RESUMEN
BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.
Asunto(s)
Proteína BRCA2/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteína de Replicación A/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/deficiencia , Proteína BRCA2/genética , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Daño del ADN/efectos de los fármacos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN/efectos de los fármacos , Humanos , Hidroxiurea/farmacología , Proteína Homóloga de MRE11/deficiencia , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Mutación , Fosforilación , ARN Interferente Pequeño , Recombinasa Rad51/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
We revisit the dynamics of a gene repressed by its own protein in the case where the transcription rate does not adapt instantaneously to protein concentration but is a dynamical variable. We derive analytical criteria for the appearance of sustained oscillations and find that they require degradation mechanisms much less nonlinear than for infinitely fast regulation. Deterministic predictions are confirmed by stochastic simulations of this minimal genetic oscillator.
Asunto(s)
Regulación de la Expresión Génica , Modelos Genéticos , Transcripción Genética , Ritmo Circadiano/genética , ADN/genética , ADN/metabolismo , Regiones Promotoras Genéticas , ARN/genética , ARN/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp(179)-Pro(180) bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.
Asunto(s)
Ciclofilinas/química , Heparina/química , Heparitina Sulfato/química , Modelos Moleculares , Oligosacáridos/química , Isomerasa de Peptidilprolil/química , Animales , Basigina/química , Basigina/metabolismo , Sitios de Unión/fisiología , Dominio Catalítico/fisiología , Adhesión Celular/fisiología , Quimiotaxis/fisiología , Ciclofilinas/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Integrinas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/metabolismo , Péptidos/química , Péptidos/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Linfocitos T/metabolismoRESUMEN
This paper introduces a novel molecular description--topological (2D) fuzzy pharmacophore triplets, 2D-FPT--using the number of interposed bonds as the measure of separation between the atoms representing pharmacophore types (hydrophobic, aromatic, hydrogen-bond donor and acceptor, cation, and anion). 2D-FPT features three key improvements with respect to the state-of-the-art pharmacophore fingerprints: (1) The first key novelty is fuzzy mapping of molecular triplets onto the basis set of pharmacophore triplets: unlike in the binary scheme where an atom triplet is set to highlight the bit of a single, best-matching basis triplet, the herein-defined fuzzy approach allows for gradual mapping of each atom triplet onto several related basis triplets, thus minimizing binary classification artifacts. (2) The second innovation is proteolytic equilibrium dependence, by explicitly considering all of the conjugated acids and bases (microspecies). 2D-FPTs are concentration-weighted (as predicted at pH=7.4) averages of microspecies fingerprints. Therefore, small structural modifications, not affecting the overall pharmacophore pattern (in the sense of classical rule-based assignment), but nevertheless triggering a pKa shift, will have a major impact on 2D-FPT. Pairs of almost identical compounds with significantly differing activities ("activity cliffs" in classical descriptor spaces) were in many cases predictable by 2D-FPT. (3) The third innovation is a new similarity scoring formula, acknowledging that the simultaneous absence of a triplet in two molecules is a less-constraining indicator of similarity than its simultaneous presence. It displays excellent neighborhood behavior, outperforming 2D or 3D two-point pharmacophore descriptors or chemical fingerprints. The 2D-FPT calculator was developed using the chemoinformatics toolkit of ChemAxon (www.chemaxon.com).
