No description available.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 130-155). Also available on the Internet.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

When proteins are unable to fold properly in the endoplasmic reticulum (ER), the resultant formation of misfolded proteins causes stress of the ER. Cells with ER stress often have a higher abundance of reactive oxygen species (ROS). Previous studies suggest that ROS could aggravate ER stress by further disrupting the ER protein folding process. More recent studies suggest that the unfolded protein response signaling pathways activated by ER stress could lead to the production of ROS. Such studies lead to the hypothesis that ER stress could be promoted by ROS, and vice versa. The aim of the present study is to test the above hypothesis by studying how ROS could be generated in ER-stressed cells. This is followed by investigating if ROS could increase or decrease the level of ER stress in cells. Finally, the extent of ER stress induced cell death in the presence and absence of ROS is assessed. The treatment of HeLa cells with tunicamycin (Tm), a common ER-stress inducing agent, resulted in the elevation of intracellular ROS that could be detected with the ROS-reactive probe dichlorodihydrofluorescein (DCF), but not dihydroethidium which is relatively specific towards superoxide anion. The Tm-induced elevation of ROS could be prevented by co-incubation of cells with thiol reductants such as dithiothreitol and N-acetylcysteine but not with the free radical scavenger ascorbate. The tunicamycin-induced elevation of ROS level could also be prevented by the over-expression of catalase in HeLa. These data is consistent with the idea that hydrogen peroxide is a major form of ROS produced in Tm-treated cells. In addition to elevation of ROS level, HeLa cells treated with tunicamycin also resulted in the phosphorylation of PERK and eIF2α, and the splicing of XBP-1. In the presence of cycloheximide to inhibit protein synthesis so as to deplete protein substrates for folding in the ER, tunicamycin-induced ER stress was greatly minimized as was evident by the absence of both the phosphorylation of PERK and splicing of XBP-1. However, the phosphorylation of eIF2α and elevation of DCF-detectable ROS remained unaffected. The cycloheximde-resistant phosphorylation of eIF2α could be prevented when cells were co-treated with thiol reductants, or upon the over-expression of catalase. These data suggest that the production of ROS in Tm-treated cells does not require the presence of ER stress as a prerequisite. Furthermore, the ROS so produced could induce phosphorylation of eIF2α without the need to cause ER stress in the first place. The quenching of ROS through the use of thiol reductants, or the over-expression of catalase, had no effect on inhibition of protein synthesis in cells treated with tunicamycin. However, the extent of cell death was significantly increased. The data obtained in this study is not consistent with the idea that ROS is a downstream product of ER stress, capable of inducing more ER-stress by a feedback mechanism. Therefore, a mutually enhancing effect between ER stress and ROS may not exist. The ROS found in stressed cells may serve to extend cellular survival under the condition of continuous stress. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Endoplasmic Reticulum (ER) stress signal is a cellular response to various insults including abnormal protein folding load, activating the unfolded protein response. Under severe ER stress, apoptosis will occur in most cell types. Interestingly, this does not happen in a disease model for Metaphyseal chondrodysplasia type Schmid (MCDS), where ER stress was activated in the hypertrophic zone of the growth plate where mutant collagen X proteins that cannot be folded correctly is expressed. Instead of normal progression from proliferating chondrocytes (PCs) to hypertrophic chondrocytes (HCs) and conversion to bone, HCs in MCDS mice undergo re-differentiation to PCs as a survival strategy due to an activation of ER stress. Transcription factors are known to be important in regulating differentiation. p53 family members, as transcription factors, are known to play important roles in developmental processes including cellular reprogramming, thus, we hypothesize that the ectopic expression of key transcription factors, p53 and TAp63, which are activated by ER stress is involved in HC re-differentiation. p53 is normally expressed in late PCs, Pre-HCs, and upper HCs, while TAp63 is expressed in PCs and Pre-HCs suggesting they may have roles in chondrocyte differentiation. p53 activated under ER stress in HCs are nuclear localized in MCDS mice, but did not invoke the apoptotic programme. In this project, using quantitative analyse to study the expression level of p53 and p63 isoforms, it was confirmed that p53 and TAp63γ are in part transcriptionally activated upon ER stress. From functional study by inactivating p53 in MCDS mice, it was shown that p53 alone was not sufficient to mediate re-differentiation. Given that TAp63γ isoforms is also highly upregulated upon ER stress, and the negative regulator, ΔNp63, is downregulated, this combination of change in gene expression also need to be considered. Furthermore, known regulators of p53 and p63 activity such as ASPP1 and iASPP are also differentially expressed in HCs, and are altered upon activation of ER stress favouring cell survival. Thus, it would be important to evaluate the combination of TAp63 in the re-differentiation process from conditional inactivation of p63 or in combination with p53 to gain a clearer understanding of the contribution and relationship of these transcription factors in the survival strategy of stressed HCs. / published_or_final_version / Biochemistry / Master / Master of Philosophy

alpha-TEA (alpha-tocopherol ether linked acetic acid) has been shown to induce apoptosis in human prostate, ovarian and breast cancer cells in culture and in xenograft models by promoting pro-apoptotic pathways and inhibiting anti-apoptotic pathways. Studies investigated the ability of alpha-TEA to induce apoptosis in human hematological malignant cell lines Jurkat, Raji and U266, representing T cell leukemia, B cell lymphoma and multiple myeloma, respectively. The three cell lines were cultured in the presence of different concentrations of alpha-TEA for different time periods, and examined for apoptosis by annexin V – FITC analyses, DAPI staining, and western blotting for poly (ADP-ribose) polymerase cleavage. alpha-TEA induced apoptosis in all three cell lines in a dose and time dependent manner. Levels of pro-apoptotic molecules DR5, c-Jun N-terminal protein kinase (JNK), C/EBP homologous protein (CHOP), caspase 9, and caspase 3 were upregulated in alpha-TEA treated cells in comparison to vehicle controls. Caspase 8 was activated in Jurkat and U266 cells but not in Raji cells. Apoptosis and pro-death signaling mediators were blocked by ceramide inhibitor, desipramine. The anti-apoptotic nuclear factor kappa B (NF-[kappa]B) signaling pathway was down-regulated in alpha-TEA treated Raji and U266 cells. Combinations of omega-3 fatty acid docosahexaenoic (DHA) and alpha-TEA significantly enhanced apoptosis in Jurkat cells in comparison to single treatments and vehicle control. In summary, alpha-TEA induced apoptosis in the malignant hematological cell lines is via shared and distinct pathways. ASMase/ceramide-mediated JNK activation and endoplasmic reticulum (ER) stress mitochondrial dependent apoptosis are involved in alpha-TEA induced apoptosis in the three cell lines; however, the cell lines exhibit cell type-specific responses to alpha-TEA: activation of death receptor/caspase 8 pathway is involved in Jurkat cells, suppression of NF-[kappa]B signaling is involved in Raji cells, and the U266 cells share both of these pathways for the induction of apoptosis. / text

Luman (LZIP/CREB3) is a basic leucine zipper (bZIP) transcription factor that has been linked to the endoplasmic reticulum (ER) stress response. In the event of ER stress, Luman is proteolytically cleaved, or ‘activated’, through regulated intramembrane proteolysis (RIP), resulting in an amino-terminal fragment that translocates to the nucleus to activate transcription of downstream unfolded protein response (UPR)-related genes. The general mode of activation of the key signal transducers of the UPR appears to be an alteration of their oligomeric states. Structural and functional similarities to these proteins suggest that Luman may be activated in a similar manner. In this thesis, we demonstrate through in vitro and in vivo studies that Luman can form homodimers in the cell. Through the use of mutagenesis, we show that Luman dimerization is mediated through the leucine zipper and we provide evidence that Luman dimerization is required for its transcription activation function. However, we found that Luman dimerization is not required for its activation by proteolytic cleavage.

No description available.

In eukaryotes, the endoplasmic reticulum is the site where folding of secretory proteins and the assembly of multimeric cell surface receptors take place. These processes are mediated by molecule chaperones that include the ER membrane bound chaperone calnexin and the sequence related calreticulin. Using a PCR strategy, a homologue for the mammalian calnexin/calreticulin family, CNE1, was isolated in S. cerevisiae. The CNE1 gene product, Cne!p, is an integral membrane glycoprotein of the ER. Disruption of the CNE1 gene did not lead to inviable cells or to gross effects on the levels of secreted wild type proteins. However, in CNE1 disrupted cells, there was an increase in the cell-surface expression of a normally intracellularly retained temperature sensitive mutant of the $ alpha$-pheromone receptor, Ste2-3p. In addition, an increase in the secretion of heterologously expressed mammalian $ alpha sb1$-antitrypsin was also observed in CNE1 disrupted cells. In order to study calnexin function in another genetically manipulable organism, a Schizosaccharomyces pombe calnexin homologue was sought. Using a similar PCR strategy, a S. pombe calnexin homologue, $cnx1 sp+$, was identified. The $cnx1 sp+$ gene product, Cnx1p, was shown to be a calcium binding type I integral membrane glycoprotein. Unlike the sequence related S. cerevisiae CNE1 gene, the $cnx1 sp+$ gene was essential for cell viability. Full length Cnx1p was able to complement the $cnx1 sp+$ gene disruption but full length mammalian calnexin could not. The ER lumenal domain of Cnx1p, which was secreted from cells, was capable of complementing the $cnx1::ura4 sp+$ lethal phenotype. Both wild type PI M1 (Val 213) $ alpha sb1$-antitrypsin and the ER retained PI Z variant were expressed in S. pombe cells. As in mammalian cells, wild type $ alpha sb1$-antitrypsin was normally secreted whereas the PI Z variant was retained intracellularly. Rescue of the secretion defective phenotype of the PI Z variant occurred in