Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Our laboratory has recently reported that lecithin:cholesterol acyltransferase (LCAT) deficient mice are hypersensitive to insulin and resistant to diet-induced obesity, particularly in the LDL receptor (LDLR) knockout background. These phenotypes are linked to hepatic endoplasmic reticulum (ER) stress, which we showed is elevated basally and highly inducible in LDLR deficient mice. While in LCAT/LDLR deficient mice, ER stress is normalized basally and its diet-induction is attenuated. Mechanistically, we show here that excess free cholesterol (FC), in part from the bile, accumulates in the ER membrane of LDLR deficient mice. In contrast, LCAT/LDLR deficient mice have reduced levels of ER membrane FC and are resistant to cholesterol diet-induced elevations, in part from increased INSIG-1 expression and cholesterol esterification by ACAT2. Our analysis has led to the first report of cholesterol-induced hepatic ER stress in vivo and the identification of ER FC levels as a critical indicator of ER stress susceptibility.

Endoplasmic reticulum (ER) stress is the condition that unfolded or misfolded proteins accumulated in the ER which leads to the solubility stress. ER can activate the unfolded protein response (UPR) to restore the ER homeostasis. JAB1 was originally identified as the coactivator of AP-1 transcription factor. JAB1 was then discovered to mediate the cyclin-dependent-kinase inhibitor p27kip1 nuclear exportation and degradation. Previous studies demonstrate that ER stress may affect the regulation of JAB1, but the mechanism is still unknown. In this study, we want to investigate how JAB1 is regulated in ER stress. We applied tunicamycin, a protein N-glycosylation inhibitor, as the ER stress inducer. Western blot and reverse transcription PCR revealed that treatment with tunicamycin for 48 hours in Hep3B induced ER stress and repressed JAB1 protein and mRNA expression. Serial deletion of the JAB1 promoter activity assay revealed that the region from -405 bp to -223 bp may be responsive in the tunicamycin-induced ER stress. Computational prediction suggested that there are several candidate factors may join the regulation of JAB1 in this region. Site-directed mutation of JAB1 promoter assay revealed that the tunicamycin-induced ER stress repressed JAB1 promoter activity through the sites at -342/-338 and -331/-327 in JAB1 promoter. Chromatin immunoprecipitation assay suggested that tunicamycin-induced ER stress repressed the JAB1 promoter activity through increasing the SP1 and DNMT3b binding to the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter. Methylation specific PCR showed that the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter were methylated in tunicamycin-induced ER stress. Taken together, we demonstrated that tunicamycin-induced ER stress repressed the JAB1 gene expression in Hep3B through increasing the binding of SP1 and DNMT3b to the SP1 binding sites and inducing promoter methylation to repress JAB1 expression.

Our laboratory has recently reported that lecithin:cholesterol acyltransferase (LCAT) deficient mice are hypersensitive to insulin and resistant to diet-induced obesity, particularly in the LDL receptor (LDLR) knockout background. These phenotypes are linked to hepatic endoplasmic reticulum (ER) stress, which we showed is elevated basally and highly inducible in LDLR deficient mice. While in LCAT/LDLR deficient mice, ER stress is normalized basally and its diet-induction is attenuated. Mechanistically, we show here that excess free cholesterol (FC), in part from the bile, accumulates in the ER membrane of LDLR deficient mice. In contrast, LCAT/LDLR deficient mice have reduced levels of ER membrane FC and are resistant to cholesterol diet-induced elevations, in part from increased INSIG-1 expression and cholesterol esterification by ACAT2. Our analysis has led to the first report of cholesterol-induced hepatic ER stress in vivo and the identification of ER FC levels as a critical indicator of ER stress susceptibility.

Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Alzheimer’s Disease and other neurodegenerative diseases have been linked to dysfunction in proteostasis in the endoplasmic reticulum (ER). The ER provides an exclusive environment for protein synthesis and folding, which is vital to the cellular function. Under normal conditions, the synthesis and degradation of proteins remain in balance. During aging or during pathological states, disturbances of ER occur and consequently the failure of protein homeostasis. The cells rely on a system, the unfolded protein response (UPR), which regulates the homeostasis by three ER sensors: PERK, ATF6, and IRE-1. Perturbations of ER function result in UPR. In physiological condition, the cell may overcome the insult and regain homeostasis. However, prolonged or chronic UPR activates apoptotic pathways and may cause cell death. The sigma-1 receptor (Sig-1R) is a 25 kD polypeptide and a chaperone protein concentrated at the mitochondria-associated ER membrane domain (MAM). The Sig-1R plays significant roles governing calcium signalling, mitochondrial function, oxidative stress, protein chaperoning and ER stress. Results of this investigation demonstrate that immortalized mouse embryonic fibroblasts (MEFs) derived from Sig-1R-/—(KO) mice have higher baseline activation in all three branches of the UPR in the absence of ER stress compared to MEFs derived from Wild-type mice. Despite this increase in baseline activation, the PERK and ATF6 pathways have a significantly blunted response to acute stress. Rescue experiments by expressing the Sig-1R in KO MEFs did not recover the WT MEFs phenotype. Primary Sig-1R KO MEFs did not show baseline ER stress, but did show inhibited recovery following treatment with the acute ER stressor DTT. Overall, our data suggests that Sig-1R is important for the reestablishment of proteostasis following acute stress.

The aggregation of misfolded proteins in the endoplasmic reticulum (ER) is a pathological trait shared by many neurodegenerative disorders. This aggregation leads to the persistent activation of the unfolded protein response (UPR) and ultimately apoptosis due to ER stress. Cannabinoids, such as tetrahydrocannabinol (THC) and cannabidiol (CBD), have been reported to be neuroprotective in in vitro and in vivo models of neurodegeneration through their antioxidant and anti-inflammatory properties. However, little is known about the role of these cannabinoids in the context of ER stress. STHdhQ7/Q7 cells were treated with the ER stress inducer thapsigargin (TG) and cannabinoids in three different experimental paradigms to investigate the effect of 2.5 µM THC and 1 µM CBD monotreatment and cotreatment on ER stress-induced cell death. The mouse striatal neurons survived significantly more when THC or CBD was given before TG exposure. To further investigate this experimental paradigm, the gene and protein expression of UPR proteins was measured to determine the effect of cannabinoid pre-treatment on cell survival through ER stress modulation. A significant increase in the gene expression of the ER chaperone GRP78 and the ER-resident neurotrophic factor MANF in pre-treated samples suggest that with THC or CBD pre-treatment, the protein folding capacity of the cell is improved. Additionally, a decrease in the ER-mediated apoptotic markers such as BIM and caspase 12 with THC or CBD pre-treatment provides further evidence that cannabinoid pre-treatments are neuroprotective through ER stress modulation. These data suggest that prior cannabinoid monotherapy prepares the cell for future insults to the ER. Understanding the role of ER stress in the neuroprotective properties of THC and CBD provides insight into the therapeutic potential of cannabinoids and the role of ER dysfunction in various neurodegenerative disorders. / Thesis / Master of Health Sciences (MSc) / With the worldwide ageing population increasing, finding new treatments for illnesses that affect the elderly is crucial. Disorders such as Parkinson’s and Alzheimer’s disease mainly affect older individuals and are caused when brain cells stop working or when brain cells die. These disorders share some common causes. One is the inability to fold proteins properly. The cellular process that is responsible for protein folding and the changes that occur within that process are studied in this project. Also, the impact of the cannabinoids THC and CBD, a major component of cannabis, on the protein folding process is studied. This project found that using cannabinoids before the protein folding system is disrupted helps brain cells survive. This study is a step in understanding how THC and CBD are helpful in brain cell survival in patients suffering from diseases that damage brain cells.

Master of Science / Department of Biochemistry / Gerald R. Reeck / Armet is a bifunctional protein widely distributed in animal species, vertebrate and invertebrate. It is an evidently part of the Unfolded Protein Response (UPR) and promotes survival in cells that are under endoplasmic-reticulum (ER) stress. It has also been found as a secreted protein with neurotrophic activity. The crystal and solution structures of human Armet show it is a helix-rich protein with two domains linked through a flexible linker region. In this study, immunofluorescence staining was used to verify Armet’s localization in ER and Golgi apparatus in MBA-MD-231 cells. Evidence for calcium binding by Armet was obtained by circular dichroism spectroscopy (the binding of calcium appeared to decrease helix content), by differential scanning calorimetry (binding of calcium resulted in a less structured protein) and two-dimensional (1H-15N HSQC) nuclear magnetic resonance spectroscopy. A difference HSQC spectrum of Armet, with and without calcium, showed peaks of increased intensity, of decreased intensity and of perturbed chemical shift. There were about 30 such peaks in total. Several of these affected amino acid residues appeared to form a cluster of negatively charged side chains that could possibly form a binding site for a calcium ion. Heterogeneity of three types was observed in recombinant Armet expressed in E. coli cells. Two bands of slightly different mobility were observed in SDS gels run in the absence of reducing agent. These may represent alternate arrangements of disulfide bonds, as previously reported by other investigators but not explained. Further, in the absence of reducing agent, a faint ladder was formed by human Armet, indicating formation of disulfides between Armet molecules. Oligomers with sedimentation coefficient greater than the monomeric protein, in the absence of reducing agent, disappeared in the presence of a reducing agent. Finally, minor species of mass differences of 98 and 180 with respect to the main protein component were observed by MALDI-TOF mass spectrometry. These studies provide a more thorough characterization of Armet than has been previously available and set the scene for future investigations of the binding of organic ligands to the protein.

MCDS is an autosomal dominant disorder, with a mild dwarfed phenotype and is caused by mutations in collagen X. The majority of the mutations identified so far are localized almost exclusively within the NC1 domain, which is responsible for trimerization of the collagen X protein. Little is known about the onset of MCDS, but recently, up-regulation of ER stress has been suggested as an important mechanism promoting the MCDS phenotype. Several studies have shown that the mutated collagen X protein is retained within the ER triggering the UPR, which has proved to be the key pathway responsible for the pathogenesis of the MCDS phenotype. In order to study the consequences of the expressing the MCDS-causing COL10A1p.N617K mutation at the molecular level, we selected HeLa cells as an appropriate cell line for the characterisation of the UPR response, by showing that the three branches of the UPR can be activated by ER stress inducing conditions in a similar manner to that seen in vivo in the MCDS growth plate. Importantly we have also shown that HeLa cells can be transduced with the collagen X cDNA constructs and will express, fold and secrete collagen X into the supernatant.Having established the cellular model for MCDS studies we demonstrated for the first time direct evidence for the retention of mutant collagen X within the ER. Moreover, we demonstrated that the mutant collagen X was degraded via a proteasomal pathway. Nevertheless, the level of ER stress induced by expression of mutant collagen X, based on BiP induction at the protein level, was disappointingly low. We therefore directly compared the level of ER stress induced by the COL10A1p.N617K mutation with that of the chondrodysplasias-causing MATN3p.V194D mutation. The ER stress induced by the matrillin mutation was far greater than that caused by the mutant collagen X. We showed that general protein synthesis was reduced in cells expressing either of the mutant proteins, most likely by the mechanism associated with the phosphorylation of eIF2alpha. Moreover, we showed the mutant matrilin-3 protein was also retained specifically in the ER. However, we could find no evidence for either proteasomal or autophagic/lysosomal degradation of mutant matrilin 3.We tested a broad range of ER stress-relieving compounds on cells expressing mutant collagen X and matrilin 3. Carbamazepine, which was previously shown to reduce ER stress in alpha1-antitripsin deficiency, reduced ER stress in cells expressing the mutant collagen X (but not matrilin 3) by way of enhanced proteasomal degradation of the retained protein. This drug should now be tested in vivo against the MCDS mouse to determine its capacity to reduce disease severity.The results presented within this thesis have contributed to the understanding of how cells deal with mutant collagen X and matrilin-3 proteins. We have identified a potential therapeutic compound that may be of use in the treatment of MCDS. Furthermore, the data presented support the concept that generic approaches to relieving ER stress may not be suitable for treating a broad range of diseases. Treatments may need to be tailored not only in a gene-specific manner but also may need to be tailored to address the differing consequences of different mutations in the same gene.

Type 2 diabetes is characterized by insulin resistance and pancreatic β-cell failure. Insulin resistance leads to increased insulin demand, which can lead to increased proinsulin misfolding in the endoplasmic reticulum (ER). The accumulation of the misfolded proteins in the ER can cause ER stress, which can lead to pancreatic β-cell dysfunction. Cells respond to ER stress by the unfolded protein response (UPR), which increases protein folding capacity and causes degradation of misfolded proteins. Using a pancreatic β-cell model of induced misfolded proinsulin expression (proinsulin-C96Y tagged with GFP) we discovered that one of the most highly induced genes was stromal cell-derived factor 2 like 1 (SDF2L1). SDF2L1 is an ER localized soluble protein with an as yet unknown function. In this thesis I examined the potential role of SDF2L1 in pancreatic β-cells in ER stress conditions.