Abstract
ADAM-15, with known zymogen, secretase, and disintegrin activities, is a catalytically active member of the ADAM family normally expressed in early embryonic development and aberrantly expressed in various cancers, including breast, prostate and lung. ADAM-15 promotes extracellular shedding of E-cadherin, a soluble ligand for the HER2/neu receptor, leading to activation, increased motility, and proliferation. Targeted downregulation of both ADAM-15 and HER2/neu function synergistically kills breast cancer cells, but to date there are no therapeutic options for decreasing ADAM-15 function or expression. In this vein, we have examined a unique string of guanine-rich DNA within the critical core promoter of ADAM-15. This region of DNA consists of seven contiguous runs of three or more consecutive guanines, which, under superhelical stress, can relax from duplex DNA to form an intrastrand secondary G-quadruplex (G4) structure. Using biophysical and biological techniques, we have examined the G4 formation within the entire and various truncated regions of the ADAM-15 promoter, and demonstrate strong intrastrand G4 formation serving to function as a biological silencer element. Characterization of the predominant G4 species formed within the ADAM-15 promoter will allow for specific drug targeting and stabilization, and the further development of novel, targeted therapeutics.
Highlights
The ‘A Disintegrin And Metalloproteinase’ (ADAM, alluding to their relationship to snake venom and involvement with sperm and fertility) protein family is over 20 members strong, approximately half of which have functional enzymatic activity [1,2]
Putative G4 formation in this full-length wild-type (FL WT) sequence was examined in the absence and presence of either
The core promoter of ADAM-15 encompassed within ~225 base pairs upstream of the TSS
Summary
The ‘A Disintegrin And Metalloproteinase’ (ADAM, alluding to their relationship to snake venom and involvement with sperm and fertility) protein family is over 20 members strong, approximately half of which have functional enzymatic activity [1,2]. We have identified a unique region of guanine-rich DNA in the critical core promoter of ADAM-15. This region DNA consists of seven contiguous runs of three or more consecutive guanines within a region 40 nucleotides in length comprising the core promoter, known to have a multiple binding sites for the transcriptional factor Sp1 and to be involved in regulating gene expression [15]. 15+ base pair regions of guanine-rich DNA are preferentially clustered ±0.5 kb from the transcriptional start site [16] and within oncogenic promoters [17], including many representatives of the six hallmarks of cancer [18]. The current works define the role of G4 formation in the ADAM-15 promoter through luciferase constructs, and elucidates the structures of the predominating isoforms through various biophysical techniques including CD, chemical footprinting, select mutations and the polymerase stop assay
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