A1M: a heme and radical binding protein. A study on structure, function and mechanisms

Abstract: This thesis describes functional and structural studies of the human protein α1-microglobulin (A1M), an evolutionarily well-conserved 26 kDa plasma and tissue protein mainly synthesized in the liver. A1M belongs to the lipocalin protein family. Lipocalins share a very similar structur, an 8-stranded β-barrel forming a hydrophobic pocket. A1M has reductase and radical scavenging properties. A1M also binds the iron-containing heme-group, a lipophilic small compound found in many proteins and enzymes, but toxic in free form. A truncated form of A1M (t-A1M) catalyzes degradation of the heme-group. These properties give A1M antioxidative properties in human tissues. The research focus in this thesis has been to study the protection mechanisms of A1M with a special focus on the structure, and to investigate its protection properties in vivo. The results show that radiation-induced cell damage induces reactive oxygen species (ROS) and that ROS mediates a so-called bystander effect (a spreading of damage to cells not directly hit by the radiation). A1M inhibited the bystander effect and by using site-directed mutagenesis it was shown that this protection is dependent on a cysteine side-chain in position 34, and regulated by three lysine residues in position 92, 118 and 130. Furthermore, when A1M binds heme, the cysteine in position 34, a histidine in position 123, and the three lysine-residues in position 92, 118 and 130 are involved in co-ordination of the iron-atom in heme. The results support a model in which two heme-groups are bound simultaneously. Molecular simulation suggests binding of the first heme group inside the lipocalin pocket, followed by a structural shift that allows binding of a second heme group, with lower affinity, near the opening of pocket. Finally, the in vivo therapeutic effects of A1M were investigated using a sheep model of the pregnancy disease preeclampsia. Starvation induced preeclampsia-like symptoms in the pregnant ewes manifesting in hemolysis and subsequent damage to the placenta and kidneys. A1M was well tolerated and displayed positive therapeutic effects.