Browsing by Subject "ER stress"

Now showing 1 - 12 of 12

Open Access
Characterization of a novel IRE1 substrate pact and interacting miRNAS
(2022-06) Doğan, Aslı Ekin
The double-stranded RNA-dependent protein kinase activator A (PACT) anchors the RNAinduced silencing complex (RISC) to the endoplasmic reticulum (ER)’s membranous platform where RISC nucleation occurs and thus, PACT plays a key role in microRNA (miR)-mediated translational repression. Previous studies have shown that ER stress leads to PACT phosphorylation while simultaneously inducing changes in the expression of many miRs. Here, we demonstrate that PACT is phosphorylated by the ER-resident Inositol-requiring enzyme-1 (IRE1), a bifunctional kinase/endoribonuclease (RNase), both under ER stress and no stress conditions. While the role of IRE1 as a stress-induced RNase driving the unfolded protein response (UPR) is well understood, the function or the target(s) of its kinase activity have remained unexplored. Findings of this thesis show that IRE1- mediated phosphorylation of PACT regulates mature miR-181c levels, which suppresses the expression of key regulators of mitochondrial biogenesis (mitobiogenesis). Phosphorylation by IRE1 causes PACT-mediated suppression of mitobiogenesis and respiration. Partial PACT-deficiency in mice leads to enhanced mitobiogenesis during brown fat activation in cells and mice. Furthermore, cardiopulmonary bypass-induced ischemia/reperfusion injury downregulates PACT protein expression in human hearts while simultaneously inducing mitobiogenesis. Collectively, these findings demonstrate PACTmiR- 181c signaling axis is a key regulator of mitochondrial biogenesis and energetics.
Open Access
The effects of ER stress on glial cells: evidence from both in vivo and in vitro models
(2022-01) Mutlu, Duygu
Consuming high fat diet for long periods of time increases neuroinflammation, which may result in cognitive decline and loss in memory formation. Excessive free fatty acid influx stresses the ER and mitochondria, two important organelles taking part in protein folding and energy production. The ER responds to stress in part by activating Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) pathway. It has been shown that PERK pathway activation inhibits mitophagy (autophagy of mitochondria) in macrophages. Since microglia are the immune cells of the Central Nervous System (CNS), first I investigated the effects of high fat diet in the cortex of wild type (C57BL/6) and ApoE -/- mice by looking at microglial marker Iba1. Western Blot analysis showed no significant effects of diet and genotype on Iba1 level. I also found that there is no correlation between Iba1 and GFAP (astrocyte marker) levels for these mice. Brain contains many other types of cells and in order to effects of ER stress directly on the microglia, I moved on to the BV2 mouse microglial cell line. There were differential effects of ER stress induction with thapsigargin and palmitate treatments. An increase in ER stress markers such as CHOP and p-IRE1 has been observed with both treatments. While CHOP protein levels could not reach significance, there was an increasing numerical trend. p-IRE1 was marginally significant for both treatments (p=0.087 for thapsigargin and p=0.061 for 100 M palmitate). Mitophagy indicators (Pink1 and p62) were assessed with Western Blot analysis after successful mitochondria isolation from BV2 cells. The data indicated that p62 marginally increased with both palmitate (p=0.61) and thapsigargin (p=0.1) treatments. Following both treatments, very subtle effects of ER stress were observed. This suggests that further experiments examining optimal dosage and duration need to be performed. Overall, the induction of ER stress appears to induce mitophagy and alter microglia, which likely leads to altered cellular and synaptic function.
Open Access
Enhanced expression of HNF4α during intestinal epithelial differentiation is involved in the activation of ER stress
(Wiley, 2020) Tunçer, S.; Sade-Memişoğlu, A.; Keşküş, Ayşe Gökçe; Sheraj, I.; Sheraj, G.; Akyol, G.; Banerjee, S.
Intestinal epithelial cells are derived from stem cells at the crypts that undergo differentiation into transit‐amplifying cells, which in turn form terminally differentiated enterocytes as these cells reach the villus. Extensive alterations in both transcriptional and translational programs occur during differentiation, which can induce the activation of cellular stress responses such as ER stress‐related unfolded protein response (UPR) and autophagy, particularly in the cells that are already committed to becoming absorptive cells. Using an epithelial cell model of enterocyte differentiation, we report a mechanistic study connecting enterocyte differentiation to UPR and autophagy. We report that differentiated colon epithelial cells showed increased cytosolic Ca2+ levels and activation of all three pathways of UPR: inositol‐requiring enzyme 1 (IRE1), protein kinase RNA‐like ER kinase, and activating transcription factor 6 (ATF6) compared to the undifferentiated cells. Enhanced UPR in the differentiated cells was accompanied by the induction of autophagy as evidenced by increased ratio of light chain 3 II/I, upregulation of Beclin‐1, and downregulation of p62. We show for the first time that mechanistically, the upregulation of hepatocyte nuclear factor 4α (HNF4α) during differentiation led to increased promoter binding and transcriptional upregulation of two major proteins of UPR: X‐box binding protein‐1 and ATF6, implicating HNF4α as a key regulator of UPR response during differentiation. Integrating wet‐lab with in silico analyses, the present study links differentiation to cellular stress responses, and highlights the importance of transcription factor signaling and cross‐talk between the cellular events in the regulation of intestinal cell differentiation.
Open Access
Identification of a novel substrate of IRE1 in lipotoxic stress response
(2022-02) Yıldırım, Zehra
Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is a ribonucleic acid (RNA)-binding protein (RBP) that controls translation of select messenger RNAs (mRNAs). I discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. I show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP-deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. The results presented in my thesis provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggest IRE1 inhibition could be developed as a promising new therapeutic strategy to counteract atherosclerosis.
Open Access
Impacts of high-fat diet and genotype on blood-brain barrier and synaptic integrity in mouse cerebral cortex: an exploration of perk pathway
(2024-09) Şeker, Büşranur
High-fat diet intake can induce hyperlipidemia and result in cognitive decline by causing endoplasmic reticulum stress, decreased blood-brain barrier, and synaptic integrity. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is one of the arms of the unfolded protein response, which is activated by endoplasmic reticulum stress. The PERK inhibits the global protein translation while allowing the translation of certain proteins that are involved in inflammation and apoptosis. Due to its apoptotic properties, it is thought that the PERK pathway causes neurodegeneration. To study the effects of hyperlipidemia, a high-fat diet-fed Apoe knock-out mice model (Apoe-/-) is appropriate. Knocking out the Apoe in mice makes the animal model more prone to high-fat diet-induced hyperlipidemia. In the cerebral cortex of these animals, endoplasmic reticulum stress, blood-brain barrier, and synaptic integrity markers are checked at protein and mRNA levels. No changes are observed in the PERK pathway markers besides phosphorylated eukaryotic Initiation Factor 2. Additionally, there is a significant increase in blood-brain barrier marker Claudin-5 levels in Apoe-/- mice fed with a high-fat diet. There is also no significant change in synaptic integrity markers. In the second part, the effects of the PERK pathway inhibition are checked with integrated stress response inhibitor and GSK2606414 in the high-fat diet-fed Apoe-/- mice cerebral cortex. There are no significant alterations in BBB and synaptic integrity when the animals are injected with inhibitors. In conclusion, this study investigates the effects of high-fat diet induced hyperlipidemia in the cerebral cortex of Apoe-/- mice on ER stress, blood-brain barrier, and synaptic integrity. In the cerebral cortex region, the PERK pathway-related ER stress is not observed, and synaptic integrity remained unchanged while the blood-brain barrier is affected. Moreover, the effects of the PERK pathway inhibition are researched, and there is no inhibition effect observed in the cerebral cortex region.
Open Access
Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth
(Wiley-VCH Verlag, 2020-11) Hamid, S. M.; Çıtır, M.; Terzi, E. M.; Çimen, İ.; Yıldırım, Zehra; Doğan, Aslı Ekin; Kocatürk, B.; Onat, Umut Inci; Arditi, M.; Weber, C.; Traynor‐Kaplan, A.; Schultz, C.; Erbay, E.
The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P3) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P3 levels in macrophages. The modulation of cellular PI(3,4,5)P3/PIP2 ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.
Open Access
Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression
(EMBO Press, 2022-02-22) Yıldırım, Zehra; Baboo, S.; Hamid, S.M.; Doğan, Asli E.; Tufanlı, Ö.; Robichaud, S.; Emerton, C.; Diedrich, J.K.; Vatandaşlar, H.; Nikolos, F.; Gu, Y.; Iwawaki, T.; Tarling, E.; Ouimet, M.; Nelson, D.L.; Yates, J.R.; Walter, P.; Erbay, E.
Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.
Open Access
Investigating hyperlipidemia-driven organelle stress and neuroinflammation on the mouse cerebral cortex: insights into the intervention of perk pathway
(2024-09) Kızıldağ, Fulya
Deficits in the metabolism of lipids called hyperlipidemia have been linked to a higher risk of developing neurodegenerative diseases. Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) signaling is crucial in cellular homeostasis. Abnormalities in the PERK have been associated with neurodegeneration. Mitophagy and the PERK pathway emphasize how cellular stress responses are regulated to preserve cellular homeostasis and mitochondrial quality control. The activity of main mitophagy regulators, such as Parkin and PINK1 (PTEN-induced kinase 1), is regulated by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) by PERK. If lipid metabolism is at a high level, abnormalities in the mitochondria and endoplasmic stress (ER) emerge. During the ER stress activation, the PERK pathway is induced, and mitophagy is blocked, causing an enhancement in the neuroinflammation. The underlying molecular mechanism by which hyperlipidemia impacts the PERK pathway and mitophagy in the cerebral cortex, as well as the relationship between mitophagy and neuroinflammation, is not fully understood. In this study, Apoe-/- and C57BL/6 mice were given a chow or western diet to stimulate hyperlipidemia. Moreover, western diet-fed Apoe-/- mice were injected with PERK inhibitors, GSK2606414 and Trans-ISRIB, intraperitoneally for six weeks to suppress the PERK pathway. This study explores the effects of hyperlipidemia on the PERK pathway, inflammatory and mitophagy markers in the cerebral cortex of chow and western diet-fed C57BL/6 and Apoe-/- mice and investigates whether the inhibition of the PERK pathway can change the levels of inflammatory and mitochondrial markers in the cerebral cortex of hyperlipidemic mice subjects. mRNA and protein expression levels of mitophagy and inflammatory markers were assessed using the RT-qPCR and western blot, respectively. PERK pathway activation under hyperlipidemia conditions was not determined. Nevertheless, significant alterations in mitophagy markers and inflammation were detected in Apoe-/- mice apart from the diet. Furthermore, significant alterations were not seen in the PERK pathway markers; however, mitophagy was stimulated, and some inflammation markers were significantly decreased mildly at the cortical tissue of WD-fed Apoe-/- mice administrated with PERK pathway inhibitors, GSK2606414 and Trans-ISRIB. Besides, no statistically significant changes were observed in the transcript levels of the inflammatory markers. Taken together, hyperlipidemia did not cause the PERK pathway to be activated in the cerebral cortex of mice; nevertheless, it mildly altered inflammation and caused mild effects of the dysregulation of the mitochondria by hyperlipidemia independent from the PERK pathway. Furthermore, although the PERK pathway was not inhibited by the administration of PERK pathway inhibitors, mitophagy was induced, and inflammation was decreased mildly. Targeting the PERK pathway with GSK2606414 and Trans-ISRIB inhibitors from the cerebral cortex would not be a therapeutic approach for neurodegenerative diseases.
Open Access
Lipotoxic endoplasmic reticulum stress-associated inflammation : molecular mechanisms and modification by a bioactive lipokine
(2012) Demirsoy, Şeyma
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.
Open Access
Prevention of fatty acid-induced inflammasome activation by a bioactive lipokine
(2014-08) Koyuncu, Seda
Exposure to excess lipids such as fatty acids and cholesterol leads to cellular stress, release of reactive oxygen species (ROS), inflammation dysfunction and death. These responses have important role in the pathogenesis of chronic metabolic and inflammatory diseases such asobesity, diabetes and atherosclerosis. Recent studies show that fatty acids also cause the formation of inflammatoryprotein complexes that are called inflammasome. Inflammasome promotesthe activation of caspase-1 protein and cleavage of inactive interleukin-1 beta(IL-1β) and interleukin-18 (IL-18) into their active, secreted forms.Two signals are required for the activation of inflammasome. The first signal (also known as priming step) is needed for the inducing the expression ofthe proinflammatory factors pro-IL-1β and pro-IL-18 andthe nucleotide-binding oligomerization domain receptor (NOD-like receptor) family, pyrin domain containing-3 (NLRP3) proteins through the activation of nuclear factor kappa beta (NF-κB),a transcription factor. The second signal is needed for the activation of caspase-1 and formation of the inflammasome complex. Saturated free fatty acids such as palmitate are known to cause the activation of inflammasome through generation of mitochondrial ROS as a second signal for inflammasome complex formation. In this study, I investigated the effect of palmitoleate, a bioactive monounsaturated fatty acidpreviously shown to counteract lipid-induced ER stress and enhance insulin sensitivity, on palmitate-induced activation of inflammasome complex. I observed that palmitoleate lead to a significant reduction in palmitate-stimulatedIL-1β transcription. Moreover, it reduced the expression of palmitate-stimulated secondary proinflammatory factors such as tumor necrosis factor alpha(TNF-a).Palmitoleate also diminished the palmitate induced activation of caspase-1, maturation and secretion of IL-1β in macrophages. To further understand the mechanism of the protective role of palmitoleate on palmitate induced inflammasome activation,I analyzed the effect of palmitoleate on palmitate-induced mtROS in macrophages cells were analyzed. These studies showed palmitoleate decreased palmitate induced mitochondrial oxygen species in macrophages. Moreover, I investigated the effect of palmitoleate on palmitate-induced inactivation of 5' AMP-activated protein kinase (AMPK)controls autopaghy and clearance of dysfunctional mitochondria, thereby reducing mtROS formation. Palmitoleate blocked the suppression of AMPK phosphorylation that was suppressed by palmitate, suggesting PAO's impact on mtROS secretion and inflammasome activation occurs by this upstream mechanism. Saturated fatty acids like palmitate are also known to cause endoplasmic reticulum (ER) stress, whichbe counteracted by palmitolate treatment. When ER stress happens, cells try to solve stress by activating the unfolded protein response (UPR). There are three arms of the UPR are Protein Kinase R-resemble like protein kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating factor 6 (ATF6). In this thesis, contribution of PERK and IRE1 on palmitate-induced activation of inflammasome was investigated. First, the relation between PERK and IRE1 with palmitate-stimulated mtROS was analyzedusing PERK- and IRE1- deficient mouse embryonic fibroblast cells (MEFs).The results of this study show PERK leads to a marked reduction in the formation of mtROS while IRE1 enhanced palmitate induced mtROS.Furthermore, palmitoleate suppressed palmitate-induced autophosphorylation of IRE1 while there was no effect of palmitoleate on palmitate-induced phosphorylation of PERK. In summary, palmitate-induced inflammasome activation can be inhibited by palmitoleate in boththe priming step and the second step.Palmitoleate blocks palmitate-induced suppression of phosphorylation of AMPK, leading to its reactivation and subsequent reduction in mtROS formation. Palmitate is a well-known inducer of ER stress, which can be counteracted by palmitoleic acid. Moreover, the IRE1 branch of the UPR has been shown to control inflammasome activationby regulating of mtROS production under lipotoxicity. The outcome of my studies show that the UPR branches initiated by PERK and IRE1 regulate mtROS production and therefore, palmitoleate may also block inflammasome activation induced by palmitate through this alternative mechanism.These findings imply that palmitoleate may have therapeutic applications inthe management of diseases where inflammasome activation has been shown to play a causal role such as obesity, diabetes and atherosclerosis.
Open Access
The role of lipid-induced integrated stress response in metaflammation and atherosclerosis
(2019-07) Onat, Umut İnci
Chronic inflammation resulting from metabolic overloading of organelles (such as the endoplasmic reticulum (ER) and mitochondria that control cellular homeostasis) is a major cause of metabolic disorders including diabetes, obesity and atherosclerosis. ER is an organelle that plays a critical role in cellular metabolism through biosynthesis of lipids, protein maturation and secretion, and calcium storage. Furthermore, a stressed endoplasmic reticulum maintains cellular homeostasis by initiating a conserved stress response pathway that is known as Unfolded protein response (UPR). UPR is activated in response to diverse stimuli that disrupts ER functions and serves asva pro-survival mechanism to regain ER homeostasis. However, in prolonged or severe ER stress, chronic UPR can promote inflammation and apoptosis. Activated UPR, inflammation and necrosis are observed in and causally associated with atherosclerosis. UPR has three branches, one of which is initiated by the protein kinase RNA (PKR) like ER kinase (PERK) and signals to eukaryotic initiation factor 2a (eIF2a). This signaling arm of the UPR is also part of a larger, translational control pathway known as the integrated stress response (ISR). Activation of ISR has been observed in atherosclerosis and could promote atherosclerosis To study the contribution of ISR to atherogenesis, I took advantage of three small molecule inhibitors that can modulate this pathway. I also used a chemical-genetic approach, known as the Adenosine triphosphate (ATP) analog sensitive kinase (ASKA) technology, to interrupt PERK kinase activity. With these multiple tools, I was able to specifically interfere with ISR signaling at multiple molecular nodes in order to study the role of lipid-induced ISR in inflammation, inflammasome activation and atherosclerosis. I discovered that during lipid-induced ER stress, PERK to Activating transcription factor 4 (ATF4) signaling resulted in transcriptional induction of a mitochondrial protease, Lon protease 1 (LONP1), which degrades PTEN induced putative kinase 1 (PINK1) and blocks Parkin-mediated mitochondria clearance (or mitophagy). This in turn causes an increase in mitochondrial reactive oxygen species (ROS) production, inflammasome activation and pro-inflammatory cytokine secretion such as interleukin-1b (IL-1b) in both mouse and human macrophages. I also discovered that these inhibitors are also effective in reducing hyperlipidemia-induced inflammasome activation in Apolipoprotein E-deficient (Apoe/- ) mice and consequently, in preventing atherosclerosis progression. These results point out that intercepting with ISR signaling in hypercholestrolemia can be considered as a novel therapeutic approach that could be developed against atherosclerosis.
Open Access
The role of Protein Kinase R in lipotoxicity
(2013) Yağabasan, Büşra
Endoplasmic reticulum (ER) is a central organelle for cellular homeostasis through its myriad of functions including protein and lipid biosynthesis, protein folding and secretion and calcium homeostasis. When protein folding or secretion is disrupted, ER elicits a unique signaling response initiated at its membranes called the unfolded protein response (UPR). UPR attempts to restore cellular homeostasis and survival via reducing unfolded protein levels, however, if this cannot be achieved or the stress is prolonged the UPR could lead to apoptosis. Three specific ER membrane proteins, inositol-requiring enzyme-1 (IRE1), Protein Kinase R-resemble like ER kinase (PERK) and activating transcription factor 6 (ATF6), act as ER stress sensors and initiate distinct but interlaced signaling pathways to restore ER homeostasis. Recently, studies demonstrated that over nutrition, especially high amount of saturated fatty acids or cholesterol in the circulation, leads to the induction of ER stress in metabolic tissues, resulting in the activation of UPR signaling pathways. Furthermore, ER stress was shown to play a causal role in the pathogenesis of metabolic diseases such as obesity, insulin resistance, type 2 diabetes and atherosclerosis. Interferon inducible double strand RNA activated protein kinase R (PKR) is also known to be activated during ER stress. Recent studies showed it can be activated by lipids during ER stress in cells and in metabolic tissues of obese mice. Genetic ablation or inhbition of PKR enhances systemic glucose homeostasis and insulin sensitivity in obesity in rodent models. However, it is not known how PKR becomes activated by overnutrition or by ER stress. In fact, many of the specific cellular components and molecular mechanisms in lipid induced cellular stress or death, namely lipotoxicity, is not completely understood. PKR is one of the serine/threonine kinase that is known to be activated during lipid induced ER stress, but only a few specific downstream substrates are known and these fall short of explaining PKR’s role in lipotoxicity in chronic metabolic disease pathogenesis. PKR also plays a crucial role in activation of inflammasomes through interacting with the inflammasome components. There is a gap in our knowledge regarding PKR’s specific molecular actions in nutrient-induced inflammation and metabolism in chronic metabolic diseases. In this thesis study, my major goal was to develop specific tools to modulate PKR’s activity and search for its specific substrates in lipotoxicity and its role in mediating lipid-induced ER stress response. For this purpose, I developed a novel chemical-genetic approach to specifically modify PKR’s kinase activity during ER stress. In this approach, the bulky sidechain of a gatekeeper amino acid (such as methionine) in the ATP binding cavity of PKR has been altered to a smaller side-chain amino acid (such as glycine) in order to slightly enlarge the cavity to accommodate bulky ATP analogs (activating or inhibiting). This mutant of the PKR has been named the analog sensitive kinase allele (ASKA) of PKR and was shown to utilize normal ATP as well as the bulky ATP analogs in kinase reactions. Furthermore, I demonstrated the specific inhibition of PKR kinase with the inhibitory, bulky ATP analogs such as4-Amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine (NAPP1) or 4-Amino- 1-tert-butyl-3-(1’-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NMPP1). In order to move one step closer to identification of potential PKR substrates, I also optimized kinase reactions for immunoprecipitated PKR ASKA mutant and visualized several potential downstream substrates in my initial experiments Finally, I studied a unique relationship between two ER stress related kinases IRE1 and PKR in lipid induced ER stress conditions. I observed specific inhibition of IRE1’s endoribonuclease activity with an inhibitor, but not its kinase activity, completely blocks PKR activation by lipids. These findings strongly support hat IRE1’s RNAse activity is necessary for PKR kinase activation by lipids. This function of IRE1 RNAse domain is novel and unsuspected. The future goals of this research should be directed to discovering the RNA mediators of IRE1-PKR coupling and understanding their role in mediating the inflammatory and metabolic pathologies associated with chronic metabolic diseases. In conclusion, in my thesis study, I developed novel chemical-genetic approach to specifically modify PKR kinase activity that could be useful in discovering novel PKR substrates. Based on the preliminary findings in this thesis, PKR appears to have many unidentified substrates regulated during lipid induced ER stress. Furthermore, using the chemical-genetic PKR as a tool as well as several other approaches I demonstrated the existence of a unique, functional relationship between IRE1 and PKR in lipotoxicity. In addition, the results in my thesis shows that IRE1’s endoribonuclease activity is required and sufficient for PKR kinase activation by lipids. These findings and tools developed during my studies can be further utilized for analyzing the specific role of PKR in lipotoxicity, which is important for the health consequences of metabolic diseases.