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Identification of a novel inhibitor of 5-Lipoxygenase

Wednesday, September 16, 2015 — Poster Session I

3:30 p.m. – 5:00 p.m.
FAES Terrace
NEI
MOLBIO-31

Authors

  • P Subramanian
  • E Mendez
  • SP Becerra

Abstract

Arachidonate 5-lipoxygenase (5-LOX) is a member of the lipoxygenase family of enzymes that plays a key role in arachidonic acid metabolism. Arachidonic acid, upon oxygenation by 5-LOX, forms 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is further converted into proinflammatory leukotrienes such as LTB4 that can have detrimental biological effects. The PNPLA2 gene codes for PEDF-R, a lipase involved in cell survival and lipid metabolism, found in the RPE and retina. Peptides derived from the longest loop region (L159-M325) of PEDF-R were screened for the effect on soybean lipoxygenase-V (LOX-V) in vitro. We identified PEDF-R peptides E5b (I193–L232) and P1 (T210-L249) with binding affinity for LOX-V that inhibited its enzymatic activity. The LOX-V enzymatic inhibition was attenuated when PEDF-R peptides were captured specifically with a P1 binding protein, fluorescein-conjugated PEDF (Fl-PEDF). These peptides efficiently protected human retinal pigment epithelial ARPE-19 and pig primary RPE cells from oxidative stress. We investigated whether binding and inhibiting mammalian 5-LOX by these peptides are critical to protect RPE cells during oxidative stress. Peptide affinity chromatography showed that human recombinant 5-LOX binds to P1 via ionic interactions. Pull-down assays revealed that full length PEDF-R interacts with 5-LOX in a dose-response fashion. Interestingly, PNPLA2 expression decreased with oxidative stress with no effect on 5-LOX expression. Moreover, addition of P1 and PEDF-R over-expression reduced of the levels of the 5-LOX product LTB4 in ARPE-19 cells under oxidative stress. Our results support that PEDF-R is a novel inhibitor of 5-LOX.

Category: Molecular Biology and Biochemistry