RESUMEN
Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.
Asunto(s)
Anemia de Fanconi , Neoplasias , Humanos , Supervivencia Celular/genética , Cromatina/genética , Ciclina E/genética , Ciclina E/metabolismo , Daño del ADN , Reparación del ADN , Replicación del ADN/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Neoplasias/genéticaRESUMEN
Multicellular organisms have evolved elaborate strategies to sense and adapt to changes in intracellular oxygen. The canonical cellular pathway responsible for oxygen sensing consists of the von Hippel-Lindau (pVHL) tumor suppressor protein, prolyl hydroxylases (PHD), and hypoxia-inducible factors (HIFs), which together regulate expression of downstream genes involved in oxygen homeostasis. In recent years, it has become increasingly clear that oxygen regulatory mechanisms are intertwined with cellular iron-sensing pathways. Key members of these networks such as prolyl-hydroxylases, E3 ubiquitin ligase adaptor protein FBXL5, iron regulatory proteins (IRPs), and Fe-S cluster proteins require both iron and oxygen for their optimal function and/or are tightly regulated by intracellular concentrations of these molecules. Monitoring how protein interactomes are remodeled as a function of intracellular oxygen and iron levels gives insights into the nature and dynamics of these pathways. We have recently described an oxygen-sensitive interaction between FBXL5 and the cytoplasmic Fe-S cluster targeting complex (CIA targeting complex) with implications in the FBXL5-dependent regulation of IRPs. Based on this work, we present a protocol describing the induction and maintenance of hypoxia in mammalian cell cultures and a mass-spectrometry-based proteomics approach aimed at interrogating changes in interactome of key proteins as a function of intracellular oxygen and iron levels. These methods are widely applicable to understanding the dynamics of iron and oxygen signaling.
Asunto(s)
Hierro , Oxígeno , Animales , Hierro/metabolismo , Oxígeno/metabolismo , Proteómica , Hipoxia/metabolismo , Espectrometría de Masas , Mamíferos/metabolismoRESUMEN
The high complexity prevalent in biological samples requires chromatographic separations with high sensitivity and resolution to be effectively analyzed. Here we introduce a robust, reproducible and inexpensive protocol for preparation of a nano-flow reversed phase high performance liquid chromatography (RP-HPLC) columns for on-line separation of analytical peptides before introduction into and detection by a mass-spectrometer in traditional bottom-up proteomics workflows. Depending on the goal of the experiment and the chemical properties of the analytes being separated, optimal column parameters may differ in their internal or outer diameters, length, particle size, pore size, chemistry of stationary phase particles, and the presence or absence of an integrated electrospray emitter at the tip. An in-house column packing system not only enables the rapid fabrication of columns with the desired properties but also dramatically reduces the cost of the process. The optimized protocol for packing a C18 AQ (aqueous) fused silica column discussed here is compatible with a wide range of liquid chromatographic instruments for achieving effective separation of analytes.
Asunto(s)
Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Péptidos , Cromatografía Liquida , Espectrometría de Masas , Péptidos/químicaRESUMEN
The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Chaperonas Moleculares/genética , Complejos Multiproteicos/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Proteínas de Ciclo Celular/química , Proteínas F-Box/química , Células HeLa , Homeostasis , Humanos , Hierro/metabolismo , Proteínas Reguladoras del Hierro/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Chaperonas Moleculares/química , Complejos Multiproteicos/química , Oxígeno/metabolismo , Proteolisis , Factores de Transcripción/genética , Complejos de Ubiquitina-Proteína Ligasa/químicaRESUMEN
Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified α-actinin-4, a host cytoskeletal protein, as an interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co-immunoprecipitation studies, first in a coupled in vitro transcription-translation assay and then in cells either transiently co-expressing the two proteins or infected with whole IAV. Importantly, the NP-actinin-4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin-4 co-localized largely around the nucleus and also in the cytoplasmic region of virus-infected A549 cells. Silencing of actinin-4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post-infection with IAV, suggesting that actinin-4 was critical for viral replication. Furthermore, actinin-4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin-4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin-4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins.
Asunto(s)
Actinina/metabolismo , Virus de la Influenza A/fisiología , Proteínas de Unión al ARN/fisiología , Proteínas del Núcleo Viral/fisiología , Replicación Viral , Actinina/genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Proteínas de la Nucleocápside , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Activación TranscripcionalRESUMEN
Tumour suppressor genes restrain inappropriate cell growth and division, as well as stimulate cell death to maintain tissue homeostasis. Loss of function leads to abnormal cellular behaviour, including hyperproliferation of cell and perturbation of cell cycle regulation. LIMD1 is a tumour suppressor gene located at chromosome 3p21.3, a region commonly deleted in many solid malignancies. LIMD1 interacts with retinoblastoma (Rb) and is involved in Rb-mediated downregulation of E2F1-target genes. However, the role of LIMD1 in cell cycle regulation remains unclear. We propose that LIMD1 induces cell cycle arrest, utilising Rb-E2F1 axis, and show that ectopic expression of LIMD1 in A549 cells results in hypo-phosphorylation that potentiates Rb function, which correlates with downregulation of E2F1. In agreement with these observations, LIMD1 overexpression retards cell cycle progression and blocks S-phase entry, as cells accumulate in G0/G1 phase and have reduced incorporation of BrdU. Most significantly, LIMD1-dependent effects on Rb function and cell cycle are reversed on depletion of endogenous LIMD1, underscoring its centrality in Rb-mediated cell cycle regulation. Hence, our findings provide new insight into cell cycle control by Rb-LIMD1 nexus.