Structural and Functional Consequences of Binding Calcium and Heavy Metal Ions to Human 15-Lipoxygenase-2

Abstract

Lipoxygenase (LOX) enzymes catalyze the oxygenation of polyunsaturated fatty acids, such as arachidonic acid, which regulate normal homeostasis, lipid metabolism, and cell signaling in humans. However, if an excess of or aberrant peroxidation products are formed, this can promote inflammation. For example, the activity of human epithelial 15-lipoxygenase-2 (15-LOX-2) has been linked to atherosclerosis, a common form of cardiovascular disease (CVD) – a leading cause of death in North Carolina.1 15-LOX-2, like other human LOXs, can bind divalent calcium ions (Ca2+) and translocate from the cytosol to the membrane surface. In vitro kinetic measurements have shown that 15-LOX-2 activity is accelerated at the phospholipid interface, suggesting that the interactions with membranes and lipids alter the LOX structure. Similarly, the divalent metal cadmium (Cd2+) has been denoted as a toxic heavy metal for its ability to mimic other divalent metal ions such as zinc or calcium and in turn traverse the body using transporters. This action can lead to lipid metabolism disruption and contribute to CVD. This thesis focuses on determining the binding thermodynamics of these two metals, Ca2+ and Cd2+, to 15-LOX-2 using isothermal titration calorimetry (ITC) and a series of site-directed mutations. The impact on 15-LOX-2 structure and stability upon Ca2+ binding was also analyzed by circular dichroism spectroscopy and differential scanning calorimetry. The cumulative data support that Cd2+ interacts with human 15-LOX-2 at the native Ca2+-binding sites and may play an important role in lipid metabolism through its dysregulation of LOX function. Future studies will be aimed at studying the structural consequences of binding these metals using high-resolution techniques such as X-ray crystallography and cryogenic electron microscopy (cryo-EM).