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.

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Type 2 diabetes (T2D) is a growing health-care and economic burden. Obesity is a risk factor for developing T2D, but the underlying molecular mechanisms are not well understood. However, mechanisms such as lipotoxicity, endoplasmic reticulum stress and inflammation are becoming increasingly well-recognised in obesity, and may underlie the development and progression of T2D. A central player in these mechanisms is Protein Kinase R (PKR), proposed to have a role within nutrient- and pathogen-sensing pathways, and is activated by ER stress and lipotoxicity. A small molecule inhibitor Compound-16, adenoviral vectors and RNAi techniques in BRIN-BD11 rodent pancreatic β-cells, were used to demonstrate that PKR knockdown affords significant protection against palmitate-induced cell death. Furthermore, PKR knockdown potentiates palmitoleate cytoprotection during lipotoxicity, suggesting the cytotoxic and cytoprotective actions of long-chain fatty acid species may function via the PKR signalling pathway. The use of a novel 1.1B4 human pancreatic β-cell line has shown that important differences exist between human and rodent cell responses to fatty acids in vitro. In 1.1B4 cells, long-chain saturated and monounsaturated fatty acids do not provide increasing protection as their chain-length increases, in contrast to rodent cell models. Furthermore, methyl-saturated fatty acid species are well tolerated, and methyl-monounsaturated fatty acids are cytoprotective to 1.1B4 β-cells. TXNIP overexpression in an INS-TXNIP β-cell model has a proapoptotic role in conditions of glucotoxicity, but not glucolipotoxicity. Furthermore, in this cell model, succinate is cytoprotective against glucotoxicity, but not glucolipotoxicity. By contrast in 1.1B4 β-cells, succinate significantly protects against apoptosis induced by both glucotoxic and glucolipotoxic conditions. Chronic inflammation has been implicated in the development and progression of T2D. At the centre of this response is the pro-inflammatory cytokine IL-1β. The cellular origin of IL-1β is unclear, but IL-1β secretion has been linked to activation of the NLRP3 inflammasome, recently implicated in pancreatic β-cell death in T2D. Results suggest that IL-1β is secreted by INS-TXNIP and 1.1B4 pancreatic β-cells under lipotoxic conditions, thus offering a potential role for targeted IL-1β therapy in T2D.

MCDS is an autosomal dominant skeletal dysplasia disorder caused by mutations in collagen X. In most cases, mutations in collagen X result in a misfolded protein which is retained within the ER of hypertrophic chondrocytes, causing increased ER stress. It has previously been demonstrated that increased ER stress causes hypertrophic chondrocytes to de-differentiate in an attempt to avoid the stress. The altered differentiation results in reduced cell hypertrophy and impaired vascular invasion accounting for reduced bone growth. The presence of increased ER stress in hypertrophic chondrocytes is sufficient to cause the MCDS pathology; therefore reducing ER stress may be beneficial in terms of improving the associated pathology. The autophagy enhancing drug carbamazepine (CBZ) has been shown to be capable of reducing ER stress in cells expressing the MCDS-causing p.N617K collagen X mutation. I show in this thesis that CBZ treatment reduced ER stress in HeLa cells transiently expressing a further 3 MCDS-causing collagen X mutations. I have also demonstrated that CBZ treatment induced the degradation of mutant collagen X proteins either through autophagy or proteasomal degradation depending on the nature of the mutation. The drug was tested in vivo using the p.N617K collagen X mouse model of MCDS. In MCDS mice, CBZ reduced the severity of the disease pathology based on histological analyses, restored hypertrophic chondrocyte differentiation toward normal, increased long bone growth rates and decreased the severity of the hip dysplasia. Gene expression analyses on RNA isolated from microdissected hypertrophic chondrocytes revealed that CBZ shifted the pattern of hypertrophic differentiation markers in MCDS mice toward the wild-type pattern, most likely through its stimulation of gene expression associated with intracellular proteolytic pathways. The results presented in this thesis have contributed to the identification of a potential treatment strategy for MCDS- the stimulation of intracellular proteolysis of mutant collagen X. CBZ is FDA approved for the use of epilepsy and bipolar disorder and has a strong safety record in humans. Therefore CBZ could be a potential treatment strategy for MCDS.

Perturbations that derail the proper folding and assembly of proteins in the endoplasmic retriculum (ER) cause misfolded protein accrual in the ER – a toxic condition known as ER stress. The Unfolded Protein Response (UPR) is a signaling system evolved to detect and rectify ER stress. The work I present herein pertains to the most ancient member of the ER stress transducers, IRE1. ER stress stimulates IRE1 to activate a UPR-dedicated transcription factor called XBP1 in metazoans (or HAC1 in yeast) to bolster the productive capacity of the ER and purge misfolded proteins from the ER. To activate XBP1/HAC1, IRE1 cleaves XBP1/HAC1 mRNA twice to eliminate an inhibitory intron using a dormant nuclease function in its cytoplasmic effector region (IRE1cyto). My focus was to understand the mechanism of XBP1/HAC1 activation by IRE1, the regulation of IRE1 function and the manipulation of IRE1 signaling output using chemical tools. To better understand IRE1 mechanism, I determined the crystal structure of IRE1cyto bound to ADP. Structural and mutational analyses uncovered a probable novel IRE1 nuclease active site, allowing a catalytic mechanism of RNA cleavage to be inferred. Further genetic and biophysical experiments revealed that the ordered sequence of events: autophosphorylation, nucleotide binding and dimerization; orchestrates the assembly of the IRE1 nuclease active site to potentiate nuclease function. The flavanol quercetin was identified in a chemical screen as a potent stimulator of IRE1 nuclease output. To understand the mechanism of action of quercetin, I determined the crystal structure of IRE1cyto in complex with quercetin and ADP. Quercetin docked to a novel ligand binding site, termed the Q-site, at the interface of IRE1 dimers. Biophysical and genetic analyses revealed that quercetin engagement of the Q-site promotes IRE1 dimerization, thereby enhancing IRE1 nuclease activity. To gain insight on how IRE1 recognizes RNA, I performed bioinformatic analysis to identify a conserved sequence element in XBP1/HAC1 mRNA (termed XBP1mini) that may compose a higher-order structure recognized by IRE1. I developed an RNA production scheme to generate XBP1mini RNA for structural and biophysical studies. Preliminary X-ray diffraction studies indicate that XBP1mini may indeed adopt an ordered crystallizable tertiary structure.

Activation of the unfolded protein response follows induction of endoplasmic reticulum (ER) stress, resulting in widespread inhibition of protein expression. FLIP protein is particularly sensitive to stresses that perturb protein translation; as such, a reduction in FLIP is likely an early outcome of ER stress. Due to the anti-apoptotic role of FLIP, it is anticipated that potential decreases in FLIP would bring about an increase in sensitivity to death receptor stimuli and anoikis, a form of anchorage-dependent cell death. It was hypothesized that induction of ER stress results in downregulation of FLIP expression, resulting in sensitization of resistant tumour cells to death receptor stimuli and anoikis. From this hypothesis, it was determined that induction of ER stress through treatment of cells with brefeldin sensitized tumour cells to Fas-mediated cell death and anoikis. Moreover, over-expression of FLIP appeared to protect against ER stress-induced sensitization to cell death.

Perturbations that derail the proper folding and assembly of proteins in the endoplasmic retriculum (ER) cause misfolded protein accrual in the ER – a toxic condition known as ER stress. The Unfolded Protein Response (UPR) is a signaling system evolved to detect and rectify ER stress. The work I present herein pertains to the most ancient member of the ER stress transducers, IRE1. ER stress stimulates IRE1 to activate a UPR-dedicated transcription factor called XBP1 in metazoans (or HAC1 in yeast) to bolster the productive capacity of the ER and purge misfolded proteins from the ER. To activate XBP1/HAC1, IRE1 cleaves XBP1/HAC1 mRNA twice to eliminate an inhibitory intron using a dormant nuclease function in its cytoplasmic effector region (IRE1cyto). My focus was to understand the mechanism of XBP1/HAC1 activation by IRE1, the regulation of IRE1 function and the manipulation of IRE1 signaling output using chemical tools. To better understand IRE1 mechanism, I determined the crystal structure of IRE1cyto bound to ADP. Structural and mutational analyses uncovered a probable novel IRE1 nuclease active site, allowing a catalytic mechanism of RNA cleavage to be inferred. Further genetic and biophysical experiments revealed that the ordered sequence of events: autophosphorylation, nucleotide binding and dimerization; orchestrates the assembly of the IRE1 nuclease active site to potentiate nuclease function. The flavanol quercetin was identified in a chemical screen as a potent stimulator of IRE1 nuclease output. To understand the mechanism of action of quercetin, I determined the crystal structure of IRE1cyto in complex with quercetin and ADP. Quercetin docked to a novel ligand binding site, termed the Q-site, at the interface of IRE1 dimers. Biophysical and genetic analyses revealed that quercetin engagement of the Q-site promotes IRE1 dimerization, thereby enhancing IRE1 nuclease activity. To gain insight on how IRE1 recognizes RNA, I performed bioinformatic analysis to identify a conserved sequence element in XBP1/HAC1 mRNA (termed XBP1mini) that may compose a higher-order structure recognized by IRE1. I developed an RNA production scheme to generate XBP1mini RNA for structural and biophysical studies. Preliminary X-ray diffraction studies indicate that XBP1mini may indeed adopt an ordered crystallizable tertiary structure.

Activation of the unfolded protein response follows induction of endoplasmic reticulum (ER) stress, resulting in widespread inhibition of protein expression. FLIP protein is particularly sensitive to stresses that perturb protein translation; as such, a reduction in FLIP is likely an early outcome of ER stress. Due to the anti-apoptotic role of FLIP, it is anticipated that potential decreases in FLIP would bring about an increase in sensitivity to death receptor stimuli and anoikis, a form of anchorage-dependent cell death. It was hypothesized that induction of ER stress results in downregulation of FLIP expression, resulting in sensitization of resistant tumour cells to death receptor stimuli and anoikis. From this hypothesis, it was determined that induction of ER stress through treatment of cells with brefeldin sensitized tumour cells to Fas-mediated cell death and anoikis. Moreover, over-expression of FLIP appeared to protect against ER stress-induced sensitization to cell death.

Pt(II) based anticancer drugs—cisplatin, carboplatin, and oxaliplatin—are widely used in the treatment of a variety of cancers. Unfortunately, the clinical efficacy of these drugs is currently hindered by the development of undesirable side effects and resistance during treatment. The molecular mechanisms underlying these effects are still unclear. For decades, research has focused on DNA as the main cellular target of Pt(II) compounds. However, there is increasing interest in proteins as alternative targets of Pt(II) and contributors to cytotoxic and resistance mechanisms of cisplatin. In this work, I utilize Pt(II) compounds that have been functionalized to participate in the azide-alkyne cycloaddition ‘click’ reaction to study protein targets of platinum reagents. First, I describe the use of an azide-modified Pt(II) compound to fluorescently label and isolate Pt(II)-bound bovine serum albumin in vitro. Additionally, we discover that Pt(II) compounds form monofunctional adducts on BSA that can crosslink to DNA oligonucleotides. I then use the click-functionalized Pt(II) compound, azidoplatin, to enrich for Pt(II)-bound proteins in Saccharomyces cerevisiae using a biotin-streptavidin pull-down. I identified 152 proteins that are significantly enriched in AzPt-treated samples by LC-MS/MS analysis. A subset of these proteins are involved in proteostasis and ER stress, which I confirm is induced in both AzPt- and cisplatin-treated yeast. Of interest was the identification of the ER protein folding chaperone protein disulfide isomerase (PDI), which I observe is inhibited by Pt(II) binding in vitro. Finally, I investigate PDI activity in human cancer cell lines HeLa and MDA-MB-468 following treatment with Pt(II) compounds. Extracts from platinum-treated MDA-MB-468 cells show significant PDI inhibition at low concentrations of Pt(II), and these cells appear to have constitutive activation of the unfolded protein response. PDI activity in extracts from platinum-treated HeLa cells is inhibited only at high concentrations of Pt(II), and HeLa cells do not show significant XBP1 mRNA splicing during Pt(II) treatment. Additionally, MDA-MB-468 cells are nearly three times as sensitive to Pt(II) compounds than HeLa cells. From these data, I hypothesize that basal ER stress increases sensitivity to PDI inhibition by Pt(II) binding and that this interaction enhances Pt(II)-induced cell death. / 10000-01-01

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22314号 / 医博第4555号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 Shohab YOUSSEFIAN, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM