RESUMEN
Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila the Imd pathway detects Gram-negative bacterial infections through recognition of diaminopimelic acid (DAP)-type peptidoglycan and activation of the NF-κB precursor Relish, which drives robust antimicrobial peptide gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and Lys-63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. Here, we demonstrate that Imd is rapidly Lys-63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 and Ubc13-Uev1a, in conjunction with the E3 ligase Diap2. Lys-63-ubiquitination activates the TGFß-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of Lys-63 chains and the addition of Lys-48 polyubiquitin. This ubiquitin-editing process results in the proteasomal degradation of Imd, which we propose functions to restore homeostasis to the Drosophila immune response.
Asunto(s)
Proteínas de Drosophila/inmunología , Inmunidad Innata , Quinasas Quinasa Quinasa PAM/inmunología , Transducción de Señal/inmunología , Ubiquitinación/inmunología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/inmunología , Quinasas Quinasa Quinasa PAM/genética , Poliubiquitina/genética , Poliubiquitina/inmunología , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/inmunología , Ubiquitinación/genéticaRESUMEN
B cell CLL/lymphoma 11A (BCL11A) is a transcription factor and regulator of hemoglobin switching that has emerged as a promising therapeutic target for sickle cell disease and thalassemia. In the hematopoietic system, BCL11A is required for B lymphopoiesis, yet its role in other hematopoietic cells, especially hematopoietic stem cells (HSCs) remains elusive. The extensive expression of BCL11A in hematopoiesis implicates context-dependent roles, highlighting the importance of fully characterizing its function as part of ongoing efforts for stem cell therapy and regenerative medicine. Here, we demonstrate that BCL11A is indispensable for normal HSC function. Bcl11a deficiency results in HSC defects, typically observed in the aging hematopoietic system. We find that downregulation of cyclin-dependent kinase 6 (Cdk6), and the ensuing cell-cycle delay, correlate with HSC dysfunction. Our studies define a mechanism for BCL11A in regulation of HSC function and have important implications for the design of therapeutic approaches to targeting BCL11A.
Asunto(s)
Proteínas Portadoras/genética , Senescencia Celular/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/patología , Proteínas Nucleares/genética , Animales , Quinasa 6 Dependiente de la Ciclina/biosíntesis , Proteínas de Unión al ADN , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas RepresorasRESUMEN
In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein-L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal ((462)LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.
Asunto(s)
Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Papillomavirus Humano 16/metabolismo , Transporte de Proteínas/fisiología , Sustitución de Aminoácidos , Animales , Línea Celular , Femenino , Humanos , Luciferasas , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7(39-98) localized mostly to the nucleus. The GST-11E7 and GST-11cE7(39-98) were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.