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.

The unfolded protein response (UPR) is a well conserved mechanism in eukaryotes that protects organisms from the damaging effects of endoplasmic reticulum (ER) stresses. Activation of the UPR will lead to two outcomes. It first attempts to restore cellular functions by enhancing protein folding capacity, inhibiting protein synthesis and promoting degradation of harmful proteins (the pro-survival pathway). However, if the stressful conditions are prolonged or severe, apoptosis will be induced (the pro-apoptotic pathway). The present study suggests that an isoform of the cellular protein Zhangfei (ZF, CREBZF) is linked to the pro-apoptotic pathway in the UPR by using DNA, protein and cell viability analyses. This isoform is known as the short-tail ZF (stZF). We demonstrated that stZF can be induced by prolonged ER stress. The protein of stZF is stable under ER stress and it has the ability to promote programmed cell death in the early stage of apoptosis through the induction of CHOP, a protein that plays a key role in the pro-apoptotic pathway of the UPR.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24531号 / 医科博第145号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 松田 秀一, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Vascular stiffening develops with both hypertension and aging, and is a strong predictor of end-organ damage. Excessive deposition of collagen by vascular smooth muscle cells (VSMCs) can lead to decreased compliance of vessels such as the aorta. The IRE1α arm of the unfolded protein response is activated in cells with a secretory phenotype due to its role in augmenting protein folding capacity. We hypothesize that by a similar mechanism, VSMCs transitioning to a collagen-secreting phenotype in response to TGF-β1 require the activation of IRE1. Inhibition of this pathway is hypothesized to reduce collagen secretion and hence prevent the development of fibrosis in the aorta. Methods: Collagen deposition by VSMCs in vitro was measured using immunoblotting and a Picrosirius Red-based colorimetric assay. Western blot and qRT-PCR were used to assess the expression of ER stress markers. Ex vivo culture of aortic rings was also performed to determine the effect of 4µ8c on TGF-β1-induced vascular stiffening. 12-14 week old male spontaneously hypertensive rats were divided into three treatment groups: 1) No treatment, 2) L-NAME (50 mg/L), and 3) L-NAME and the IRE1α inhibitor 4µ8c (2.5 mg/kg/day i.p.). Aortic compliance after 18 days of treatment was measured ex vivo using a wire myograph to construct tension-diameter curves. Results: Inhibition of IRE1α endonuclease activity by 4µ8c reduced collagen production in VSMCs stimulated with TGF-β1 or Ang II. A decrease in the expression of the collagen-associated chaperones PDI, GRP78 and GRP94 was observed. Aortic rings treated with TGF-β1 developed vascular stiffening, which was improved by co-treatment with 4µ8c. SHRs treated with L-NAME for 18 days developed aortic stiffening, which was prevented by daily injections of 4µ8c. Conclusions: Our data suggest that inhibition of the IRE1α pathway can reduce vascular stiffening and fibrosis by disrupting the collagen biosynthesis pathway in VSMCs. / Thesis / Master of Science (MSc)

The random-coil model has been dominant for unfolded proteins since the 1950’s; however, some experiments showed that the unfolded proteins were biased toward specific conformations in conflict with the random-coil model. Recently, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancement (PRE) were applied to obtain a large amount of structural information on unfolded proteins. Typically, these data were interpreted in a framework of random-coil ensembles with a good agreement between experimental data and theoretical predictions. In this thesis, it was tested whether locally organized nonrandom ensembles could describe this agreement equally as well. Using a complete set of RDC and PRE data for denatured ubiquitin, it was revealed that there was no distinguishable difference between random-coil ensembles and ensembles containing 50% native structure. Thus, while it is important to measure as many RDCs or PRE as possible, even the best datasets may be insensitive to local organization in unfolded proteins.

No description available.

In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This triggers a series of signaling pathways known as the unfolded protein response (UPR). The response helps to both enhance protein folding capacity and initiate mis-folded protein degradation, reducing E.R. stress. Alternatively, misfolded proteins are degraded and nutrients are recycled through autophagy. Thus, E.R. homeostasis depends on both UPR and autophagy. However, if E.R. stress is not resolved, UPR and autophagy can also cause apoptosis by mechanisms that are not fully understood. In chicken embryo fibroblasts, gamma-aminobutyric acid receptor-associated protein or GABARAP (a protein involved in autophagy) can promote apoptosis in conditions of prolonged starvation (Maynard et al. 2015). In these conditions, the down-regulation of GABARAP by shRNA/RNA interference reduces the expression of the pro-apoptotic CHOP (CAAT-enhancer-binding protein homologous protein) transcription factor (a marker of E.R. stress) and enhances cell survival. This suggests that elevated levels of autophagy compromises E.R. homeostasis and promotes the expression of CHOP in UPR lethal pathways. While GABARAP induction and processing/activation has been linked to the expression of CHOP upon prolonged starvation (Maynard et al. 2015), nothing is known about the pathway mediating CHOP expression and the relationship with other pathways of the UPR in cells with GABARAP mis-expression. Understanding these pathways will allow us to determine if GABARAP is a general determinant of E.R. stress or acts specifically on the expression of CHOP to control cell survival. Elucidating mechanisms which are involved in E.R. stress and the cellular transition between pro-survival to pro-apoptotic roles can allow understanding of processes associated with several pathological conditions like cancer and neuro-degenerative diseases. Additionally, establishing a role for GABARAP tumor suppressor in the control of the UPR and cell fate is also important. / Thesis / Bachelor of Science (BSc) / In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This activates both the Unfolded Protein Response (UPR) and Autophagy as both processes help to reduce E.R. stress. GABARAP, a protein involved in autophagy, has been shown to be involved in the promotion of apoptosis in conditions of prolonged starvation as its downregulation reduces apoptosis and CHOP expression (Maynard et al. 2015). However, how GABARAP regulates apoptosis remains unknown. Here, we investigate if GABARAP mis-expression affects multiple pathways in the UPR relieving global E.R. stress or if its specifically involved in blocking CHOP expression.

Die Unfolded Protein Response (UPR) ist ein in Eukaryoten konservierter Signalweg, der durch Akkumulation von un-/fehlgefalteten Proteinen im endoplasmatischen Retikulum (ER) aktiviert wird um die Proteinhomöostase zu gewährleisten. In <i>Saccharomyces cerevisiae</i> wird die UPR durch den ER-Stresssensor Ire1p und den bZIP Transkriptionsfaktor Hac1p, oder XBP1 in höheren Eukaryoten, reguliert. In dieser Arbeit konnten die Homologe der zentralen UPR-Regulatoren im biotrophen Pilz <i>Ustilago maydis</i> charakterisiert und die UPR als essenzieller Koordinator der pathogenen Entwicklung identifiziert werden. Die Komplementation der <i>∆HAC1</i> Mutante durch <i>cib1</i> (Homolog von <i>HAC1</i>) und umfassende Expressionsanalysen zeigten, dass die Regulationsmechanismen der UPR in <i>U. maydis</i> weitestgehend konserviert sind, das Spektrum der regulierten Zielgene jedoch sekretierte Virulenzfaktoren beinhaltet die für die pathogene Entwicklung notwendig sind. So konnten durch <i>in silico</i> Vorhersage möglicher Cib1 Bindestellen (UPRE) mit pit1/pit2 und tin1-1 drei bereits charakterisierte Effektorgene als direkt regulierte UPR-Zielgene identifiziert werden. Die gezielte Deletion des vorhergesagten UPREs führt zu einer Aufhebung der ER-Stress induzierten und Cib1 abhängigen Expression von pit2 und verringert die Virulenz signifikant. Darüber hinaus konnte gezeigt werden, dass eine funktionelle UPR sowohl notwendig für eine verstärkte Expression wie auch für die korrekte Prozessierung des Pit2-Effektors innerhalb des ERs ist. Im Gegensatz zur Bäckerhefe <i>S. cerevisiae</i> und den filamentösen Ascomyceten <i>Aspergillus niger</i> und <i>Trichoderma reseei</i> kodiert die ungespleißte XBP1 mRNA in höheren Eukaryoten für einen negativen Regulator der UPR. Mit den vorliegenden Untersuchungen in <i>U. maydis</i> konnte erstmals für niedere Eukaryoten gezeigt werden, dass die ungespleißte cib1 mRNA für einen negativen Regulator kodiert, der darüber hinaus eine bislang unbeschriebene und vermutlich konservierte Funktion in der Antwort auf ER-Stress besitzt. Die genaue Kontrolle der UPR-Aktivität ist Voraussetzung für die korrekte Ausführung der verschiedenen Schritte innerhalb der pathogenen Entwicklung von <i>U. maydis</i>. Während eine vorzeitige UPR-Aktivierung zur Inhibition des zur Pflanzeninfektion notwendigen filamentösen Wachstums führt, ist die gezielte Aktivierung der UPR nach erfolgreicher Penetration der Pflanzenoberfläche und ihre andauernde Aktivität während des Wachstums <i>in planta</i> notwendig für die pathogene Entwicklung. Die direkte Interaktion zwischen Cib1 und dem Entwicklungsregulator Clp1 während dieser Entwicklungsphase führt zur Stabilisierung von Clp1 und der Modulation der Cib1 abhängigen Genexpression. Auf diese Weise wird die Proliferation <i>in planta</i> ermöglicht und eine erhöhte ER-Stressresistenz vermittelt. Zusammenfassend zeigen die gewonnenen Ergebnisse, dass die UPR in <i>U. maydis</i> als Kontrollpunkt dient, um die zelluläre Physiologie, den Entwicklungsverlauf und die Sekretion von Effektoren aufeinander abzustimmen.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically devastating pathogen affecting the global swine industry. Since the emergence of the virus in the late 1980s, vaccination strategies aimed to control the virus have not been very effective. Current commercial vaccines are generally protective against homologous or closely-related strains but ineffective at conferring heterologous protection against genetically-diverse strains of the virus. Consequently, emergence of variant and sometime more pathogenic strains of PRRSV continues in global swine herds. As such, there is a need for better understanding of the molecular mechanisms involved in the replication of the virus. In order to better understand the molecular mechanisms of host responses to PRRSV replication, we first sought to evaluate the ability of the virus to induce stress granules (SGs) during PRRSV infection. SGs are intracellular, cytoplasmic aggregates of RNA-binding proteins (RBPs) and mRNA. Formation of SGs is observed upon cellular stress and ultimately function to arrest cellular translation to promote cellular survival until the stress has been remedied. Indeed, several viruses have been shown to modulate the SG pathways to facilitate viral replication and even suppress the host's immune response. However, it is currently unknown whether PRRSV modulates the SG response. First, we used confocal microscopy and fluorescent in situ hybridization (FISH) to determine the distribution of known SG marker proteins and cellular mRNAs. Our findings revealed that PRRSV induces a potent SG response at late time points post-infection, and that SGs were closely associated with viral replication complexes (VRCs). Subsequently, we demonstrated that SGs are dispensable for viral replication, as short hairpin RNA (shRNA)-mediated knockdown of critical SG components (G3BP1 and G3BP2) did not affect viral replication. Interestingly, we found that the PRRSV-induced SGs are formed in a PERK-dependent manner. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Further investigation into the PERK signaling pathway revealed that PRRSV induces a significant amount of ER stress upon the cell during viral infection, and that exogenous stress significantly impaired the ability of the virus to replicate in MARC145 cells. We also showed that PRRSV potently induces all three signaling branches of the UPR, including PERK. While PERK knockdown had no effect on cell viability or viral replication, it significantly upregulated the mRNA expression of interferon-β and interferon stimulated genes (ISGs). The results from our studies suggest a critical role for PERK in regulating the host innate immune response to PRRSV infection. Only with a better understanding of the underlying molecular mechanisms of PRRSV replication will we be able to rationally design more effective vaccines against the virus. / Doctor of Philosophy / Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically-devastating disease in the global swine industry. Annually, PRRSV is estimated to cause more than $600 million in economic losses to the swine industry in the United States alone. Current commercial vaccines against the virus are not effective against the diverse field strains largely due to the extreme heterogeneity of the virus. PRRSV is also able to potently suppress several aspects of the host's immune response and therefore establish a persistent infection. The underlying mechanisms of PRRSV-mediated immune suppression are not well understood. Therefore, in this dissertation we decided to investigate the molecular mechanisms of host responses to PRRSV infection. We first investigated the ability of the virus to induce stress granules (SGs). SGs are important intracellular regulatory components that modulate many aspects of the host's cellular processes, and have even been shown to play roles in regulating viral replication and controlling immune responses to viral infection. We demonstrate that PRRSV not only induces SGs, but that the PRRSV-induced SGs are closely associated with viral replication complexes (VRCs) within infected cells. The PRRSV-induced SGs were dispensable for viral replication. PRRSV-induced SGs were previously shown to form in a PERK dependent manner. Therefore, in the second part of this dissertation research, we decided to investigate the PERK signaling pathway during PRRSV infection. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Our results showed that PRRSV potently induces ER stress and all three signaling branches of the UPR, including PERK. Furthermore, we revealed that PERK may play an important role in regulating the type I interferon response to PRRSV infection. The results from our studies will aid in understanding the underlying molecular mechanism of PRRSV replication which will help rationally design the next generation of more effective vaccines against this devastating swine pathogen.

Motor neuron disorders are a heterogeneous group of diseases characterized by the selective degeneration of motor neurons leading to muscle wasting and atrophy. Amyotrophic Lateral Sclerosis (ALS) is the most common amongst these disorders and is characterized by the selective loss of both upper and lower motor neurons in the brain and spinal cord. 20% of familial cases of ALS are caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1), a ubiquitously expressed enzyme responsible for scavenging superoxide radicals. The exact mechanisms underlying mutant SOD1-mediated neurotoxicity are unknown. Misfolded mutant SOD1 accumulates in the cytosol and mitochondrial intermembrane space (IMS) indicating the involvement of unfolded protein responses in ALS pathogenesis. Unfolded protein responses (UPRs) are complex signal transduction cascades which detect perturbations in protein folding and couple them to the expression of protein quality control machinery thereby allowing individual compartments to adapt to stress. In the cytosol, this study has shown that HspB8 was upregulated by SOD1 mutants, where it induced the clearance of aggregates by macroautophagy. This is a protective mechanism, as overexpression of HspB8 suppressed mutant-SOD1 mediated toxicity. In contrast, HspB8 mutants were impaired in macroautophagy and are toxic to NSC-34 cells. The mechanisms for the IMS-UPR have not been previously identified. To address this issue, a model for the accumulation of misfolded mutant SOD1 within the IMS was created and candidate proteins involved in protein quality control within the IMS were explored at the transcriptional level and at the level of protein expression. Preliminary results revealed some possible candidates that may have a role in the adaptation to mitochondrial stress. Interestingly, increased mitophagy was also found in IMS-G93A expressing cells, advocating the central role of macroautophagy in eliminating protein aggregates and damaged mitochondria in SOD1-FALS.