RESUMEN
C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd(-/-) dendritic cells, but not those lacking PKCα, PKCß, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd(-/-) cells, and Prkcd(-/-) mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD , Candida albicans/inmunología , Ratones , Ratones Noqueados , Proteína Quinasa C-delta/metabolismo , Transducción de Señal , Quinasa SykRESUMEN
The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state.
Asunto(s)
Proteínas Bacterianas/química , Enzimas Reparadoras del ADN/química , Proteínas de Unión al ADN/química , ADN/química , Modelos Moleculares , Complejos Multiproteicos/química , Thermotoga maritima/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cristalografía , ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae , Alineación de Secuencia , Análisis de Secuencia de ADN , Homeostasis del Telómero/genéticaRESUMEN
Bacteria have developed multiple complex mechanisms ensuring an adequate response to environmental changes. In this context, bacterial cell division and growth are subject to strict control to ensure metabolic balance and cell survival. A plethora of studies cast light on toxin-antitoxin (TA) systems as metabolism regulators acting in response to environmental stress conditions. Many of those studies suggest direct relations between the TA systems and the pathogenic potential or antibiotic resistance of relevant bacteria. Other studies point out that TA systems play a significant role in ensuring stability of mobile genetic material. The evolutionary origin and relations between various TA systems are still a subject of a debate. The impact of toxin-antitoxin systems on bacteria physiology prompted their application in molecular biology as tools allowing cloning of some hard-to-maintain genes, plasmid maintenance and production of recombinant proteins.
Asunto(s)
Antitoxinas/metabolismo , Toxinas Biológicas/metabolismo , Fenómenos Fisiológicos Bacterianos , Farmacorresistencia Bacteriana/fisiología , Biología MolecularRESUMEN
Staphylococcal cysteine proteases are implicated as virulence factors in human and avian infections. Human strains of Staphylococcus aureus secrete two cysteine proteases (staphopains A and B), whereas avian strains express staphopain C (ScpA2), which is distinct from both human homologues. Here, we describe probable reasons why the horizontal transfer of a plasmid encoding staphopain C between avian and human strains has never been observed. The human plasma serine protease inhibitor α(1)-antichymotrypsin (ACHT) inhibits ScpA2. Together with the lack of ScpA2 inhibition by chicken plasma, these data may explain the exclusively avian occurrence of ScpA2. We also clarify the mechanistic details of this unusual cross-class inhibition. Analysis of mutated ACHT variants revealed that the cleavage of the Leu383-Ser384 peptide bond results in ScpA2 inhibition, whereas hydrolysis of the preceding peptide bond leads to ACHT inactivation. This evidence is consistent with the suicide-substrate-like mechanism of inhibition.