Cellular Pro-Hepdidin Processing and Cleavage

Background: Hepcidin, which is referred to as `iron hormone`, is a circulating peptide that is produced by the liver in response to iron loading and inflammatory stimuli. Hepcidin overproduction is associated with the anaemia of inflammation, which is a common problem for patients with acute and chronic infections and tumours. Inhibition of intestinal iron absorption and reduction of macrophage iron release are the main functions of hepcidin. Circulating hepcidin is a 25 amino acid peptide which is produced as pro-84 aminoacid pre-pro-peptide, from which the biologically active C-terminus is cleaved during or after secretion. Although regulation of hepcidin production has latterly attracted great scientific attention, little is known about cellular pro-hepcidin processing and secretory mechanisms. Hypothesis & Aims: Based on our preliminary observations, we propose that hepcidin -like other known hormones- is secreted from hepatocytes in a regulated manner and not via constitutive pathways. The study of the secretory pathway of pro-hepcidin will lead to valuable insights into the regulation of iron metabolism. Further, the secretory pathway of hepcidin offers a potential target for pharmaceutical intervention. The aim of the proposed experimental work is to identify specific endo-peptidases that are implicated in pro- hepcidin processing. Biochemical, cell biological and histological studies are outlined to define and characterise this pathway of hormone activation, which occurs mainly in the liver. Predictions based on sequence homologies, have attributed pro-hepcidin cleavage to furin -a KEX2- like serine protease, which is the main endo-protease implicated in constitutive protein secretion. Pro-hepcidin peptidase activity will be studied in hepatic tissue extracts using artificial chromagenic and fluorogenic substrates. We propose to purify pro-peptide convertases implicated in pro-hepcidin cleavage from liver by chromatography. Further, candidate Kexin-2 like serine proteases will be tested for pro-hepcidin peptidase activity in vitro using recombinant pro-hepcidin. The biological relevance of such candidate peptidases that are ubiquitously expressed like furin or pro-protein convertase (PC)5/6 will be further investigated by cell biological assays. The tissue distribution and sub-cellular localisation of pro-hepcidin will be compared with that of candidate PCs and furin by two colour immuno-staining. Of those candidate pro-hormone convertases that co-localise with pro-hepcidin, co-trafficking studies will be carried out using an FK-binding protein fusion protein assay in cells over-expressing the pro-hepcidin and peptidases. Finally, animal studies in furin knock out mice might give valuable insights into the functional relationship between hepcidin production and furin in mammals. Implications of the Project: The proposed studies are designed to investigate the co-secretory processing of pro- hepcidin in cells and tissues. Dissecting the molecular mechanisms implicated in these cellular processes will improve our understanding of hepatic hormone secretion and maturation in general and may therefore extend to mechanisms underlying diabetes and obesity.

Background: Hepcidin, which is referred to as "iron hormone", is a circulating peptide that is produced by the liver in response to iron loading and inflammatory stimuli. Hepcidin overproduction is associated with the anaemia of inflammation, which is a common problem for patients with acute and chronic infections and tumours. Inhibition of intestinal iron absorption and reduction of macrophage iron release are the main functions of hepcidin. Circulating hepcidin is a 25 amino acid peptide which is produced as pro-84 aminoacid pre-pro-peptide, from which the biologically active C-terminus is cleaved during or after secretion. Although regulation of hepcidin production has latterly attracted great scientific attention, little is known about cellular pro-hepcidin processing and secretory mechanisms. Hypothesis & Aims: Based on our preliminary observations, we propose that hepcidin -like other known hormones- is secreted from hepatocytes in a regulated manner and not via constitutive pathways. The study of the secretory pathway of pro-hepcidin will lead to valuable insights into the regulation of iron metabolism. Further, the secretory pathway of hepcidin offers a potential target for pharmaceutical intervention. The aim of the proposed experimental work is to identify specific endo-peptidases that are implicated in pro-hepcidin processing. Biochemical, cell biological and histological studies are outlined to define and characterise this pathway of hormone activation, which occurs mainly in the liver. Predictions based on sequence homologies, have attributed pro-hepcidin cleavage to furin -a KEX2- like serine protease, which is the main endo-protease implicated in constitutive protein secretion. Pro-hepcidin peptidase activity will be studied in hepatic tissue extracts using artificial chromagenic and fluorogenic substrates. We propose to purify pro-peptide convertases implicated in pro-hepcidin cleavage from liver by chromatography. Further, candidate Kexin-2 like serine proteases will be tested for pro-hepcidin peptidase activity in vitro using recombinant pro-hepcidin. The biological relevance of such candidate peptidases that are ubiquitously expressed like furin or pro-protein convertase (PC)5/6 will be further investigated by cell biological assays. The tissue distribution and sub-cellular localisation of pro-hepcidin will be compared with that of candidate PCs and furin by two colour immuno-staining. Of those candidate pro-hormone convertases that co-localise with pro-hepcidin, co-trafficking studies will be carried out using an FK-binding protein fusion protein assay in cells over-expressing the pro-hepcidin and peptidases. Finally, animal studies in furin knock out mice might give valuable insights into the functional relationship between hepcidin production and furin in mammals. Implications of the Project: The proposed studies are designed to investigate the co-secretory processing of pro- hepcidin in cells and tissues. Dissecting the molecular mechanisms implicated in these cellular processes will improve our understanding of hepatic hormone secretion and maturation in general and may therefore extend to mechanisms underlying diabetes and obesity.