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1.
Thromb Haemost ; 118(2): 229-250, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29378352

RESUMO

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.


Assuntos
Tromboembolia/terapia , Trombose/sangue , Trombose/terapia , Anticoagulantes/uso terapêutico , Biomarcadores/sangue , Coagulação Sanguínea , Eritrócitos/metabolismo , Fator VIII/metabolismo , Fator XII/metabolismo , Fator XIII/metabolismo , Humanos , Macrófagos/metabolismo , Países Baixos , Fenótipo , Placa Aterosclerótica/sangue , Placa Aterosclerótica/diagnóstico , Placa Aterosclerótica/terapia , Polifosfatos/metabolismo , Fatores de Risco , Transdução de Sinais , Tromboembolia/sangue , Tromboembolia/diagnóstico , Trombose/diagnóstico
2.
Oncogene ; 35(12): 1529-40, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-26165842

RESUMO

As the majority of patients with basal-like breast carcinoma present with invasive, metastatic disease that do not respond to available therapies, it is essential to identify new therapeutic targets that impact invasion and metastasis. Protease-activated receptor 1 (PAR1), a G-protein coupled receptor has been shown to act as an oncogene, but underlying mechanisms are not well understood. Here, we show that ectopic expression of functionally active PAR1 in MCF-7 cells induced a hormone-refractory, invasive phenotype representative of advanced basal-like breast carcinoma that readily formed metastatic lesions in lungs of mice. PAR1 was found to globally upregulate mesenchymal markers, including vimentin, a direct target of PAR1, and downregulate the epithelial markers including E-cadherin, as well as estrogen receptor. In contrast, non-signaling PAR1 mutant receptor did not lead to an invasive, hormone refractory phenotype. PAR1 expression increased spheroid formation and the level of stemness markers and self-renewal capacity in human breast cancer cells. We identified HMGA2 (high mobility group A2) as an important regulator of PAR1-mediated invasion. Inhibition of PAR1 signaling suppresses HMGA2-driven invasion in breast cancer cells. HMGA2 gene and protein are highly expressed in metastatic breast cancer cells. Overall, our results show that PAR1/HMGA2 pathway may present a novel therapeutic target.


Assuntos
Neoplasias da Mama/patologia , Proteína HMGA2/fisiologia , Metástase Neoplásica/fisiopatologia , Receptor PAR-1/fisiologia , Feminino , Humanos , Células MCF-7 , Fenótipo , Vimentina/metabolismo
3.
Oncogene ; 33(17): 2264-72, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-23708660

RESUMO

Matrix metalloprotease-1 (MMP1) is an important mediator of tumorigenesis, inflammation and tissue remodeling through its ability to degrade critical matrix components. Recent studies indicate that stromal-derived MMP1 may exert direct oncogenic activity by signaling through protease-activated receptor-1 (PAR1) in carcinoma cells; however, this has not been established in vivo. We generated an Mmp1a knockout mouse to ascertain whether stromal-derived Mmp1a affects tumor growth. Mmp1a-deficient mice are grossly normal and born in Mendelian ratios; however, deficiency of Mmp1a results in significantly decreased growth and angiogenesis of lung tumors. Coimplantation of lung cancer cells with wild-type Mmp1a(+/+) fibroblasts completely restored tumor growth in Mmp1a-deficient animals, highlighting the critical role of stromal-derived Mmp1a. Silencing of PAR1 expression in the lung carcinoma cells phenocopied stromal Mmp1a-deficiency, thus validating tumor-derived PAR1 as an Mmp1a target. Mmp1a secretion is controlled by the ability of its prodomain to facilitate autocleavage, whereas human MMP1 is efficiently secreted because of stable pro- and catalytic domain interactions. Taken together, these data demonstrate that stromal Mmp1a drives in vivo tumorigenesis and provide proof of concept that targeting the MMP1-PAR1 axis may afford effective treatments of lung cancer.


Assuntos
Carcinoma Pulmonar de Lewis/enzimologia , Precursores Enzimáticos/deficiência , Neoplasias Pulmonares/enzimologia , Metaloproteinase 1 da Matriz/deficiência , Neovascularização Patológica/enzimologia , Sequência de Aminoácidos , Animais , Células COS , Carcinogênese/metabolismo , Carcinoma Pulmonar de Lewis/secundário , Linhagem Celular Tumoral , Chlorocebus aethiops , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/secundário , Masculino , Metaloproteinase 1 da Matriz/química , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Transplante de Neoplasias , Sinais Direcionadores de Proteínas , Carga Tumoral
4.
Am J Physiol Gastrointest Liver Physiol ; 304(5): G516-26, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23275617

RESUMO

Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.


Assuntos
Doenças Biliares/prevenção & controle , Lipopeptídeos/farmacologia , Pancreatite/prevenção & controle , Receptor PAR-2/antagonistas & inibidores , Células Acinares/efeitos dos fármacos , Animais , Ácidos e Sais Biliares/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ceruletídeo/farmacologia , Colangiopancreatografia Retrógrada Endoscópica , Quimotripsinogênio/metabolismo , Corantes , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Cálculos Biliares/prevenção & controle , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Propídio , Tripsinogênio/metabolismo
5.
Cancer Res ; 61(15): 5933-40, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11479236

RESUMO

Proteases give cancer a defining characteristic of being able to break through extracellular matrix barriers and invade into other tissues in response to chemotactic signals. Recently, the cell surface protease-activated receptor (PAR)-1 has been shown to act as a chemokine receptor in inflammatory cells, and its expression is tightly correlated with metastatic propensity of breast cancer cells. The aim of the present study was to determine whether activation of PAR1 or the other known PARs (PAR2-4) can regulate migration and invasion of breast cancer cells. We found that the highly invasive MDAMB231 breast cancer cell line expressed very high levels of functional PAR1, PAR2, and PAR4, whereas minimally invasive MCF7 cells had trace amounts of PAR1 and low levels of PAR2 and PAR4. Despite the differences in expression, PAR2 and PAR4 acted as chemokine receptors in both invasive and minimally invasive breast cell lines. Quite unexpectedly, we found that activation of PAR1 with thrombin or the peptide agonist SFLLRN markedly inhibited invasion and migration of MDAMB231 cells when applied as a concentration gradient in the direction of cell movement. Additionally, we demonstrated that inhibition of chemotaxis was mediated through a G(i)/phosphoinositide-3-OH kinase-dependent pathway. Activation of G(i) signaling with epinephrine or wasp venom mastoparan also inhibited invasion and migration of the breast cancer cells. These findings suggest that therapeutics targeted toward G(i)-couplers that are selectively expressed in breast cancer cells could prove beneficial in halting the progression of invasive breast cancer.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Receptores de Trombina/fisiologia , Transdução de Sinais/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Fase G1/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Invasividade Neoplásica , Inibidores de Fosfoinositídeo-3 Quinase , Receptor PAR-1 , Receptores de Trombina/biossíntese , Trombina/farmacologia
6.
Mol Cell Biol ; 21(6): 2213-20, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11238954

RESUMO

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the mu-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of mu-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The mu-calpain-deficient mice are viable and fertile. The complete deficiency of mu-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta3 subunit of alphaIIbbeta3 integrin, talin, and ABP-280 (filamin). However, mu-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta3 subunit of alphaIIbbeta3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that mu-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.


Assuntos
Plaquetas/fisiologia , Calpaína/genética , Calpaína/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Transdução de Sinais , Difosfato de Adenosina/farmacologia , Animais , Antígenos CD/metabolismo , Proteínas Sanguíneas/metabolismo , Western Blotting , Calcimicina/farmacologia , Colágeno/farmacologia , Proteínas Contráteis/metabolismo , Filaminas , Inativação Gênica , Integrina beta3 , Camundongos , Camundongos Mutantes , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Talina/metabolismo , Trombina/farmacologia , Tirosina/metabolismo
7.
J Biol Chem ; 275(52): 40671-8, 2000 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-11005807

RESUMO

Platelet activation and aggregation are mediated by thrombin cleavage of the exodomain of the PAR1 receptor. The specificity of thrombin for PAR1 is enhanced by binding to a hirudin-like region (Hir) located in the receptor exodomain. Here, we examine the mechanism of thrombin-PAR1 recognition and cleavage by steady-state kinetic measurements using soluble PAR1 N-terminal exodomains. We determined that the primary role of the PAR1 Hir sequence is to reduce the kinetic barriers to formation of the docked thrombin-PAR1 complex rather than to form high affinity ground-state interactions. In addition, the exosite I-bound Hir motif facilitates the productive interaction of the PAR1 (38)LDPR/SFL(44) sequence with the active site of thrombin. This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin. This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence.


Assuntos
Receptores de Trombina/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Catálise , Dados de Sequência Molecular , Receptor PAR-1 , Receptores de Trombina/química , Trombina/farmacologia
8.
FEBS Lett ; 477(3): 199-202, 2000 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-10908720

RESUMO

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.


Assuntos
Plaquetas/metabolismo , Receptores de Trombina/metabolismo , Serina Endopeptidases/metabolismo , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Plaquetas/efeitos dos fármacos , Células Cultivadas , Humanos , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Trombina/farmacologia
9.
Biochemistry ; 39(18): 5458-67, 2000 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-10820018

RESUMO

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.


Assuntos
Plaquetas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/metabolismo , Transdução de Sinais , Difosfato de Adenosina/farmacologia , Animais , Anticorpos/farmacologia , Células COS , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Cinética , Oligopeptídeos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Receptor PAR-1 , Trombina/farmacologia , Transfecção
10.
J Biol Chem ; 275(4): 2627-35, 2000 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-10644723

RESUMO

Signal transfer between the protease-activated PAR1 thrombin receptor and membrane-associated heterotrimeric G proteins is mediated by protein-protein interactions. We constructed a yeast signaling system that resolves domain-specific functions of binding from coupling in the Galpha subunit. The endogenous yeast Galpha subunit, Gpa1, does not bind to PAR1 and served as a null structural template. N- and C-terminal portions of mammalian G(i2) and G(16) were substituted back into the Gpa1 template and gain-of-function assessed. The C-terminal third of G(16), but not of G(i2), provides sufficient interactions for coupling to occur with PAR1. The N-terminal two-thirds of G(i2) also contains sufficient determinants to bind and couple to PAR1 and overcome the otherwise negative or missing interactions supplied by the C-terminal third of Gpa1. Replacement of the N-terminal alpha-helix of G(i2), residues 1-34, with those of Gpa1 abolishes coupling but not binding to PAR1 or to betagamma subunits. These data support a model that the N-terminal alphaN helix of the Galpha subunit is physically interposed between PAR1 and the Gbeta subunit and directly assists in transferring the signal between agonist-activated receptor and G protein.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Trombina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Proteínas de Ligação ao GTP/química , Humanos , Hidrólise , Modelos Moleculares , Ligação Proteica , Ensaio Radioligante , Receptor PAR-1 , Saccharomyces cerevisiae/metabolismo , Trombina/metabolismo
12.
Biochemistry ; 38(14): 4572-85, 1999 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-10194379

RESUMO

It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases. Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain. We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts. The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82. Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.


Assuntos
Fibrinolisina/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Terapia Trombolítica , Sequência de Aminoácidos , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células COS , Sinalização do Cálcio/efeitos dos fármacos , Fibroblastos , Glicosilação , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese Insercional , Fragmentos de Peptídeos/biossíntese , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Estrutura Terciária de Proteína , Coelhos , Receptor PAR-1 , Receptores de Trombina/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Solubilidade , Ativador de Plasminogênio Tecidual/farmacologia
14.
Biochemistry ; 34(2): 481-9, 1995 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-7819240

RESUMO

The propeptide binding/activation site on the vitamin K dependent carboxylase has been localized to a region of carboxylase between residues Arg +50 and Glu +225 by photoinactivation studies using [125I]tyrosyl-labeled benzoylphenylalanine (Bpa)-containing analogs of proFIX19, a peptide containing residues -18 to +1 of factor IX. Four proFIX19 analogs with Bpa substituents at -16, -13, -7, and -6 were synthesized. These peptides were specific photoinactivators of carboxylase and were used to label a His6-carboxylase construct produced in baculovirus-infected insect cells. Fragments of the labeled carboxylase produced by V8 protease digestion were analyzed by peptide-specific antibodies and by autoradiography. The propeptide recognition site was localized to the N-terminal one-third of the 94 kDa carboxylase. This is consistent with previous studies using a carboxylase substrate affinity label, N-(bromoacetyl)-FLEELY [Kuliopulos, A., Nelson, N.P., Yamada, M., Walsh, C.T., Furie, B., Furie, B.C., & Roth, D.A. (1994) J. Biol. Chem. 269, 21364-21370], indicating that the propeptide binding site and the FLEEL binding site are both located within the N-terminal one-third of the vitamin K dependent carboxylase.


Assuntos
Carbono-Carbono Ligases , Fator IX/metabolismo , Ligases/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Ligases/antagonistas & inibidores , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fenilalanina/análogos & derivados , Fotoquímica , Ligação Proteica , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Serina Endopeptidases , Vitamina K/metabolismo
15.
J Biol Chem ; 269(33): 21364-70, 1994 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-8063763

RESUMO

A recombinant His6-tagged vitamin K-dependent gamma-glutamyl carboxylase has been produced in baculovirus-infected insect cells. The His6-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes. The His6-carboxylase was irreversibly inactivated by the N-bromoacetyl-FLEEL-125I-Y peptide substrate/affinity label under pseudo-first order conditions. This inactivation could be abolished by coincubation with a high affinity peptide substrate consistent with an active site-directed inactivation. The inactivated His6-carboxylase-Ac-FLEEL-125I-Y, purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis, was subjected to proteolytic digestions with either Glu-C or Lys-C endoproteinases. The resulting polypeptide fragments were probed with three regiospecific antibodies which recognized epitopes present at the extreme N terminus (residues -23 to -13), at the hydrophobic N-terminal region (residues 86-99), and at the hydrophilic C-terminal region (residues 661-673). The site of attachment to the 125I-affinity label is located within the first 218 amino acid residues of the 758-residue carboxylase. This is the first evidence for the involvement of either the putative membrane-anchoring hydrophobic region (residues 50-314) or possibly the N-terminal hydrophilic region (residues 1-50) in gamma-carboxylation of glutamate-peptide substrates.


Assuntos
Carbono-Carbono Ligases , Ligases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Hidrólise , Radioisótopos do Iodo , Cinética , Ligases/química , Ligases/genética , Ligases/metabolismo , Dados de Sequência Molecular , Mariposas , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
16.
Proc Natl Acad Sci U S A ; 90(10): 4611-5, 1993 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-8506307

RESUMO

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.


Assuntos
Carbono-Carbono Ligases , Ligases/genética , Sequência de Aminoácidos , Animais , Células CHO , Bovinos , Clonagem Molecular , Cricetinae , Fator IX/metabolismo , Expressão Gênica , Ligases/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Peptídeos/química , Peptídeos/imunologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transfecção
17.
Biochemistry ; 32(7): 1816-24, 1993 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-8439542

RESUMO

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and conservative transfer of the 4 beta-proton to the 6 beta-position. The 10(9.5)-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 +/- 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutation of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa-7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr-14 of 11.6 +/- 0.2 was used to estimate a local dielectric constant of 18 +/- 2 near this residue.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Esteroide Isomerases/química , Tirosina/química , Sequência de Bases , Sítios de Ligação , Cristalização , Cinética , Lisina/química , Lisina/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Nandrolona/metabolismo , Pseudomonas/enzimologia , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismo , Termodinâmica , Tirosina/genética
18.
Biochemistry ; 31(39): 9436-44, 1992 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-1390725

RESUMO

Vitamin K dependent carboxylase (carboxylase) is a membrane-associated endoplasmic reticular enzyme that catalyzes the conversion of certain glutamate residues of essential blood coagulation proteins to gamma-carboxyglutamyl (Gla) residues. A series of N-bromoacetyl-peptide substrate affinity labels based on the Gla domain of these blood-clotting proteins was synthesized, and the substrate and inactivator kinetic parameters were assessed. The most promising of these affinity peptides, N-bromoacetyl-FLEELY, was both substrate for carboxylase and an irreversible time-dependent inactivator of the enzyme, inactivating 80% of carboxylase under pseudo-first-order conditions. Addition of saturating amounts of a competing peptide substrate completely abolished the inhibitory properties of N-bromoacetyl-FLEELY, consistent with inactivation occurring at the active site. The partition ratio of inactivation/carboxylation was 1/30. The 94-kDa carboxylase was purified to 15-50% purity by a modification of a recent protocol [Wu, S.-M., Morris, D. P., & Stafford, D. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2236-2240] and covalently labeled with N-bromoacetyl-FLEEL[125I]Y. On silver-stained 10% sodium dodecyl sulfate-polyacrylamide gels, the predominant radiolabeled band was the 94,000 molecular weight species. This result independently validates that the 94-kDa protein is a carboxylase.


Assuntos
Marcadores de Afinidade/metabolismo , Carbono-Carbono Ligases , Ligases/antagonistas & inibidores , Ligases/metabolismo , Acetatos/farmacologia , Marcadores de Afinidade/síntese química , Marcadores de Afinidade/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Cinética , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Especificidade por Substrato
19.
Biochemistry ; 31(33): 7722-8, 1992 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-1510957

RESUMO

The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.


Assuntos
Carbono-Carbono Ligases , Ligases/metabolismo , Microssomos Hepáticos/enzimologia , Oxigênio/metabolismo , Animais , Bovinos , Marcação por Isótopo/métodos , Ligases/isolamento & purificação , Espectrometria de Massas/métodos , Isótopos de Oxigênio , Vitamina K 1/análogos & derivados , Vitamina K 1/metabolismo
20.
Arch Biochem Biophys ; 294(2): 327-40, 1992 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-1567189

RESUMO

The quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on which kinetic or thermodynamic parameter of an enzyme is measured, the same two mutations can interact differently in the double mutant. Additive effects of two mutations on an equilibrium constant, such as the dissociation constant of the enzyme-substrate complex (KS), occur when noninteracting residues which facilitate the same step (substrate binding) are mutated. Partially additive effects result from the cooperative interaction with the substrate of the two residues mutated, and synergistic effects result from the anticooperative interaction with the substrate of the two residues mutated. An alternative explanation for synergy is extensive unfolding of the enzyme. Antagonistic effects on an equilibrium constant such as KS result from opposing structural effects of the two mutations on substrate binding. No additional effect of the second mutation in the double mutant represents a limiting case of either partial additivity or antagonism [corrected]. The interactions of the effects of two mutations on a rate constant such as kcat have the same explanations as those given above for equilibrium constants since the binding of a rate-limiting transition state is occurring. However, due to kinetic complexity, the following exceptions and additions exist. Additive effects of two mutations on kcat occur when noninteracting residues which facilitate the same step are mutated, provided this step is rate limiting. If the affected step is not rate limiting then synergistic effects of the two mutations are observed as each mutation causes the step to become progressively more rate limiting. Additive effects on kcat also occur when the two mutations affect consecutive steps, provided one of them is rate limiting. Partially additive effects on kcat also occur when noninteracting residues facilitating consecutive, non-rate-limiting steps are mutated. These concepts, when applied to published data on double mutants of delta 5-3-ketosteroid isomerase, staphylococcal nuclease, tyrosyl-tRNA synthetase, glutathione reductase, and subtilisin, provide deeper insights into the independent, cooperative, anticooperative, or antagonistic interactions of amino acid residues in the binding of substrates, activators, and inhibitors and in promoting catalysis.


Assuntos
Enzimas/genética , Enzimas/metabolismo , Mutação , Sequência de Aminoácidos , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Cinética , Matemática , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Modelos Teóricos , Conformação Proteica , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismo , Termodinâmica , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Difração de Raios X
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