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1.
Regul Toxicol Pharmacol ; 95: 236-243, 2018 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-29574193

RESUMEN

Bevacizumab, a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is approved for treatment of metastatic colorectal cancer, nonsquamous non-small-cell lung cancer, metastatic kidney cancer, and glioblastoma. To support clinical development of the potential bevacizumab biosimilar PF-06439535, nonclinical studies evaluated structural, functional, toxicological, and toxicokinetic similarity to bevacizumab sourced from the European Union (bevacizumab-EU) and United States (bevacizumab-US). Peptide mapping demonstrated the amino acid sequence of PF-06439535 was identical to bevacizumab-EU and bevacizumab-US. Biologic activity, measured via inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells and binding to VEGF isoforms, was similar across the three drugs. In vivo similarity was demonstrated in cynomolgus monkeys administered intravenous PF-06439535 or bevacizumab-EU (0 or 10 mg/kg/dose twice weekly for 1 month; total of nine doses). Systemic exposure appeared similar and test article-related effects were limited to physeal dysplasia of the distal femur. The potential for non-target-mediated toxicity of PF-06439535 was evaluated in rats administered intravenous PF-06439535 (15 or 150 mg/kg/dose twice weekly for 15 days; total of five doses). Nonadverse higher liver weights and minimal sinusoidal cell hyperplasia were observed. Collectively, these studies demonstrated similarity of PF-06439535 to bevacizumab, supporting entry into clinical development.


Asunto(s)
Inhibidores de la Angiogénesis/toxicidad , Antineoplásicos Inmunológicos/toxicidad , Bevacizumab/toxicidad , Biosimilares Farmacéuticos/toxicidad , Inhibidores de la Angiogénesis/sangre , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos Inmunológicos/sangre , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/farmacología , Bevacizumab/sangre , Bevacizumab/farmacocinética , Bevacizumab/farmacología , Biosimilares Farmacéuticos/farmacocinética , Biosimilares Farmacéuticos/farmacología , Proliferación Celular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Macaca fascicularis , Masculino , Estructura Molecular , Tamaño de los Órganos/efectos de los fármacos , Unión Proteica , Isoformas de Proteínas/metabolismo , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/metabolismo
2.
Nucleic Acids Res ; 38(12): 4052-66, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20215441

RESUMEN

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20-Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.


Asunto(s)
Autoantígenos/química , ARN no Traducido/química , Ribonucleasa P/química , Secuencia de Aminoácidos , Autoantígenos/metabolismo , Dimerización , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Largo no Codificante , ARN no Traducido/metabolismo , Ribonucleasa P/metabolismo
3.
Nat Commun ; 13(1): 6133, 2022 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-36253406

RESUMEN

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-JUN proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-JUN multisite phosphorylation has remained elusive. Here we show that c-JUN phosphorylation by JNK exhibits defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identify the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis reveals three c-JUN phosphorylation states: unphosphorylated c-JUN recruits the MBD3 repressor, serine63/73 doubly-phosphorylated c-JUN binds to the TCF4 co-activator, whereas the fully phosphorylated form disfavours TCF4 binding attenuating JNK signalling. Thus, c-JUN phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.


Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Proto-Oncogénicas c-jun , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal
4.
Acta Obstet Gynecol Scand ; 88(5): 606-11, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19308748

RESUMEN

OBJECTIVES: To investigate a possible association between pre-eclampsia (PE) and the genotype for the angiotensin II type-1 receptor (AT1R) and the angiotensin type-2 receptor (AT2R) in various population groups. DESIGN: The study was retrospective in a case-controlled design. SAMPLES: Two hundred thirty-six pregnant women with PE/eclampsia (E) and 426 non-hypertensive pregnant women were included. METHOD: Polymorphic sites of AT1R (A1166C) and AT2R (A1675G) were amplified by polymerase chain reaction, digested with a restriction enzyme that differentiated between the alternative alleles, and analyzed. MAIN OUTCOME MEASURES: Maternal genotypes and their correlation with clinical parameters. RESULTS: The frequency of the AT2R-GG genotype (A1675G) in the PE group was significantly greater than in controls for Afro-Caribbean women (49.3% vs 26.9%, p=0.004), but the frequency difference in Asian or Caucasian women was not significant (23.0% vs 25.4%, p=0.63; 27.7% vs 14.8%, p=0.17, respectively). The highly significant difference in Afro-Caribbean women was maintained after controlling for the effects of age, BMI and parity (p=0.005). There was no significant association of the molecular variant of AT1R (A1166C) with PE in Afro-Caribbean, Caucasian or Asian women. However, in the whole PE group compared to the controls there was a higher proportion of the AT2R-GG genotype with AT1R-AC (56% vs 44%, OR 2.37; 95% CI: 1.06-5.32). In Afro-Caribbean women, the combination of AT1R-AC with AT2R-AG genotypes was significantly higher in controls compared to PE group (93.8% vs 6.3%, OR 0.11; 95% CI: 0.01-0.81). CONCLUSION: There is an association between PE/E and the GG-genotype of AT2R in Afro-Caribbean women.


Asunto(s)
Polimorfismo Genético , Preeclampsia/genética , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 2/genética , Adulto , Pueblo Asiatico , Población Negra , Índice de Masa Corporal , Estudios de Casos y Controles , Fragmentación del ADN , Femenino , Genotipo , Humanos , Paridad , Preeclampsia/etnología , Embarazo , Estudios Retrospectivos , Población Blanca
5.
Artículo en Inglés | MEDLINE | ID: mdl-18453699

RESUMEN

Gene PA1377 from Pseudomonas aeruginosa encodes a 177-amino-acid conserved hypothetical protein of unknown function. The structure of this protein (termed pitax) has been solved in space group I222 to 2.25 A resolution. Pitax belongs to the GCN5-related N-acetyltransferase family and contains all four sequence motifs conserved among family members. The beta-strand structure in one of these motifs (motif A) is disrupted, which is believed to affect binding of the substrate that accepts the acetyl group from acetyl-CoA.


Asunto(s)
Acetiltransferasas/química , Coenzima A/química , Pseudomonas aeruginosa/enzimología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Coenzima A/metabolismo , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido
7.
Biochimie ; 91(4): 484-9, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19135125

RESUMEN

Gene ACIAD1637 from Acinetobacter baylyi ADP1 encodes a 182 amino acid putative antibiotic resistance protein. The structure of this protein (termed acepita) has been solved in space group P(2) to 2.35 A resolution. Acepita belongs to the GCN5-related N-acetyltransferase (GNAT) family, and contains the four sequence motifs conserved among family members. The structure of acepita is compared with that of pita, its homologue from Pseudomonas aeruginosa. Acepita has a similar substrate profile to pita and performs a similar function.


Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , Acinetobacter/enzimología , Dominio Catalítico/fisiología , Acetiltransferasas/genética , Acinetobacter/metabolismo , Secuencia de Aminoácidos , Cristalización , Cinética , Datos de Secuencia Molecular , Estructura Molecular , Mutación/genética , Alineación de Secuencia , Especificidad por Sustrato/fisiología
8.
Biochemistry ; 46(7): 1829-39, 2007 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-17253769

RESUMEN

The gene PA4866 from Pseudomonas aeruginosa is documented in the Pseudomonas genome database as encoding a 172 amino acid hypothetical acetyltransferase. We and others have described the 3D structure of this protein (termed pita) [Davies et al. (2005) Proteins: Struct., Funct., Bioinf. 61, 677-679; Nocek et al., unpublished results], and structures have also been reported for homologues from Agrobacterium tumefaciens (Rajashankar et al., unpublished results) and Bacillus subtilis [Badger et al. (2005) Proteins: Struct., Funct., Bioinf. 60, 787-796]. Pita homologues are found in a large number of bacterial genomes, and while the majority of these have been assigned putative phosphinothricin acetyltransferase activity, their true function is unknown. In this paper we report that pita has no activity toward phosphinothricin. Instead, we demonstrate that pita acts as an acetyltransferase using the glutamate analogues l-methionine sulfoximine and l-methionine sulfone as substrates, with Km(app) values of 1.3 +/- 0.21 and 1.3 +/- 0.13 mM and kcat(app) values of 505 +/- 43 and 610 +/- 23 s-1 for l-methionine sulfoximine and l-methionine sulfone, respectively. A high-resolution (1.55 A) crystal structure of pita in complex with one of these substrates (l-methionine sulfoximine) has been solved, revealing the mode of its interaction with the enzyme. Comparison with the apoenzyme structure has also revealed how certain active site residues undergo a conformational change upon substrate binding. To investigate the role of pita in P. aeruginosa, a mutant strain, Depp4, in which pita was inactivated through an in-frame deletion, was constructed by allelic exchange. Growth of strain Depp4 in the absence of glutamine was inhibited by l-methionine sulfoximine, suggesting a role for pita in protecting glutamine synthetase from inhibition.


Asunto(s)
Acetiltransferasas/química , Aminobutiratos/química , Metionina Sulfoximina/química , Pseudomonas aeruginosa/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Aminobutiratos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Glutamato-Amoníaco Ligasa/metabolismo , Cinética , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Metionina Sulfoximina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Estereoisomerismo , Especificidad por Sustrato
9.
Biochem J ; 365(Pt 3): 731-8, 2002 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-11955282

RESUMEN

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.


Asunto(s)
Amidohidrolasas/química , Dominio Catalítico , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/enzimología , Amidohidrolasas/genética , Amidohidrolasas/aislamiento & purificación , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pseudomonas aeruginosa/genética , Alineación de Secuencia , Especificidad por Sustrato
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