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Selective degradation of BET proteins with dBET1 attenuates lipopolysaccharide‐induced pro‐inflammatory response in microglia
Author(s) -
CastroRivera Carolina Isabelle,
Yang Changjun Castro,
DeMars Kelly M.,
CandelarioJalil Eduardo Castro
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.740.4
Subject(s) - microglia , bromodomain , brd4 , chemistry , microbiology and biotechnology , protein degradation , inflammation , nitric oxide , ubiquitin , ubiquitin ligase , nitric oxide synthase , histone , pharmacology , biology , immunology , biochemistry , organic chemistry , gene
Stroke is the fifth leading cause of death in the United States and its most common type, ischemic stroke, is caused when a major blood vessel that carries oxygen and nutrients to the brain is blocked. This blockage initiates tissue death downstream resulting in inflammation and oxidative stress. Several studies have shown that microglia/macrophage neuroinflammatory responses significantly contribute to neuronal death in ischemic stroke. Bromodomain and extraterminal (BET) proteins‐ Brd2, Brd3, Brd4, and Brdt‐ bind to acetylated lysines on histones and act as a scaffold for transcription factors of several pro‐inflammatory genes important for the spread of cell death after stroke. To recreate the inflammatory process that occurs in response to ischemic stroke in vitro we subjected mouse SIM‐A9 microglia to lipopolysaccharide (LPS) stimulation and then treated with dBET1, a proteolysis‐targeting chimera (PROTAC) molecule that produces a targeted degradation of BET proteins. dBET1 is also a hybrid of the highly selective BET inhibitor, JQ1, that blocks the acetylated lysine‐binding pocket of BET proteins, complexed to a ligand for an E3 ubiquitin ligase. The PROTAC approach results in ubiquitination and subsequent degradation of the target protein by the proteasome. In this study, we did a dose‐response and a time course curve and found that 24 hours of treatment with 1 μM of dBET1 optimally decreases Brd2 and Brd4. Using Western blot and qRT‐PCR, we also found that treatment with dBET1 produced a dramatic reduction in the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), interleukin (IL)‐1β, tumor necrosis factor‐α (TNFα), IL‐6, chemokine ligand 2 (CCL‐2), and matrix metalloproteinase 9 (MMP‐9). This is the first study showing that dBET1‐mediated targeted degradation of BET proteins prevents pro‐inflammatory responses in LPS‐stimulated microglia. The data collected in vitro show promising results that suggest that in vivo studies using dBET1 may reduce the deleterious effects of stroke. Support or Funding Information This project was possible through the funding of the Neuroscience Summer Internship Program from the McKnight Brain Institute at the University of Florida. It also received funding through the University of Puerto Rico at Mayagüez's MARC U*STAR Program and the National Institutes of Health's grant 2T34GM008419‐26. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .