Publication
The innate immune system maintains internal homeostasis against hordes of ever-present pathogens. Principally, neutrophils and macrophages are recruites to sites of tissue damage to manage or slow down the rate of infection through several mechanisms, such as phagocytosis or NETosis. As the most abundant innate immune cells, they are competent at producing high concentrations of reactive oxygen species (ROS) which in turn can produce reactive electrophilic species (RES) such as lipid-derived electrophiles (LDE) through lipid peroxidation. A recent screen using REX (Reactive Electrophiles and oXidants) technologies, state-of-the-art chemical biology method, has identified a negative regulator of neutrophil apoptosis, SERPINB1 (SERine Protease INhibitor Clade B1) as a novel RES sensor. This thesis focuses on characterizing the interaction between LDEs and SERPINB1 and its downstream consequences for neutrophil homeostasis. Because RES/LDE frequently interact with cysteine residues, I hypothesize that the covalent interaction between RES and SERPINB1 will be at Cys344 which is located at the binding site for proteases, effectively resulting in SERPINB1 inhibition and subsequent apoptosis. In chapter 2, SERPINB1 is validated as a RES sensor and the site of modification is confirmed as C344 using purified recombinant proteins and T-REX in a mammalian cell model. Chapter 3 takes this finding further using Z-REX to label SERPINB1 with LDE in larval zebrafish models, exhibiting a significant decrease in neutrophil population with no effect on macrophage population. Additionally, this neutropenia phenotype can be rescued by injection of a morpholino to block expression of zebrafish neutrophil elastase (ela2/ela2l), which indicates a signaling axis between SERPINB1 and at least one protease. Overall, given the role of diseases such as neutropenia and the increasing prevalence of electrophilic motifs in approved small molecule drugs, this work promises to inform on electrophilic-fragments and targets of importance in disease treatment.