RESUMEN
Caspases form a family of proteinases required for the initiation and execution phases of apoptosis. Distinct proapoptotic stimuli lead to activation of the initiator caspases-8 and -9, which in turn activate the common executioner caspases-3 and -7 by proteolytic cleavage. Whereas crystal structures of several active caspases have been reported, no three-dimensional structure of an uncleaved caspase zymogen is available so far. We have determined the 2.9-A crystal structure of recombinant human C285A procaspase-7 and have elucidated the activation mechanism of caspases. The overall fold of the homodimeric procaspase-7 resembles that of the active tetrameric caspase-7. Each monomer is organized in two structured subdomains connected by partially flexible linkers, which asymmetrically occupy and block the central cavity, a typical feature of active caspases. This blockage is incompatible with a functional substrate binding site/active site. After proteolytic cleavage within the flexible linkers, the newly formed chain termini leave the cavity and fold outward to form stable structures. These conformational changes are associated with the formation of an intact active-site cleft. Therefore, this mechanism represents a formerly unknown type of proteinase zymogen activation.
Asunto(s)
Caspasas/metabolismo , Precursores Enzimáticos/metabolismo , Secuencia de Aminoácidos , Caspasa 7 , Caspasas/química , Cristalización , Dimerización , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Precursores Enzimáticos/química , Humanos , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Because invertebrates lack an adaptive immune system, they had to evolve effective intrinsic defense strategies against a variety of microbial pathogens. This ancient form of host defense, the innate immunity, is present in all multicellular organisms including humans. The innate immune system of the Japanese horseshoe crab Tachypleus tridentatus, serving as a model organism, includes a hemolymph coagulation system, which participates both in defense against microbes and in hemostasis. Early work on the evolution of vertebrate fibrinogen suggested a common origin of the arthropod hemolymph coagulation and the vertebrate blood coagulation systems. However, this conjecture could not be verified by comparing the structures of coagulogen, the clotting protein of the horseshoe crab, and of mammalian fibrinogen. Here we report the crystal structure of tachylectin 5A (TL5A), a nonself-recognizing lectin from the hemolymph plasma of T. tridentatus. TL5A shares not only a common fold but also related functional sites with the gamma fragment of mammalian fibrinogen. Our observations provide the first structural evidence of a common ancestor for the innate immunity and the blood coagulation systems.
Asunto(s)
Coagulación Sanguínea , Proteínas Sanguíneas/química , Evolución Molecular , Lectinas/química , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/fisiología , Cristalografía por Rayos X , Fibrinógeno/química , Cangrejos Herradura , Humanos , Inmunidad Innata , Lectinas/inmunología , Lectinas/fisiología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.
