RESUMEN

A subset of the neutralizing anti-HIV antibodies recognize epitopes on the envelope protein gp120 of the human immunodeficiency virus. These epitopes are exposed during conformational changes when gp120 binds to its primary receptor CD4. Based on chemical modification of lysine and arginine residues followed by mass spectrometric analysis, we determined the epitope on gp120 recognized by the human monoclonal antibody 559/64-D, which was previously found to be specific for the CD4 binding domain. Twenty-four lysine and arginine residues in recombinant full-length glycosylated gp120 were characterized; the relative reactivities of two lysine residues and five arginine residues were affected by the binding of 559/64-D. The data show that the epitope is discontinuous and is located in the proximity of the CD4-binding site. Additionally, the reactivities of a residue that is located in the secondary receptor binding region and several residues distant from the CD4 binding site were also altered by Ab binding. These data suggest that binding of 559/64-D induced conformational changes which result in altered surface exposure of specific amino acids distant from the CD4-binding site. Consequently, binding of 559/64-D to gp120 affects not only the CD4-binding site, which is recognized as the epitope, but appears to have a global effect on surface exposed residues of the full-length glycosylated gp120.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Mapeo Epitopo/métodos , Epítopos/química , Proteína gp120 de Envoltorio del VIH/química , Espectrometría de Masas/métodos , Acetilación , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Arginina/química , Arginina/metabolismo , Antígenos CD4/química , Antígenos CD4/metabolismo , Epítopos/metabolismo , Glicosilación , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica

RESUMEN

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.

Asunto(s)

Anticolesterolemiantes/farmacología , Azetidinas/farmacología , Antígenos CD13/metabolismo , Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , Enterocitos/efectos de los fármacos , Microvellosidades/efectos de los fármacos , Absorción/efectos de los fármacos , Animales , Anticolesterolemiantes/química , Anticolesterolemiantes/metabolismo , Azetidinas/química , Azetidinas/metabolismo , Células CACO-2 , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Membrana Celular/enzimología , Membrana Celular/metabolismo , Cromatografía de Afinidad , Colon/citología , Enterocitos/citología , Enterocitos/enzimología , Ezetimiba , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Intestino Delgado/citología , Riñón/citología , Micelas , Microvellosidades/enzimología , Microvellosidades/metabolismo , Peso Molecular , Etiquetas de Fotoafinidad , Unión Proteica , Conejos , Espectrometría de Masa por Ionización de Electrospray , Estómago/citología

RESUMEN

The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

Asunto(s)

Endopeptidasas/química , Espectrometría de Masas/métodos , Complejos Multiproteicos , Ribonucleoproteína Nuclear Pequeña U1/química , Empalmosomas/metabolismo , Cromatografía Liquida , Humanos , Isótopos , Ácidos Nicotínicos/farmacología , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Succinimidas/farmacología

RESUMEN

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.

Asunto(s)

Proteína S6 Ribosómica/fisiología , Proteínas Ribosómicas/fisiología , Ribosomas/fisiología , Levaduras/fisiología , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteína Ribosómica L10 , Proteína S6 Ribosómica/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Levaduras/genética

RESUMEN

A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.

Asunto(s)

Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Péptidos/química , Angiotensinas/química , Bradiquinina/química , Cromatografía , Cromatografía Líquida de Alta Presión , Fluoresceínas/farmacología , Humanos , Ligandos , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo