RESUMEN
Psoriasis is an inflammatory skin disease often associated with obesity. The anti-inflammatory adipokine vaspin, a suggested serine proteinase inhibitor of the serpin family, is discussed as a new link between inflammation and obesity. Here, we demonstrate that - different from healthy controls - vaspin serum levels in patients with psoriasis were body mass index independent. Moreover, we could identify keratinocytes as the major source of vaspin in skin. Vaspin expression in lesional psoriatic skin was reduced compared with uninvolved skin as shown by immunohistochemistry and RT-PCR. In aggregate, we report on the cellular source of vaspin in skin and its expression in psoriasis.
Asunto(s)
Obesidad/complicaciones , Obesidad/metabolismo , Psoriasis/complicaciones , Psoriasis/metabolismo , Serpinas/metabolismo , Secuencia de Bases , Índice de Masa Corporal , Estudios de Casos y Controles , Expresión Génica , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Queratinocitos/metabolismo , Obesidad/sangre , Obesidad/genética , Obesidad/patología , Psoriasis/sangre , Psoriasis/genética , Psoriasis/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serpinas/sangre , Serpinas/genética , Piel/metabolismo , Piel/patologíaRESUMEN
In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis. Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors. For most of the inhibitors studied, progressive loop replacement at the trypsin surface resulted in inhibitory profiles akin to factor Xa. Crystals of the variants were obtained in the presence of benzamidine (3), and could be soaked with the highly specific factor Xa inhibitor (1). Binding of the latter to X99rT results in a series of structural adaptations to the ligand, including the establishment of an "aromatic box" characteristic of factor Xa. In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the "intermediate helix", otherwise common to trypsin and factor Xa. The re-orientation is accompanied by an isomerisation of the Cys168-Cys182 disulphide bond, and burial of the critical Phe174 side-chain. In the presence of (1), a major re-organisation of the binding site takes place to yield a geometry identical to that of factor Xa. In all, binding of (1) to trypsin and its variants results in significant structural rearrangements, inducing a binding surface strongly reminiscent of factor Xa, against which the inhibitor was optimised. The structural data reveal a plasticity of the intermediate helix, which has been implicated in the functional cofactor dependency of many trypsin-like serine proteinases. This approach of grafting loops onto scaffolds of known related structures may serve to bridge the gap between structural genomics and drug design.
Asunto(s)
Factor Xa/química , Factor Xa/metabolismo , Conformación Proteica , Pliegue de Proteína , Tripsina/química , Animales , Sitios de Unión , Dominio Catalítico , Bovinos , Cristalografía por Rayos X/métodos , Factor Xa/genética , Humanos , Cinética , Ligandos , Modelos Moleculares , Estructura Molecular , Ratas , Relación Estructura-Actividad , Tripsina/metabolismo , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
Although proteases are capable of synthesizing peptide bonds, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides. This account reports on the synthetic potential of nonactivatable trypsinogen and zymogen-like enzymes designed to minimize proteolytic side reactions during peptide synthesis.