CONICET | Buscador de Institutos y Recursos Humanos

Abstract: During a systematic screening of the cytoplasmic proteomeof bacterial-infected human macrophages (n=19) we detecteda 110 KDa protein, identified by MS, antibodies and pI rangeas C23/NCL, which was down-regulated after infection (51.7 % atday 4, p=0.05). The triggering of the effect did not depend on bacterialviability suggesting an innate response to surface bacterialcomponents. Little is known about the roles in the cytosol duringinfection of this major nucleolar phospho-protein. To understand thisdown-regulation, we developed novel biochemical and proteomicassays including, protein kinase determinations. We followed putativeupstream protein candidates in a NCL pathway studying kinasesand other candidates. Several sensitive assays were developed tomonitor its level and in vivo phosphorylation state. The down-regulationwas not due to affected kinases regulating its phosphorylationstate or to enzymes modulating its isomerization. Instead, it wasdue to increased partial cleavage of the full-length protein generatingsmaller fragmented forms. The cleavage was detected withhigher sensitivity and more quantitatively with our assays comparedto immunoblots. To understand the cleavage, we studied caspaseactivation. The results suggest that the cleavage is correlated withtransient caspase activation and it could be a biomarker of apoptoticversus inflammatory signaling balance in the cytosol. It will be interestingto further study C23/NCL as a signaling hub integrating apoptotic,proliferation, nucleolar and immune/inflammatory signals bothin infected and non-infected macrophages. Our sensitive assayswill help studies in other cell types and cell compartments since themultitasking, acidic, RNA-binding and histone-binding C23/NCL isconsidered in cell cycle, gene expression and mRNA half-life studiesand also as target for anti-cancer and anti-angiogenesis treatments.It is also a cell surface receptor for microbes and other ligands.Keywords: nucleolar, cytosolic