Browsing by Subject "Protein interaction."

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Open Access
Characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum protein stress
(2011) Yılmaz, Mustafa
Family 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.
Open Access
Identification and characterization of two endoplasmic reticulum protein isoforms encoded by senescence-associated FAM134B gene
(2008) Taşdemir, Nilgün
Liver 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.
Open Access
Identification of putative protein kinase inhibitors acting on liver cancer cells
(2012) Güven, Ebru Bilget
Hepatocellular carcinoma (HCC) as a, heterogeneous, multi-step, slowprogressing disease, has very limited treatment options due to its chemoresistant nature and late diagnosis. According to World Health Organization (WHO) reports HCC is a major public health problem. Each year, 748,000 new cases appear and 696,000 people lose their lives, all due to liver cancer alone. Sorafenib, the multi-tyrosine kinase inhibitor, is still the only (Food and Drug Administration) FDA-approved drug available for treatment. Therefore, there is an urgent need for novel, target-specific drugs based on the underlying molecular mechanisms of liver carcinogenesis. In this Ph.D. dissertation, synthetic purine, purine nucleoside analogs, aminotriazole and thiadiazine derivatives were evaluated for their cytotoxic activities and mechanisms of action against liver cancer. These novel molecules were selected because of their potential kinase inhibitory activity. Protein kinases, involved in signaling pathways, are the main enzymes of target-specific drug discovery; therefore, discovery of novel, putative protein kinase inhibitors as drug candidates can be promising for the treatment of primary liver cancer. Initially, sulforhodamine B (SRB) assay was used to screen the novel smallmolecules for their cytotoxic activities against breast, colon and liver cancer cell lines. Active molecules, then, were further exploited on a panel of HCC cell lines. The differential IC50 (half-maximal inhibitory concentration) values obtained, might indicate that these small-molecules interfere with cell signaling; since, these cell lines have individual characteristics of cell signaling activities. Further investigations are envisaged for the identification of the molecular mechanisms that these putative kinase inhibitors are involved in. Among the 228 newly synthesized putative kinase inhibitors, 3 smallmolecules were identified as promising anti-cancer agents against liver cancer with druggable cytotoxic activities and remarkable kinase inhibition potentials. The purine analogue, AUM32, and the purine nucleoside analogue, AUM42, were revealed as pro-senescence therapeutic agents in liver cancer. Both drug candidates were shown to initiate senescence-induced cell death and the underlying mechanism was confirmed for AUM42 as the induction of p15(INK4b) and the correlated decrease in Rb phosphorylation. Synthetic 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivative, ClAT-TM, is the other effective small-molecule with kinase inhibition potential. Upon ClATTM treatment, liver cancer cells experience a growth inhibition accompanying with dramatic morphological changes. Rounded, swollen and eventually detached cells were shown to be arrested in the G2/M stage of the cell cycle and eventually undergo apoptosis. Moreover, ROS (reactive oxygen species) accumulation and the activation of JNK signaling pathway were found as associated with the mechanism of action ClAT-TM cytotoxicity.
Open Access
Identification of the interacting domain of p53 family members with P33ING1 protein
(2002) Dinçel, Deniz
p53 is a tumor suppressor gene, which is mutated, in about 50% of human cancers. The product of p53 gene encodes a sequence specific transcription factor. The genes transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation and DNA damage repair. Two proteins, p63 and p73 members of p53 family, show striking homology to p53. p53 protein interacts with several viral and cellular proteins and these interactions are important in the regulation and dysregulation of the functions of p53. Another gene named, ING1 was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p24ING1, one of the protein product of ING1, was shown to enhance the growth suppressor functions of p53. Furthermore a physical association between p53 and p33ING1, another ING1 transcript, proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 family proteins and p33ING1 using in vitro techniques in order to determine the region(s) of p53 family proteins and p33ING1 that enabled this interaction. As a preliminary step for the study, the wild-type p53 cDNA and its several deletion mutant constructs were used in GST pulldown assays and Far Western assays with purified GST-p33 protein to map the interacting region on p53 protein. New deletion mutant constructs of p53 protein were created and cloned into expression vectors for the detailed analysis of the interacting domain of p53 protein. Also the other members of p53 protein family, p63 and p73 were examined in vitro for interaction with p33ING1. Deletion mutants of these proteins were created and cloned into expression vectors for protein-protein interaction assays. The results of this study shows that p53 protein interacts with p33ING1 and suggests that oligomerization domain of p53 protein is needed for this interaction. In addition, for the first time, it was showed that p63 and p73 proteins interact with p33ING1 and in p63 the C-terminus region is the primary determinant region, involved in these interactions with p33ING1.
Open Access
Identification of the role of p33ING1 protein in the cellular activities of p53 tumor suppressor protein
(1999) Emre, N. C. Tolga
p53 is a tumor suppressor gene which is mutated in about 50% of human cancers. The product of p53 gene encodes a sequence-specific transcription factor. The genes are transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation (via apoptosis or cell cycle arrest) and DNA damage repair. p53 protein interacts with several viral and cellular proteins and these interactions are important in the regulation and dysregulation of the functions of p53. Another gene, named ING1 (for "Inhibitor of Growth 1"), was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p33ING1, the protein product of ING1, was shown to enhance the growth suppressive functions of p53. Furthermore, a physical association between p53 and p33ING1 proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 and p33ING1 using in vitro methods in order to determine the region of p53 that enabled this interaction. As a preliminary step for the study, the ING1 cDNA was amplified from total cDNA of a cell line by PCR and cloned into expression vectors. The recombinant p33ING1 protein was overexpressed in E.coli and purified as a fusion protein with GST. The wild-type p53 cDNA and its several deletion mutant constructs were subcloned into a suitable expression vector to enable subsequent in vitro transcription-translation reactions. In vitro transcription-translation products of these constructs were used in GST pulldown assays with purified GST p53 protein to map the interacting region on the human p53 protein. The results of the study suggest that the primary determinant on p53 protein in its interaction with p33ING1, is the C-terminal domain while there may be other regions that are involved in the interaction.
Open Access
Molecular analysis of senescence-associated protein phosphatases DUSP10 and MTMR11
(2009) Gülay, Suna Pelin
Liver cancer is the fifth most common cancer in the world. Until recently, tumor cells were thought to proliferate indefinitely. In a previous study, our group showed spontaneous induction of replicative senescence in p53- and p16INK4a-deficient HCC (hepatocellular carcinoma) cell clones. The gene expression profiling was later done for these different clones, in an attempt to find novel therapeutic targets in HCC. Since protein kinases are known to be very important in disease formation and carcinogenesis, their partners in signaling, protein phosphatases should also be important in these processes. Hence analysis and targeting of protein phosphatases genes with differential expression between immortal and senescent clones might prove beneficial for HCC therapeutics. Among the phosphatase genes with differential expression patterns, we focused on two most upregulated genes in senescent clones with respect to immortal clones, DUSP10 and MTMR11. After gathering detailed information on these genes and their products by bioinformatics analysis, we confirmed the upregulation of the two genes in our senescent clones compared to our immortal clones by semi-quantitative RT-PCR. We then checked DUSP10 and MTMR11 expression in HCC and breast cancer cell lines to see if a differential expression of these genes are observed in different subtypes of these cell lines. Other experiments on MTMR11 focused on discovery of novel transcripts of this gene in HCC and breast cancer cell lines and checking the amounts of different transcripts in different subtypes of these cell lines, to form a bridge between MTMR11 transcript variants and carcinogenesis, however we did not observe differential expression. Two microarray studies comparing non-tumor and HCC tissues have listed MTMR11 as upregulated in HCC. Hence, upregulation of this gene in senescent clones may not be significant in hepatocarcinogenesis or replicative senescence, and further experiments should be performed. Considering DUSP10, we checked the subcellular localization of this protein in HCC cell lines by immunostaining, to see if the two subtypes (well-differentiated and poorlydifferentiated) of HCC cell lines differed in DUSP10 localization. We observed some cell lines having only nuclear or only cytoplasmic DUSP10, whereas most had both nuclear and cytoplasmic DUSP10. This lead the way for us to explore the factors that may be important in changing this protein’s localization, as this may be a type of regulation on this protein, and may change during carcinogenesis or upon induction of senescence. For this purpose, we checked to see if DUSP10 changed its localization in aging MRC-5 cell passages compared to young, proliferating ones and in premature senescence-induced cells compared to normal ones. Interestingly, it was found that upon replicative senescence induction, but not premature senescence, DUSP10 localized more to the cell nucleus which indicated a connection between DUSP10 localization and replicative senescence. We also checked to see if DUSP10 changed its localization upon disruption of the MAPK pathways it participates in, by kinase inhibitor experiments. Interestingly, it was found that DUSP10 localized significantly more to the cell nucleus upon inhibition of JNK pathway but not p38 pathway, in well-differentiated subtype of HCC cell lines. DUSP10 localization did not change significantly in poorly-differentiated subtype of HCC cell lines. Although JNKs, which seem to regulate DUSP10 through its localization according to this study, act as oncogenes in HCC, the significance of the change in DUSP10 localization should be characterized further before stating that DUSP10 can be a putative tumor suppressor. However, our other results indicate a relationship between DUSP10 localization and replicative senescence, which is promising because DUSP10 has emerged from our group’s microarray data as a replicative senescence-associated gene, and this connection should be analyzed further.
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.