The SRP and SRP receptor have long been recognized as essential components of the protein translocation machinery in higher eukaryotes. The biochemical studies discussed in this thesis demonstrate that the signal recognition particle (SRP) mediated transport of proteins across the mammalian endoplasmic reticulum requires the participation of guanine ribonucleotides, in a capacity distinct from their role in polypeptide elongation. The requirement for guanine ribonucleotides during translocation was detected by experimentally separating the synthesis and transport phases of the translocation reaction. Here, the initial targeting of ribosomes to the membrane required SRP and an SRP receptor, but not GTP. However, the insertion of the nascent chain into the membrane required the presence of both SRP and SRP receptor, as well as, GTP. Further biochemical characterization of the initially targeted translocation intermediate demonstrated that SRP remains bound to targeted nascent signal sequences, unless GTP is present. The SRP-receptor catalyzed displacement of SRP from ribosomes was GTP-dependent both with intact membranes and with the purified SRP receptor preparations. GTP specific binding localized to the α subunit of the receptor by photoaffinity labeling and by probing nitrocellulose blots of the receptor with GTP. In addition, an analysis of the α subunit primary sequence revealed elements which are similar, yet not identical, to guanine ribonucleotide binding site consensus sequence elements. These results, taken together, indicate that the SRP receptor represents a novel class of GTP binding protein and is responsible for the guanine ribonucleotide mediated displacement of SRP from nascent signal sequences. A more detailed biochemical investigation of the GTP hydrolysis cycle of the SRP receptor demonstrated that the affinity between SRP and the SRP receptor is substantially greater in the presence of bound GTP and that the subsequent hydrolysis of bound GTP by SRα is necessary to recycle SRP to the cytoplasm. Purified SRP receptor was shown to hydrolyze GTP slowly. However, the GTP hydrolysis rate was substantially increased when both the SRP receptor and SRP were present in equimolar quantity. SRP does not hydrolyze GTP under these assay conditions. Moreover, free SRP was found not to compete effectively with SRP-ribosome complexes for the receptor, implying that the conformation of SRP is altered upon binding to a signal sequence. This result suggests that the affinity between SRP and the SRP receptor may be exquisitely regulated in order to prevent futile GTP hydrolysis cycles from occurring in the absence of secretory protein synthesis. Furthermore, the demonstration that the SRP receptor is a GTP binding protein provides fundamental insight into the mechanism of protein translocation. The displacement of SRP appears to be tightly coupled to the membrane insertion of nascent signal sequences. The membrane inserted intermediate in nascent chain translocation can be characterized by i) a resistance to extraction from the membrane with either EDTA or 0.5M KOAc; ii) an insensitivity to protease digestion, even after dissolution of the membrane with nonionic detergent. These results indicate that SRP displacement allows the nascent chain to interact with an additional membrane bound, protein component of the cellular translocation apparatus. Once in contact with this additional component, the nascent chain is shown to be capable to transverse the membrane bilayer in the absence of ribonucleotide hydrolysis or the continued elongation of the polypeptide. Thus, the results are incompatible with postulated mechanisms of protein translocation requiring that energy be derived from the continued elongation of the nascent polypeptide or from the direct interaction of a hydrophobic signal sequence with the lipid bilayer.

The temperature-sensitive α-factor receptor (Ste2-3p) and arginine permease (Can1tsp) were found to provide the model substrates for quality control of plasma membrane proteins in Saccharomyces cerevisiae. When the ste2-3 mutant cells were grown at 34°C, Ste2-3p failed to accumulate at the plasma membrane and was delivered to the vacuole for degradation without traversing the plasma membrane. Upon reaching the vacuole, cytoplasmic domains of both Ste2p and Ste2-3p appeared within the vacuolar lumen. Four stp mutants were identified to suppress temperature-sensitive defects in both Ste2-3p and Can1tsp. The stp22 and STP26 mutations also caused missorting of vacuolar protein carboxypeptidase Y, and a subset of vacuolar protein sorting mutants (vps) suppressed ste2-3 mutation. In the stp22 mutant, both Ste2p and Ste2-3p accumulated in the prevacuolar compartment (PVC) and on the plasma membrane. Three independent mutations that bypassed the phenotype of stp22Δ mutant were identified and mapped to the SNF8 locus, and they were found to affect a single amino acid residue (G209D). The mutant protein, Snf8bpp, but not Snf8p, was able to compensate for the lack of functional Stp22p and to restore PVC-to-vacuole trafficking. The order of function for some VPS genes involved in PVC-to-vacuole traffic (class E) was determined by using this special snf8bp allele. In addition, a PtdIns 4-kinase encoded by the PIK1 gene was found to be involved in Ste2-3p trafficking, possibly affecting the PVC function.

The nonapoptotic form of regulated cell death known as ferroptosis is an attractive target for combating numerous diseases. Ferroptosis is an iron-dependent death of cells by lipid peroxidation. Pharmacological inhibition of anti-ferroptotic pathways is a promising therapeutic avenue for treatment of cancer, and death by ferroptosis has been implicated in numerous neurodegenerative and ischemia-reperfusion-driven diseases. Therefore, demystifying the dynamics of lipid peroxidation in this cell death process opens a window to understanding disease processes and how to treat them. This dissertation makes use of ferroptosis-modulating compounds as chemical probes to elucidate the roles of different subcellular membranes in ferroptotic lipid peroxidation. Chapters two and three explore the structure-activity-distribution relationship of fatty acids and the ferroptosis inducer FINO2, respectively, and together demonstrate the endoplasmic reticulum as a driver of lipid peroxidation in ferroptosis. Chapter two makes use of stimulated Raman scattering imaging, while chapter three uses confocal fluorescence imaging. Chapter four shifts gears to focus on development of FINO2 as a drug lead, performing structure activity relationship analysis to increase the potency and pharmacological properties of the analogs. Altogether, this work answers questions about how cells die by ferroptosis, and provides footwork for how we can better modulate ferroptosis against cancer and other illnesses.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20008号 / 農博第2192号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5017(農学部図書室) / 33104 / 京都大学大学院農学研究科農学専攻 / (主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13161号 / 論医博第2148号 / 新制||医||1029(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 大森 孝一, 教授 松本 智裕, 教授 秋山 芳展 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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No description available.

Increased secretion of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein, by the liver causes hypertriglyceridemia, which is a major risk factor for the development of atherosclerosis. The rate of VLDL-secretion from the liver is determined by its controlled transport from the endoplasmic reticulum (ER) to the Golgi. The ER-to-Golgi transport of newly synthesized VLDL is a complex multi-step process and is mediated by the VLDL transport vesicle (VTV). Once a nascent VLDL particle is synthesized in the lumen of the ER, it triggers the process of VTV-biogenesis and this process requires coat complex II (COPII) proteins that mediate the formation of classical protein transport vesicles (PTV). Even though, both VTV and PTV bud off the same ER at the same time and require the same COPII proteins, their cargos and sizes are different. The VTV specifically exports VLDL to the Golgi and excludes hepatic secretory proteins such as albumin and the size of the VTV is larger (~ 100 -120 nm) than PTV to accommodate VLDL-sized particles. These observations indicate (i) the existence of a sorting mechanism at the level of the ER; and (ii) the involvement of proteins in addition to COPII components. This doctoral thesis is focused on identification of proteins regulating VLDL sorting into the VTV and involved in the biogenesis of the VTV. In order to identify proteins present exclusively in VTV, we have characterized the proteome of VTV, which suggest CideB (cell death-inducing DFF45-like effector b) and SVIP (small VCP/P97 interacting protein) as candidates, present in VTV but excluded from PTV. We further confirmed the finding by performing co-immunoprecipitation studies and confocal microscopy studies. CideB, a 26-kDa protein was found to interact with apolipoprotein iv B100 (apoB 100), the structural protein of VLDL. Moreover, CideB interacts with two of the COPII components, Sar1 and Sec24. VTV generation was examined after blocking CideB by specific antibodies and by silencing CideB in rat primary hepatocytes. Knockdown of CideB in primary hepatocytes showed significant reduction in VTV generation, however, CideB was concentrated in VTV as compared with the ER suggesting its functional role in the sorting of VLDL into the VTV. SVIP, a small (~ 9-kDa) protein was found to interact with Sar1, a COPII component that initiates the budding of vesicles from ER membrane. SVIP has sites for myristoylation and we found increased recruitment of SVIP on ER membrane upon myristic acid (MA) treatment. Sar1 that lacks sites for myristoylation also is recruited more on ER upon myristoylation indicating that SVIP promotes Sar1 recruitment on ER. Additionally, our data suggest that Sar1 interacts with SVIP and forms a multimer that facilitates the biogenesis of VTV. Interestingly, silencing of SVIP reduced the VTV generation significantly. Conversely, incubation with MA increased the VTV budding, suggesting recruitment of SVIP on ER surface facilitates the VTV budding. We conclude that SVIP recruits Sar1 on ER membrane and makes an intricate COPII coat leading to the formation of a large vesicle, the VTV. Overall, the data presented in this thesis, determines the role of CideB and SVIP in regulating VLDL sorting and VTV biogenesis.

The mechanisms by which mitochondria convert nutrients into cellular energy have been described in intricate detail, and yet, the regulation and compartmentalization of such metabolic pathways are poorly understood. As a result, the underlying causes of mitochondrial dysfunction and bioenergetic deficiency in diseases such as amyotrophic lateral sclerosis (ALS) remain elusive. To address this longstanding gap in the field, we first sought to understand how the metabolism of glucose and glucose-derived pyruvate are regulated in the cell. Previous research has suggested that this metabolic regulation is mediated by specialized lipid raft domains of the endoplasmic reticulum (ER) in close contact with mitochondria, referred to as mitochondria-associated ER membranes (MAM). Using density gradient ultracentrifugation and immunoblotting techniques, we found that MAM domains play a role in the compartmentalization of glycolysis by recruiting and promoting the interaction of specific glycolytic enzymes. We then performed a series of bioenergetic, proteomic, and lipidomic analyses to determine how the establishment of ER-mitochondria contact sites at MAM affects the biology of mitochondria attached at these domains. We observed a novel distinction between mitochondria in contact with ER-MAM domains (MER) and those that are free from the ER (FM), with MER displaying a higher capacity for pyruvate-driven respiration and FM being specialized for fatty acid-driven energy production. Finally, using cell and mouse models of ALS with mutations in superoxide dismutase 1 (SOD1), we found that the glycolytic deficiency in ALS is a direct consequence of the progressive disruption of MAM structure and function, which thereby hinders the use of glucose-derived pyruvate as a mitochondrial fuel. This triggers a shift in mitochondrial substrate from pyruvate to fatty acids that, when sustained over time, contributes to the death of motor neurons and the progression of this fatal disease. Overall, this work aims to advance our understanding of metabolic compartmentalization, mitochondrial substrate specificity, and the relevance of both to ALS etiology.

<p> Cellular cholesterol homeostasis is a fundamental and highly regulated process. Transcription factors known as sterol regulatory element binding proteins (SREBP) are responsible for the expression of many genes involved in the uptake and biosynthesis of cholesterol. SREBP activation and lipid dysregulation has been associated with cellular endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). Our lab has previously reported a relationship between ER stress and SREBP activation causing lipid dysregulation and hepatic steatosis. This project was designed to elucidate the mechanism of ER stress-induced SREBP activation and determine its relationship with cellular pathologies associated with ER stress and lipid accumulation. My research has examined the mechanism by which ER stress activates SREBP-2 in various cell lines, including epithelial and macrophage cells. This research revealed that (1) ER stress-induced SREBP-2 activation is not dependent on caspases and occurs through the conventional sterol-mediated proteolytic pathway; (2) the mechanism of ER stress-induced SREBP-2 activation is sensitive to changes in ER calcium; (3) ER stress is associated with SREBP-2 activation and lipid dysregulation in a model of renal injury; and ( 4) ER stress-induced SREBP activation in vitro is not associated with lipid accumulation in macrophage foam cells. </P> <p> This project has also offered me the opportunity to further enhance our understanding of the mechanism by which ER stress causes SREBP activation in a sterolindependent manner. </P> / Thesis / Doctor of Philosophy (PhD)