RESUMEN
Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.
Asunto(s)
Activación Plaquetaria , Adhesividad Plaquetaria , Dominios y Motivos de Interacción de Proteínas , Precursores de Proteínas/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Adulto , Animales , Sitios de Unión , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Humanos , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/genética , Adhesividad Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/genética , Adhesividad Plaquetaria/fisiología , Unión Proteica/genética , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Transfección , Factor de von Willebrand/genética , Factor de von Willebrand/farmacologíaRESUMEN
The carcinogenic potential of naveglitazar, a gamma-dominant peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual agonist, was evaluated in a two-year study in F344 rats (0, 0.3, 1.0, or 3.0 mg/kg, males; 0, 0.1, 0.3, or 1.0 mg/kg, females). Increased mortality in male rats of the high-dose group was related to cardiac-associated lesions, neoplasms, and undetermined causes. Degeneration and hypertrophy of the myocardium occurred with dose-responsive increased incidence and severity. Neoplasms with increased incidence included sarcomas in male rats and urinary bladder neoplasms in female rats. Most sarcomas in male rats occurred in the adipose tissue of the subcutis and were diagnosed as fibrosarcomas, with fewer liposarcomas and other histologic types. Non-neoplastic changes in adipose tissue included expansion of adipose tissue in multiple sites, alterations in cytoplasmic vesicular pattern in brown and white fat, increases in stroma and mesenchymal cells, and fibrosis. The severity of chronic progressive nephropathy was decreased in a dose-responsive manner in males, and hyperplasia and neoplasia of the mammary gland were decreased in incidence in females. The adverse effects of cardiotoxicity and increased incidence of neoplasms occurred with dose-responsive incidence and/or severity, and a no-effect level for these effects was not achieved in this study.
Asunto(s)
Pruebas de Carcinogenicidad/métodos , Neoplasias Experimentales/inducido químicamente , Fenilpropionatos/toxicidad , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Genitales/patología , Histocitoquímica , Hiperplasia/inducido químicamente , Masculino , Neoplasias Experimentales/patología , PPAR alfa/agonistas , PPAR gamma/agonistas , Ratas , Ratas Endogámicas F344 , Sarcoma/inducido químicamente , Sarcoma/patología , Urotelio/patologíaRESUMEN
To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas/análisis , Anticuerpos Catalíticos/análisis , Benchmarking , Sitios de Unión , Técnicas Biosensibles/estadística & datos numéricos , Glutatión Transferasa/análisis , Cinética , LigandosRESUMEN
Naveglitazar, a gamma-dominant peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual agonist, was tested for carcinogenicity in F344 rats in a 2-year study. Changes in urine composition and urothelial morphology were characterized in a companion 18-month investigative study. A significant increase in neoplasms of the bladder occurred only in females of the high-dose group (14/60) in the carcinogenicity study. Trends toward increased cell proliferation in the urothelium were noted in both sexes at all time points evaluated in the 18-month study. Group means for urothelial mitogenesis were increased statistically significantly only in high-dose females at 12 and 18 months. Urothelial hyperplasia occurred in high-dose females at 18 months. Morphologic changes in the urothelium at earlier time points were limited to hypertrophy and decreased immunolabeling of the superficial cells for cytokeratin 20 (a marker of terminal differentiation in urothelial cells) in both males and females. No treatment-related changes in urinary parameters, including urinary sediments, were associated with the occurrence of urothelial proliferation. Urinary pH was unaffected by treatment in both males and females, but expected diurnal changes were demonstrated. Collectively, these data indicate that naveglitazar was associated with hypertrophic and proliferative effects on the urothelium, but a link with changes in urinary parameters was not demonstrated.
Asunto(s)
PPAR gamma/agonistas , Fenilpropionatos/toxicidad , Neoplasias de la Vejiga Urinaria/inducido químicamente , Vejiga Urinaria/efectos de los fármacos , Urolitiasis/inducido químicamente , Urotelio/efectos de los fármacos , Animales , Calcio/orina , Pruebas de Carcinogenicidad , Proliferación Celular/efectos de los fármacos , Cristalización , Relación Dosis-Respuesta a Droga , Femenino , Hiperplasia/inducido químicamente , Hiperplasia/patología , Longevidad/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas F344 , Urinálisis , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/patología , Urolitiasis/patología , Urotelio/patología , Urotelio/ultraestructuraRESUMEN
The cellular microenvironment impacts how signals are transduced by cells and plays a key role in tissue homeostasis. Although pH is generally well regulated, there are a number of situations where acidosis occurs and our work addresses how low pH impacts cell association of insulin-like growth factor-I (IGF-I) in the presence of IGF binding protein-3 (IGFBP-3). We have previously shown that IGF-I cell binding was enhanced in the presence of IGFBP-3 at low pH and now show that this binding is IGFBP-mediated as it is inhibited by Y60L-IGF-I, a mutant with reduced affinity for the IGF receptor (IGF-IR), and unaffected by insulin, which binds but not IGFBPs. Using surface plasmon resonance (SPR), we show that direct binding between IGF-I and IGFBP-3 is pH sensitive. Despite this, the key step in the process appears to be IGFBP-3 cell surface association as Long-R(3)-IGF-I, a mutant with reduced affinity for IGFBPs, shows a similar increase in cell association at pH 5.8 in the presence of IGFBP-3 but does not exhibit pH-dependent binding by SPR. Further, analysis indicates a large increase in low-affinity binding sites for IGF-I in the presence of IGFBP-3 and an elimination of IGF-I enhanced binding when a non-cell associating mutant of IGFBP-3 is added in place of IGFBP-3. That the IGFBP-3-mediated binding localizes IGF-I away from IGF-IR is suggested by triton-solubility testing and indicates additional complexities to IGF-I regulation by IGFBP-3. Identifying the pH-dependent binding partner(s) for IGFBP-3 is a necessary next step in deciphering this process.
Asunto(s)
Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Animales , Sitios de Unión/fisiología , Bovinos , Agregación Celular/fisiología , Línea Celular , Concentración de Iones de Hidrógeno , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/genética , Cinética , Sustancias Macromoleculares/metabolismo , Mutación/genética , Unión Proteica/fisiología , Transducción de Señal/fisiología , Solubilidad , Propiedades de SuperficieRESUMEN
In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.
Asunto(s)
Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Sulfonamidas/antagonistas & inhibidores , Técnicas Biosensibles , Calorimetría , Inhibidores de Anhidrasa Carbónica/clasificación , Variaciones Dependientes del Observador , Unión Proteica , Investigadores , Sulfonamidas/clasificación , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/normasRESUMEN
The kinetics of enzymatic surface-initiated polymerization of PHB on gold surface has been examined by SPR and the resultant polymer layers characterized by AFM and FT-IR spectrometry. The immobilized enzyme catalyzed surface-initiated polymerization of 3HB-CoA, resulting in the formation of a polymer brush on the surface. The rate of polymer growth from the surface was monitored by SPR in real-time. Polymer growth as measured by the increase in the resonance angle showed no apparent lag phase during the polymerization reaction. SPR analysis also revealed that the thickness of the polymer film could be controlled by varying the initial enzyme density on the surface. The average thicknesses of the PHB film after polymerization reaction were 95, 45 and 15 nm for the surfaces that were treated with 0.5, 0.3 and 0.1*10(-6) M of enzyme, respectively. The binding of PHA synthase at different concentration to the mixed SAMs and subsequent polymerization.
Asunto(s)
Poliésteres/química , Poliésteres/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Cupriavidus necator/enzimología , Cupriavidus necator/genética , Enzimas Inmovilizadas , Oro , Microscopía de Fuerza Atómica , Ácido Nitrilotriacético/análogos & derivados , Compuestos Organometálicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Silicio , Resonancia por Plasmón de SuperficieRESUMEN
Rebinding of dissociated ligands from cell surface proteins can confound quantitative measurements of dissociation rates important for characterizing the affinity of binding interactions. This can be true also for in vitro techniques such as surface plasmon resonance (SPR). We present experimental results using SPR for the interaction of insulin-like growth factor-I (IGF-I) with one of its binding proteins, IGF binding protein-3 (IGFBP-3), and show that the dissociation, even with the addition of soluble heparin in the dissociation phase, does not exhibit the expected exponential decay characteristic of a 1:1 binding reaction. We thus consider the effect of (multiple) rebinding events and, within a self-consistent mean-field approximation, we derive the complete mathematical form for the fraction of bound ligands as a function of time. We show that, except for very low association rate and surface coverage, this function is nonexponential at all times, indicating that multiple rebinding events strongly influence dissociation even at early times. We compare the mean-field results with numerical simulations and find good agreement, although deviations are measurable in certain cases. Our analysis of the IGF-I-IGFBP-3 data indicates that rebinding is prominent for this system and that the theoretical predictions fit the experimental data well. Our results provide a means for analyzing SPR biosensor data where rebinding is problematic and a methodology to do so is presented.