The novel prolyl hydroxylase-2 inhibitor caffeic acid upregulates hypoxia inducible factor and protects against hypoxia.

BACKGROUND & AIMS: Hypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a protective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors. METHODS: An established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out. RESULTS: Caffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1α protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1α protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Conventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group. CONCLUSIONS: This study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia.

Production of functional recombinant prolyl hydroxylase-2 enzyme in insect cells for small molecule inhibitor screening studies.

Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates the hypoxia inducible factor (HIF) which then targets it for degradation in oxygenated cells. Inhibition of the activity of the PHD2 enzyme under hypoxic environmental conditions acts to upregulate HIF. Thus, PHD2 inhibitors may serve as a promising treatment for HIF-dependent diseases. In this study, recombinant PHD2 protein was successfully expressed using a baculovirus-insect cell expression secretory system. PHD2 was purified and in combination with bacterially expressed functional von Hippel Lindau protein-elongin B-elongin C (VBC) protein complex was used to successfully develop a fluorescence-based PHD2 activity assay. Myricetin was identified as a novel potent PHD2 inhibitor by high-throughput screening of a natural compound library. Further studies showed that treatment of human neuroblastoma SH-SY5Y cells with Myricetin increased HIF-1α protein levels. These results indicate that the insect cell expression system is capable of producing highly active recombinant PHD2 protein from which a fluorescence-based activity assay can be developed for high-throughput screening applications.

Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors.

Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant protein in Pichia pastoris. The pPICZα-LGMN expression plasmid was constructed and transformed into Pichia pastoris strain and positive recombinants were identified. Fermentation conditions were optimized and it was found that Legumain was most highly expressed under pH 6 culture conditions. In addition, the enzyme activity of the purified Legumain was tested using a fluorogenic substrate (Z-Ala-Ala-Asn-AMC) assay and the optimum pH for the autocatalytic activation of recombinant Legumain was very acidic at a pH value of 3. The recombinant protein was then used to screen a library of compounds and small molecule 1773 (Terramycin) was shown to effectively inhibit Legumain enzyme activity. These results indicate that the Pichia pastoris expression system can produce highly active recombinant Legumain protein allowing it to be used for High-throughput screening (HTS) applications.