Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics

  • 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.

Download full text files

Export metadata

Additional Services

Share in Twitter Search Google Scholar
Author:Katharina Schlag, Dieter SteinhilberORCiDGND, Michael KarasGND, Bernd L. Sorg
Parent Title (English):The FEBS Journal 287.2020
Place of publication:Oxford [u.a.]
Document Type:Article
Date of Publication (online):2020/02/24
Date of first Publication:2020/02/24
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2020/10/27
Tag:5-lipoxygenase;; DNA G-quadruplex; DNA pulldown; DNA-protein-interactions; labelfree quantitative mass spectrometry
Page Number:19
First Page:4481
Last Page:4499
Institutes:Biochemie, Chemie und Pharmazie
Fachübergreifende Einrichtungen / Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - Namensnennung-Nicht kommerziell - Keine Bearbeitung 4.0