN

The fundamental importance of thrombin in biology and medicine has made it one of the most extensively studied of all proteases. Thrombin performs essential functions in vertebrate biology as the central enzyme involved in blood coagulation and platelet aggregation, and as a mitogen and secretagogue for a variety of cell types. Thrombin is synthesized in the liver and secreted into the general circulation in an inactive zymogen form (prothrombin), a complex multidomain glycoprotein that is activated to yield thrombin at sites of vascular injury by limited proteolysis following upstream activation of the coagulation cascade. Thrombin shares its general architecture and catalytic mechanism with those of pancreatic trypsin, the prototypical digestive serine protease. However, the specificity of thrombin toward substrates and cofactors, as well as its spatiotemporal regulation by effectors and inhibitors, is directed by features of the molecule that distinguish it from relatively nonspecific serine proteases like trypsin. Structural and functional studies have demonstrated the presence of surface loops that partially occlude the active site and make specific contacts with residues adjacent to the scissile bond of substrates. Specificity toward macromolecular substrates and cofactors is additionally enhanced by anion-binding exosites that are spatially distinct from the active site. More than five decades of multidisciplinary research on thrombin have produced an abundance of functional and structural information and provided a robust framework for understanding the role of thrombin in vertebrate biology.


Asunto(s)
Trombina/química , Trombina/fisiología , Animales , Sitios de Unión , Hemostasis , Humanos , Alineación de Secuencia , Serina Endopeptidasas/química , Trombina/biosíntesis
16.
Proc Natl Acad Sci U S A ; 103(43): 15794-9, 2006 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-17043233

RESUMEN

We have isolated and sequenced several cDNAs derived from the sea squirt Ciona intestinalis that encode vitamin K-dependent proteins. Four of these encode gamma-carboxyglutamic acid (Gla) domain-containing proteins, which we have named Ci-Gla1 through Ci-Gla4. Two additional cDNAs encode the apparent orthologs of gamma-glutamyl carboxylase and vitamin K epoxide reductase. Ci-Gla1 undergoes gamma-glutamyl carboxylation when expressed in CHO cells and is homologous to Gla-RTK, a putative receptor tyrosine kinase previously identified in a related ascidian. The remaining three Gla domain proteins are similar to proteins that participate in fundamental developmental processes, complement regulation, and blood coagulation. These proteins are generally expressed at low levels throughout development and exhibit either relatively constant expression (Ci-Gla1, gamma-glutamyl carboxylase, and vitamin K epoxide reductase) or spatiotemporal regulation (Ci-Gla2, -3, and -4). These results demonstrate the evolutionary emergence of the vitamin K-dependent Gla domain before the divergence of vertebrates and urochordates and suggest novel functions for Gla domain proteins distinct from their roles in vertebrate hemostasis. In addition, these findings highlight the usefulness of C. intestinalis as a model organism for investigating vitamin K-dependent physiological phenomena, which may be conserved among the chordate subphyla.


Asunto(s)
Coagulación Sanguínea , Ciona intestinalis/metabolismo , Proteínas/metabolismo , Vitamina K/metabolismo , Ácido 1-Carboxiglutámico/metabolismo , Secuencia de Aminoácidos , Animales , Ciona intestinalis/química , Ciona intestinalis/efectos de los fármacos , Ciona intestinalis/genética , Secuencia Conservada , Expresión Génica/genética , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas/química , Proteínas/genética , Alineación de Secuencia , Vitamina K/farmacología
17.
J Neurochem ; 95(4): 1177-87, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16271051

RESUMEN

Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long-term phenotypic changes. We and others have recently reported that commercial-grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re-investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical-grade recombinant human thrombin (rh-thr) and compared it with a commercial-grade plasma-derived bovine thrombin (pb-thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin-6 and tumour necrosis factor-alpha release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb-thr induced robust responses in all variables tested. In contrast, rh-thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species-specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co-stimulatory effects. In addition, these results suggest that previous studies examining thrombin's activation of microglia should be cautiously re-interpreted.


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Microglía/efectos de los fármacos , Serina Endopeptidasas/farmacología , Trombina/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antígenos CD40/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Interleucina-6/metabolismo , Ratones , Microglía/fisiología , Óxido Nítrico/metabolismo , Serina Endopeptidasas/clasificación , Trombina/clasificación , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismo
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