TACKLING THE MOST FREQUENT WILSON DISEASE CAUSING MUTATIONWITH ALPHA-CRYSTALLIN DERIVED PEPTIDE

Mutations in the copper (Cu) transporter gene ATP7B are the primary cause of Wilson Disease (WD) associated with high liver Cu and death of hepatocytes.The recessive ATP7B-H1069Q mutation alone is the most frequent cause of WD in the white population (~60%). In hepatocytes, ATP7B resides within the Trans Golgi Network (TGN), where it mediates the incorporation of Cu into newly synthesized cuproenzymes. On increased Cu levels, ATP7B moves from the TGNto post-Golgi vesicles and Plasma Membrane (PM) to excrete toxic Cu via bile. ATP7B-H1069Q is not exported from the ER to the TGN, because partially misfolded. However, this mutant maintains a residual Cu transfer activity, most likely sufficient to prevent the disease if the protein would be correctly located in the TGN. Our previous results describe the rescue of localization and trafficking response to Cu overload of ATP7B-H1069Q in human Hep-G2 hepatocytesand COS-7 cells by the cytosolic holdasealpha?-crystallin (CRYAB or Hsp-B5)(1). Recent studies have identified potential sequences within the crystallin domain of both alphaA- and alphaB- crystallin with remarkable chaperone and anti-apoptotic activities. In particular, the peptide (residues 73 to 92) in alphaB-crystallin can act as mini-chaperone in vitro; protects Human Lens Epithelial cells (HLE) from stress induced apoptosis and inhibits Selenite-induced Cataracts after intraperitoneal injection in rats (2,3). Thus, the aim of the study is to develop pharmacologically interesting peptides of CRYAB acting as chaperones for the folding, localization and activity of ATP7B-H1069Q in tissue cultured cells. Our current results show that the CRYAB peptide 73-92 (Pept1) and the control peptide bearing inverted sequence (Pept2) are taken up by COS-7 cells and don't affect cell viability. Interestingly, onlyPept1 is able to correct the localization of ATP7B-H1069Q from the ER to the TGN and the post-TGN trafficking response to toxic Cu, as well as CRYAB full length.Moreover, FLIM analysis reveals that Pept1 interacts with ATP7B-H1069Q. This resultis strongly supported by the co-immunoprecipitation of Pept1 with ATP7B-H1069Q, as revealed by mass spectrometry analysis.