Zinc induces hephaestin expression via a PI3K-CDX2 dependent mechanism to regulate iron transport in intestinal Caco-2 cells.

Zinc stimulates intestinal iron absorption via induction of divalent metal ion transporter (DMT1) and hephaestin (HEPH). While the increase in DMT1 is mediated via a PI3K/IPR2 axis, the mechanisms of Zn-induced HEPH expression downstream of PI3K remain elusive. In the current study we probed the role of Caudal-related homeobox transcription factor-2 (CDX2) on Zn-induced HEPH expression. Zn treatment of Caco-2 cells increased CDX2 phosphorylation and HEPH protein and mRNA expression. siRNA-silencing of CDX2 inhibited Zn-induced HEPH expression. LY294002, an antagonist of PI3K inhibited Zn-induced phosphorylation of CDX2, and downstream HEPH expression. These results suggest that increased expression of HEPH in intestinal cells following Zn treatment is mediated via a PI3K-CDX2 pathway.

Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli.

Apical membrane antigen 1 (AMA1) is a surface protein of Plasmodium sp. that plays a crucial role in forming moving junction (MJ) during the invasion of human red blood cells. The obligatory presence of AMA1 in the parasite lifecycle designates this protein as a potential vaccine candidate and an essential target for the development of novel peptide or protein therapeutics. However, due to multiple cysteine residues in the protein sequence, attaining the native fold with correct disulfide linkages during the refolding process after expression in bacteria has remained challenging for years. Although several approaches to obtain the refolded protein from bacterial expression have been reported previously, achieving high yield during refolding and proper functional validation of the expressed protein was lacking. We report here an improved method of refolding to obtain higher quantity of refolded protein. We have also validated the refolded protein's functional activity by evaluating the expressed AMA1 protein binding with a known inhibitory peptide, rhoptry neck protein 2 (RON2), using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).