IRE1'nin iki farklı enzimatik aktivitelerinin biyolojik hedeflerinin ve rollerinin incelenmesi

The unfolded protein response (UPR) is composed of complex and intricate series of highly regulated pathways geared to resolve the accumulation of misfolded proteins and to maintain cellular homeostasis. Endoplasmic reticulum (ER) is an organelle that regulates the protein folding. Disregulation of the mechanisms underlying ER folding capacity can activate ERrelated UPR pathways (UPRER). The inositol-requiring enzyme-1 (IRE1) is an ER stress sensor and proximal regulator of the UPRER. IRE1 harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1's kinase substrate(s) and their function are unknown. This study was undertaken with the objectives of (i) discovering novel IRE1 kinase targets and (ii) deciphering the pathways regulated by IRE1 during ER stress and related pathophysiological conditions, (iii) investigating the mechanisms underlying how bioactive lipids resolve saturated fatty acidinduced ER stress. For the first part, I found out that phosphorylation of sphingosine 1-phosphate (S1P) lyase (SPL), a S1P degrading enzyme, is induced upon IRE1 kinase activation and this phosphorylation leads to inhibition of SPL's enzymatic activity. Consequently, SPL inactivation by IRE1 results in the accumulation of cellular S1P levels. Mitochondria is another important organelle and have their own UPR system (UPRmt) to regain organelle proteostasis. However, the link between UPRER and UPRmt is unclear. My experiments further showed that increased S1P levels potentiate UPRmt signaling in an IRE1 kinase activity-dependent manner. As a result of these experiments, we present IRE1 kinase activity as an important regulator of sphingolipid metabolism linking UPRER to UPRmt. I also investigated the function of IRE1 in Kawasaki Disease (KD), a pathophysiological condition leads to coronary artery aneurysm. Upon analysis of publicly available transcrioptome data sets from KD patients, I identified a significant increase in ER stressrelated gene signatures that associated with KD progression. The single cell transcriptomics data from Lactobacillus casei cell wall extract (LCWE)- induced murine model of KD further confirmed the activation of ER stress in myeloid cells. Upon confirmation of ER stress activation, I used genetic or small molecule approaches to ablate IRE1 function in order to study IRE1's role in LCWE-induced KD progression. My data demonstrated significance decrease in caspase-1 induction and IL-1β secretion leading to inhibition of cardiovascular lesion formations that were dependent on IRE1's RNase activity. The data shows that ER stress plays a causal role in KD progression via IRE1 activity. In last part, I studied how a bioactive lipid molecule, palmitoleate, acts on IRE1 in order to remodel ER membrane and to resolve lipid induced ER stress. My findings show that saturated lipids induce IRE1 oligomerization and activation while the monounsaturated fatty acid, palmitoleate, blocks this oligomerization. The promosing results of our mice studies with IRE1 inhibition warrants future investigation to assess IRE1's therapeutic potential in metabolic and inflammatory human diseases.