Lipotoxic endoplasmic reticulum stress-associated inflammation : molecular mechanisms and modification by a bioactive lipokine

Abstract

Physiologic or pathologic processes that disturb protein folding in the endoplasmic reticulum (ER) activate a signaling pathway named the unfolded protein response (UPR). UPR promotes cell survival by reducing misfolded protein levels. The three proximal stress sensors of the UPR are known as PKR-resemble like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6), which monitor the quality of protein folding in the ER membrane and relay that information to the rest of the cell. If ER homeostasis can not be restored, prolonged UPR signaling can lead to cell death. Recent studies have shown metabolic overload, particularly high levels of fatty acids and cholesterol can induce ER stress and activate UPR signaling. These studies also demonstrated ER stress is a central mechanism that underlies the pathogenesis of metabolic diseases including obesity, type 2 diabetes, insulin resistance, atherosclerosis and hepatosteatosis. Understanding how nutrient excess activates the UPR and its novel molecular mechanisms of operation during metabolic stress could facilitate the development of novel and effective future therapeutics aiming to restore ER homeostasis. The molecular mechanisms of lipid induced activation of UPR and how the three proximal UPR stress sensors are linked to lipid metabolism and inflammation is not well understood. One of the UPR stress sensors, PERK, is a trans-membrane serine/threonine kinase with only two known downstream substrates, the eukaryotic translation initiation factor (eIF2) that controls translation initiation, and an antioxidant transcription factor, Nuclear factor eryhthroid-2-related factor-2 (Nrf2), that keeps redox homeostasis. One of the existing road blocks in studying PERK signaling has been the lack of molecular or chemical tools to regulate its activity. For my thesis studies, I developed a chemical-genetic approach to specifically modify PERK’s kinase activity. In this approach, the ATP binding pocket of a particular kinase is altered via site-directed mutagenesis in order to accommodate a bulky ATP analog that is not an effective substrate for the wild type kinase. Thus, only the mutated kinase can be targeted by the activatory or inhibitory bulky ATP analogs and this form of the kinase is referred to as ATP analog sensitive kinase (ASKA). Furthermore, I identified specific siRNA sequences that can be efficiently delivered to mouse macrophages and significantly reduce PERK expression. Both of these methods can be applied to study the direct impact of PERK activity on lipotoxic ER stress- associated inflammation. The results of the siRNA mediated PERK expression silencing experiments showed that PERK has a direct contribution to lipid-induced pro-inflammatory response in macrophages. Finally, I examined whether palmitoleate, a bioactive monounsaturated fatty acid previously shown to reduce lipid-induced ER stress and death, could also modify lipotoxic ER stress-associated inflammation. Based on the results from my experiments, palmitoleate is highly effective in preventing lipid induce inflammation. Unexpectedly, I also observed that palmitoleate could significantly block LPS-induced inflammation, too. In summary, during my thesis study I generated several useful tools including siRNA mediated knock-down of PERK and a novel chemical-genetic tool to directly and specifically modify PERK kinase activity. The findings from my studies demonstrate that PERK plays a significant role in lipid-induced inflammation, suggesting modification of PERK activity or its direct pro-inflammatory substrates could become desirable approaches to inhibit obesity-induced inflammation that contributes to the pathogenesis of diabetes and atherosclerosis. The outcome of my studies also showed that palmitoleate can significantly reduce lipotoxic-ER stress associated inflammation, which may explain its beneficial impact on both insulin resistance and atherosclerosis. Furthermore, the ATP-analog sensitive PERK mutant developed in my thesis can be coupled with proteomics to identify the full repertoire of PERK substrates during metabolic stress. In conclusion, the findings and tools developed in my thesis studies can form the basis of future studies to identify the molecular details of PERK’s involvement in lipid induced inflammation, the identification of novel PERK substrates during metabolic stress and the development of new therapeutic strategies against metabolically induced inflammation in obesity, diabetes and atherosclerosis.