RESUMEN
BACKGROUND: The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity. RESULTS AND CONCLUSION: In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.
Asunto(s)
Escherichia coli/enzimología , Sialiltransferasas/metabolismo , Animales , Bovinos , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Humanos , Chaperonas Moleculares/metabolismo , Mucinas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sialiltransferasas/genéticaRESUMEN
Mammalian cell surfaces are modified by complex arrays of glycoproteins, glycolipids and polysaccharides, many of which terminate in sialic acid and have central roles in essential processes including cell recognition, adhesion and immunogenicity. Sialylation of glycoconjugates is performed by a set of sequence-related enzymes known as sialyltransferases (STs). Here we present the crystal structure of a mammalian ST, porcine ST3Gal-I, providing a structural basis for understanding the mechanism and specificity of these enzymes and for the design of selective inhibitors.
Asunto(s)
Sialiltransferasas/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Estructura Secundaria de Proteína , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Especificidad por Sustrato , Porcinos , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.
Asunto(s)
Adenilato Quinasa/química , Quinasa 2 Dependiente de la Ciclina/química , Levaduras/genética , Adenosina Trifosfato/química , Adenilato Quinasa/genética , Secuencia de Aminoácidos , Cristalografía , Dimerización , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Fosfotransferasas/química , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Homología Estructural de Proteína , Relación Estructura-Actividad , Levaduras/químicaAsunto(s)
Regulación de la Expresión Génica/genética , Histonas/genética , Saccharomyces cerevisiae/genética , Acetilación , Acetiltransferasas/aislamiento & purificación , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Centrifugación/métodos , Histona Acetiltransferasas , Histonas/química , Histonas/metabolismo , Metilación , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fosforilación , Protamina Quinasa/aislamiento & purificación , Protamina Quinasa/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Activación Transcripcional , Ubiquitina/metabolismoRESUMEN
Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage. We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1. Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions. These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9. However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1. The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade. Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.
Asunto(s)
Proteínas Fúngicas/metabolismo , MAP Quinasa Quinasa 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transducción de Señal/fisiología , Alanina/genética , Sustitución de Aminoácidos , Sitios de Unión/genética , Sitios de Unión/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Daño del ADN/fisiología , Proteínas Fúngicas/genética , Péptidos y Proteínas de Señalización Intracelular , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas de Schizosaccharomyces pombeRESUMEN
Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.
Asunto(s)
Replicación del ADN/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transducción de Señal/fisiología , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Clonación Molecular , Daño del ADN/fisiología , Mutación , Proteínas Nucleares/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.