Browsing by Subject "Endoplasmic reticulum."
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Item Open Access Characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum protein stress(Bilkent University, 2011) Yılmaz, MustafaFamily with sequence similarity 134, member B (FAM134B) is a replicative senescence associated gene, previously identified in studies of our group as a result of microarray analysis in spontaneously senescent clones of Huh7 hepatocellular carcinoma cell line and their immortal counterparts. Originating from this finding, this study primarily focused on characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum stress. At the beginning, the relationship between senescence and FAM134B was experimented by inducing premature senescence in Huh7 cells. Adriamycin or TGF-β induced premature senescence did not result in amplification of FAM134B gene expression, suggesting that upregulation of FAM134B expression in spontaneous replicative senescence is not directly associated with a senescence phenotype. Then, FAM134B mRNA and protein levels were analyzed in both well- and poorly-differentiated HCC cell lines. Results showed that FAM134B expression is greater in poorly-differentiated cell lines, which represent advanced and metastatic HCC in vitro. On the other hand, our studies on the relationship between FAM134B and endoplasmic reticulum (ER) stress showed that FAM134B is an ER stress response gene, whose expression is upregulated by induction of ER stress with chemicals, such as thapsigargin, tunicamycin or DTT. Therefore, high protein and mRNA levels of FAM134B in poorly-differentiated cell lines are linked to the presence of a basal level ER stress response in this group of cell lines. Furthermore, overexpression studies in Huh7 cells indicated that FAM134B cannot trigger an ER stress response or autophagic response in these cells. However, FAM134B was detected as an effector in cellular response, when ER stress is artificially induced by thapsigargin or tunicamycin treatments. FAM13B4 overexpression in Huh7 resulted in increased sensitivity to thapsigargin or tunicamycin induced apoptosis. Moreover, increased FAM134B expression was also associated with decreased proliferative capacity in response to ER stress induction with the same chemicals. Consequently, FAM134B was suggested to affect the severity of stress in the ER when ER stress is started with an inducer. In addition, our tissue based experiments revealed that FAM134B is expressed in the brain and liver. Taken together, FAM134B might be an important protein contributing to the liver tissue damage and pathogenesis of HCC.Item Open Access Identification and characterization of two endoplasmic reticulum protein isoforms encoded by senescence-associated FAM134B gene(Bilkent University, 2008) Taşdemir, NilgünLiver cancer is the fifth most common cancer in the world. Until recently, tumor cells were known to have the capacity to proliferate indefinitely. In a previous study, we showed the spontaneous induction of replicative senescence in p53- and p16INK4a-deficient HCC (hepatocellular carcinoma) cells. In a follow-up study, we have analyzed the Affymetrix expression profiling of the senescent and immortal HCC clones that we had established. Among the genes with differential expression pattern, in this study, we have focused on a novel gene, FAM134B (family with sequence similarity 134, member B), which is significantly up-regulated (p-value=1.097E-06) in our senescent clones with respect to their immortal counterparts. FAM134B gene is located on human chromosome 5p15.1 near a LOH region, and its protein product has not yet been characterized. To begin with, we confirmed the up-regulation of FAM134B in our senescent clones as compared to our immortal clones by RT-PCR analysis. As a next step, meta-analysis of HCC microarray data indicated that the expression of FAM134B gene is progressively down-regulated in non-metastatic and metastatic HCC as compared to normal liver. Thus, we decided to characterize the protein product of this gene. Two known forms of transcripts were used to construct FLAG-tagged expression plasmids (encoding two isoforms with predicted molecular weights of 30 and 55 kDa). Immuno-staining experiments performed after transient ectopic expression indicated that both short and long isoforms of FAM134B-encoded protein localize to the endoplasmic reticulum (ER). Both protein isoforms co-localized with calnexin, a well known ER-chaperon. Thus, it appears that senescent cells over-express FAM134B-encoded ER protein isoforms, while cancer cells are deficient in their expression. We have also performed gain-of-function studies by stable ectopic expression of these two protein isoforms in an HCC cell line and addressed the potential role(s) of these isoforms in senescence and ER-stress. Our studies indicated that over-expression of these proteins did not have a ‘causative’ role in induction of senescence and did not affect the rate of cell proliferation. We also did not observe any changes in the responses of cells over-expressing these two protein isoforms to ER-stress induced via tunicamycin treatment. Therefore, FAM134B gene may be performing a yet unidentified function in senescent cells. All in all, we have identified two FAM134B-encoded proteins that localize to the ER, the function and the senescence association of which need further investigation.Item Open Access Lipotoxic endoplasmic reticulum stress-associated inflammation : molecular mechanisms and modification by a bioactive lipokine(Bilkent University, 2012) Demirsoy, ŞeymaPhysiologic 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.