Variations in the NBN/NBS1 gene and the risk of breast cancer in non-BRCA1/2 French Canadian families with high risk of breast cancer

<p>Abstract</p> <p>Background</p> <p>The Nijmegen Breakage Syndrome is a chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, and increased frequency of cancers. Familial studies on relatives of these patients indicated that they also appear to be at increased risk of cancer.</p> <p>Methods</p> <p>In a candidate gene study aiming at identifying genetic determinants of breast cancer susceptibility, we undertook the full sequencing of the <it>NBN </it>gene in our cohort of 97 high-risk non-<it>BRCA1 </it>and -<it>BRCA2 </it>breast cancer families, along with 74 healthy unrelated controls, also from the French Canadian population. <it>In silico </it>programs (ESEfinder, NNSplice, Splice Site Finder and MatInspector) were used to assess the putative impact of the variants identified. The effect of the promoter variant was further studied by luciferase gene reporter assay in MCF-7, HEK293, HeLa and LNCaP cell lines.</p> <p>Results</p> <p>Twenty-four variants were identified in our case series and their frequency was further evaluated in healthy controls. The potentially deleterious p.Ile171Val variant was observed in one case only. The p.Arg215Trp variant, suggested to impair NBN binding to histone γ-H2AX, was observed in one breast cancer case and one healthy control. A promoter variant c.-242-110delAGTA displayed a significant variation in frequency between both sample sets. Luciferase reporter gene assay of the promoter construct bearing this variant did not suggest a variation of expression in the MCF-7 breast cancer cell line, but indicated a reduction of luciferase expression in both the HEK293 and LNCaP cell lines.</p> <p>Conclusion</p> <p>Our analysis of <it>NBN </it>sequence variations indicated that potential <it>NBN </it>alterations are present, albeit at a low frequency, in our cohort of high-risk breast cancer cases. Further analyses will be needed to fully ascertain the exact impact of those variants on breast cancer susceptibility, in particular for variants located in <it>NBN </it>promoter region.</p

Determining Spatial Organisation and Localization of a Proposed Enterobactin Metabolon in Escherichia coli

To obtain scarce iron from their environment, bacteria synthesize and secrete high affinity siderophores that chelate ferric ions. Enterobactin is a catecholate siderophore produced by Escherichia coli. Synthesized in the cytoplasm by the concerted action of seven enzymes, EntCBDAEF and EntH, enterobactin is then transported to the periplasm by the inner-membrane protein EntS. We hypothesize that the Ent proteins assemble in a large multiprotein complex to enhance the metabolic flux of the intermediates and prevent their diffusion, and that this assembly localizes at the inner membrane where it interacts with EntS for secretion. The overarching goal of this thesis is to gather further structural evidence of interactions among the biosynthetic enzymes, to determine the manner in which the proposed assembly interacts with the transporter channel, and to initiate characterization of EntS. Herein we demonstrate an interaction between the first two enzymes of the pathway, EntC and EntB. Through in vivo crosslinking with formaldehyde, we captured intracellular complexes comprising both proteins. These results were supported by two-hybrid assays, whereas automated docking of the crystal structures of EntC and EntB resulted in a model where the active sites of EntC and EntB are oriented in apposition and connected by an electropositive surface potentially capable of channeling the negatively charged precursor of enterobactin, isochorismate. We then built on these research outcomes, which suggested that other Ent proteins could interact simultaneously and form complexes in which both EntC and EntB were detected. Using in vivo crosslinking with formaldehyde, we confirmed the presence of proteins EntA and EntE in these higher-order complexes. We also initiated work towards identification of EntS cytosolic interactors, where using an engineered ascorbate peroxidase enzyme fused in frame with EntS as the bait, proximity labeling assays revealed prey proteins localizing within 10 nm of EntS. Finally, we undertook extraction and isolation of EntS in a detergent micelle and in synthetic lipid nanodiscs. We identified two detergents and two copolymers that were efficient in extracting and solubilizing EntS, as well as the optimized conditions for successful extraction. The results obtained also suggest that EntS might assemble as a dimer