Investigating Dnase1L3's role in inflammation and redesigning the enzyme to be used as a therapeutic.

Abstract

Systemic lupus erythematosus (SLE) affects 1.5 million Americans. Lupus is a debilitating autoimmune disorder in which patients form antibodies that react to self-antigens. Anti-dsDNA antibodies bind to DNA, forming immune complexes (ICs) and deposit on organs. Over time accumulation of the ICs can lead to lupus nephritis, which will ultimately require a kidney transplant. Removing and breaking down the immune complexes from circulation and deposits would be beneficial in preventing further damage to organs. Some patients with SLE have a deficiency in Dnase1L3 (Dnase1-Like 3) enzyme caused by a pathogenic mutation in the Dnase1L3 gene. Dnase1L3 digests extracellular DNA in the peripheral circulation. In contrast to Dnase1, Dnase1L3 possesses the unique activity of digesting DNA complexed with other biological molecules in the form of microparticles and immune complexes; this activity is essential in preventing the creation of antibodies against self-antigens. Dnase1L3 degrades antigenic DNA complexes and is a potential therapeutic for SLE. Dnase1L3 has also been implicated in having a role in releasing inflammatory cytokines, but specific inhibitors block this action; it is still unclear what Dnase1L3 function is inhibited by these inhibitors. We have developed a unique bacterial expression system for Dnase1L3 and solved the X-ray crystal structure of the core enzyme without the C-terminal domain. I plan to determine the structure of the full-length enzyme bound to DNA to better understand the mechanism of action of Dnase1L3. To minimize any potential off-target side effects from a Dnase1L3 therapeutic, I am also investigating the inhibitors to elucidate the Dnase1L3 role that is blocked in the inflammatory pathway. The long-term goal of this project is to produce the most effective therapeutic for lupus nephritis.