5-Lipoxygenase (5-LO) is the initial
enzyme in the biosynthesis of
leukotrienes, which are mediators involved in pathophysiological conditions such as
asthma and certain
cancer types. Knowledge of
proteins involved in 5-LO pathway regulation, including gene regulatory
proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter-interacting
proteins, obtained by
DNA pulldown and label-free quantitative mass spectrometry.
Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter
DNA interactors. Through statistical analysis, 66
proteins were identified as specific ALOX5 promotor
binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel-like factors (KLF13 and KLF16) and six other zinc finger
proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the
proteins hnRNPD and hnRNPK, which are, together with the
protein MAZ, known to interact with
DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger
proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form
DNA G-quadruplex structures, which may play a functional role in ALOX5 gene regulation.