RESUMEN

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

Asunto(s)

Ácido Aspártico Endopeptidasas/metabolismo , Catepsina D/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/química , Catepsina D/química , Catepsina E/genética , Catepsina E/metabolismo , Extractos Celulares/química , Línea Celular , Cerámica , Endopeptidasas , Humanos , Metaloendopeptidasas/metabolismo , Ratones , Microesferas , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Pepstatinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Especificidad por Sustrato , Suecia , Transfección

RESUMEN

The Alzheimer beta-amyloid peptide (Abeta) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Abeta polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Péptidos/metabolismo , Priones/metabolismo , Piridonas/farmacología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/ultraestructura , Western Blotting , Colorantes/farmacología , Rojo Congo/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Ligandos , Unión Proteica , Piridonas/química , Albúmina Sérica/metabolismo , Espectrometría de Fluorescencia , Factores de Tiempo

RESUMEN

Phytase is a high molecular weight acid phosphatase. The structure has an alpha/beta-domain similar to that of rat acid phosphatase and an alpha-domain with a new fold.

Asunto(s)

6-Fitasa/química , Fosfatasa Ácida/química , Aspergillus/enzimología , Pliegue de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Animales , Simulación por Computador , Cristalografía por Rayos X , Cisteína , Disulfuros , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido , Programas Informáticos , Electricidad Estática

RESUMEN

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.

Asunto(s)

Endopeptidasas/metabolismo , Productos del Gen pol/metabolismo , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Aminoácidos , Proteasa del VIH , VIH-1/metabolismo , Cinética , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato

RESUMEN

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/aislamiento & purificación , Quelantes , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Endorribonucleasas/genética , Endorribonucleasas/aislamiento & purificación , Escherichia coli/metabolismo , Productos del Gen pol/aislamiento & purificación , Vectores Genéticos , VIH-1/genética , Humanos , Plásmidos , ADN Polimerasa Dirigida por ARN/genética , Ribonucleasa H

RESUMEN

The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3.

Asunto(s)

Anticodón/metabolismo , VIH-1/enzimología , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Lisina/metabolismo , ARN de Transferencia/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Cápside/metabolismo , Línea Celular , Electroforesis , VIH-1/genética , Humanos , Immunoblotting , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia de Lisina/genética , Proteínas del Núcleo Viral/metabolismo

RESUMEN

Two single site substitutions (E478----Q and H539----F) were introduced into the C-terminal RNase H domain of HIV-1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

Asunto(s)

Endorribonucleasas/metabolismo , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Aminoácidos , Análisis Mutacional de ADN , VIH-1/genética , Datos de Secuencia Molecular , ADN Polimerasa Dirigida por ARN/genética , Ribonucleasa H , Relación Estructura-Actividad