ATR/ATM-Mediated Phosphorylation of BRCA1 T1394 Promotes Homologous Recombinational Repair and G2-M Checkpoint Maintenance.

BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSB) and DNA damage-induced cell-cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response (DDR). Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single-strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G2-M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DDR and may have implications in the development or prognosis of BRCA1-associated cancers. SIGNIFICANCE: This study identifies a BRCA1 phosphorylation event critical for its DNA repair function and reveals the functional defects of several BRCA1 variants of unknown significance.

Renilla Luciferase Reporter Assay to Study 3'-UTR-Driven Posttranscriptional Regulation of OPRM1.

MOR expression levels at a specific cell type or tissue significantly contribute to its role in pain transmission and in other responses involving opioid receptors. Therefore, molecular processes regulating MOR levels have gained more and more interest. Recently, posttranscriptional regulation mechanisms have been shown to play a relevant role in influencing MOR expression levels, with polymorphisms and mutations within OPRM1 3'-UTR region impacting the differential opioid-mediated response observed within individuals. Here we report a Renilla luciferase reporter assay format suitable for dissecting the contribution of different and distinct OPRM1 3'-UTR elements to MOR expression levels in a model of glial cells, both under basal conditions and following specific treatments.

PALB2 connects BRCA1 and BRCA2 in the G2/M checkpoint response.

The G2/M checkpoint inhibits mitotic entry upon DNA damage, thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1-PALB2-BRCA2 axis that is essential for homologous recombination (HR)-based DNA doublestrand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all three proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in the checkpoint response. The BRCA1-PALB2 interaction can be important for checkpoint activation, whereas the PALB2-BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1-PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.

Evidence of Intertissue Differences in the DNA Damage Response and the Pro-oncogenic Role of NF-κB in Mice with Disengaged BRCA1-PALB2 Interaction.

The BRCA1-PALB2-BRCA2 axis plays an essential role in DNA homologous recombination repair, defect in which drives genome instability and cancer development. How cells with defects in this pathway respond to DNA damage in vivo and how tumors develop from these cells remain poorly defined. Here, we analyzed several aspects of the DNA damage response in multiple tissues of Palb2-mutant mice in which the interaction between PALB2 and BRCA1 is disengaged. Without any challenge, the mutant mice showed increased endogenous DNA damage. Following ionizing radiation, the mutant mice displayed higher levels of DNA breaks and stronger induction of p53 and p21, but continued DNA synthesis, reduced apoptosis, and accelerated tumor development. The differences in p21 induction, DNA synthesis, and apoptosis between wild-type and mutant mice were substantially more pronounced in the mammary gland than in the intestine, suggesting a potential contributing factor to the increased risk and the tissue specificity of BRCA/PALB2-associated tumor development. Moreover, the mutant mice showed higher levels of reactive oxygen species and constitutive activation of NF-κB, an antiapoptotic transcription factor inducible by both DNA damage and oxidative stress. Treatment of the mutant mice with an inhibitor of NF-κB reactivated apoptosis and delayed tumor development following radiation. Thus, our results also suggest a prosurvival and pro-oncogenic role of NF-κB in PALB2-mutant cells.Significance: This study explores novel tumor suppression mechanisms of the BRCA1-PALB2 DNA damage response pathway and implicates NF-κB activation as a protumorogenic event and possible therapeutic target. Cancer Res; 78(14); 3969-81. ©2018 AACR.

Nociceptin/orphanin FQ antagonizes lipopolysaccharide-stimulated proliferation, migration and inflammatory signaling in human glioblastoma U87 cells.

Glioblastoma is among the most aggressive brain tumors and has an exceedingly poor prognosis. Recently, the importance of the tumor microenvironment in glioblastoma cell growth and progression has been emphasized. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and endogenous ligands originating from dying cells or the extracellular matrix involved in host defense and in inflammation. G-protein coupled receptors (GPCRs) have gained interest in anti-tumor drug discovery due to the role that they directly or indirectly play by transactivating other receptors, causing cell migration and proliferation. A proteomic analysis showed that the nociceptin receptor (NOPr) is among the GPCRs significantly expressed in glioblastoma cells, including U87 cells. We describe a novel role of the peptide nociceptin (N/OFQ), the endogenous ligand of the NOPr that counteracts cell migration, proliferation and increase in IL-1ß mRNA elicited by LPS via TLR4 in U87 glioblastoma cells. Signaling pathways through which N/OFQ inhibits LPS-mediated cell migration and elevation of [Ca2+]i require ß-arrestin 2 and are sensitive to TNFR-associated factor 6, c-Src and protein kinase C (PKC). LPS-induced cell proliferation and increase in IL-1ß mRNA are counteracted by N/OFQ via ß-arrestin 2, PKC and extracellular signal-regulated kinase 1/2; furthermore, the contributions of the transcription factors NF-kB and AP-1 were investigated. Independent of LPS, N/OFQ induces a significant increase in cell apoptosis. Contrary to what was observed in other cell models, a prolonged exposure to this endotoxin did not promote any tolerance of the cellular effects above described, including NOPr down-regulation while N/OFQ loses its inhibitory role.

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1.

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.

Renilla luciferase reporter assay to study 3'UTR-driven posttranscriptional regulations of OPRM1.

The regulation of MOR expression at the level of mRNA is relevant for its role in pain transmission and in other functions involving opioid receptors. Recently, the role of the 3'UTR in the posttranscriptional regulation of MOR expression has been highlighted. Here we describe a Renilla luciferase reporter assay for the study of the effect of any selective treatment on the 3'UTR-dependent regulation of OPRM1 in a model of glial cells.

Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core.

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α (polymerase), ε (3'-5' exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.

pGOODs: new plasmids for the co-expression of proteins in Escherichia coli.

Two systems for the co-expression of proteins in Escherichia coli were designed and constructed. The first system relies on the new vector, pGOOD, which is compatible with ColE1-type plasmids and sustains efficient co-expression of soluble protein complexes. The second system is based on the pGOOD1 vector (a derivative of pGOOD), useful for the production of toxic proteins, whose synthesis can be regulated by the co-expressed LacI repressor.