טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentYehonatan Gottlieb
SubjectOrientation and Localization of Heme-Oxygenase in
Macrophages
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Dr. Meyron Holtz Esther
Full Thesis textFull thesis text - English Version


Abstract

A central step of iron recycling is the phagocytosis of senescent erythrocytes by cells of the reticuloendothelial system such as macrophages. In response to erythrophagocytosis, heme-oxygenase-1 levels are elevated, to catalyze the oxidative degradation of erythrocyte-heme to iron, CO and biliverdin. Hemeoxygenase- 1 is characterized as an endoplasmic reticulum (ER) anchored protein. It is unclear how heme released from phagocytosed erythrocytes reaches the heme-oxygenase-1 active site and whether the active site of hemeoxygenase-1 in vivo is facing the cytosol or the ER lumen. It has been shown that ER membrane is recruited to the phagosome during phagocytosis but no enrichment of heme-oxygenase-1 on the phagosomal membrane has been demonstrated.
We studied the orientation and possible phagosomal recruitment of heme-oxygenase-1 during erythrophagocytosis with fluorescent microscopy and Western blots, using fused GFP-heme-oxygenase-1 and heme-oxygenase-1-GFP constructs transfected into macrophage cell-lines. Selective membrane permeabilization and digestion of cytosolic proteins revealed that: A. Both fluorescent heme-oxygenase-1 constructs are targeted to the ER membrane, B. The heme-oxygenase-1 active site is facing the cytosol. In addition, initial results indicate a massive recruitment of endogenous heme-oxygenase-1 to the erythrophagosome. Orientation of heme-oxygenase-1 on the erythrophagosome will be indicative for the mechanism of heme transport to its degradation site.