The mammalian target of rapamycin complex 1 (mTORC1) is known to regulate lipogenesis through sterol regulatory element binding proteins (SREBPs), master regulators of cholesterol and fatty acid synthesis. Through an incompletely understood mechanism, mTORC1 triggers translocation of SREBPs, an endoplasmic reticulum (ER) resident protein, to the Golgi, where mature SREBP is proteolytically produced to activate transcription of lipogenic genes. Low ER cholesterol is a well-known trigger for SREBPs activation, which includes translocation, maturation, and transcriptional activation. The study investigated whether mTORC1 activates SREBP by limiting cholesterol delivery to the ER. The findings indicate an increase in mTORC1 activity is accompanied by lower ER cholesterol and by SREBP-2 activation, a transcription factor primarily responsible for cholesterol synthesis. A decrease in mTORC1 activity, on another hand, coincides with higher ER cholesterol and lower SERBP-2 activity. I further report that this ER cholesterol is of lysosomal origin, as blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from rising and, consequently, SREBP-2 is activated without mTORC1 activation. I identified two membrane trafficking processes, triggered by low mTORC1 activity, supply the lysosomes with cholesterol: autophagy and re-routing of endosomes to lysosomes. Indeed, a dual blockade by Atg5-/- and rab5 kept the ER cholesterol low even when mTORC1 activity was low, and resulted in SREBP-2 activation. Conversely, over-expressing Atg7, which forces autophagy, raises the ER cholesterol and suppresses SREBP-2 activity even when mTORC1 activity is high. Thus, it can be concluded that mTORC1 actively suppresses the formation of autophagosomes and promotes endosomal recycling, both of which prevents cholesterol to reach the lysosomes, thereby reducing cholesterol levels in the ER and activating SREBP-2.

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京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22633号 / 理博第4622号 / 新制||理||1664(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 平野 丈夫, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Thesis advisor: Eranthie Weerapana / Disulfide-bonding cysteine residues perform critical roles in the structural stabilization and redox regulation of protein function. Secreted proteins are often enriched for structural disulfide bonds conferring conformational stability in the oxidizing extracellular environment. The controlled formation of disulfide bonds in secreted proteins is regulated in the endoplasmic reticulum (ER) by the protein disulfide isomerase (PDI) family. To investigate disulfide-bond formation in the ER, quantitative chemical-proteomic methods were coupled to subcellular-fractionation-based ER enrichment. Cysteine reactivity studies identified highly reactive post-translationally modified cysteine residues including disulfide-bonding cysteines. Upon discovering a highly reactive population of traditionally oxidized cysteines, the percentage of oxidation for cysteines localizing to the ER was determined. Next, ER function was chemically perturbed to evaluate changes to cysteine oxidation following upregulation of the unfolded protein response (UPR). Disulfide bond formation was specifically disrupted in the ER by CRISPR-Cas9-mediated PDIA1 and PDIA4 knockout. The effects of PDI knockout on cancer cell phenotype and changes to cysteine oxidation states were evaluated. Finally, in vitro studies were performed to evaluate PDIA4 oxidase activity and identify potential PDIA4-selective inhibitors. In the future, the platforms developed within may be applied to profiling changes to cysteine oxidation in other biological systems such as other organelles and disease states. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13235号 / 論医博第2175号 / 新制||医||1037(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 竹内 理, 教授 渡邊 直樹, 教授 山下 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

N,N,N’,N’-tetrakis-(2-Pyridylmethyl) ethylenediamine (TPEN) is a heavy metal chelator with high affinity for zinc. TPEN causes important responses in mammalian eggs. For example, these eggs are arrested at the MII stage by the Endogenous Mitotic Inhibitor 2 (Emi2), which prevents activation of the Anaphase Promoting Complex (APC) and degradation of Cyclin B. By chelating zinc, TPEN inactivates Emi2 and eggs undergo spontaneous exit of meiosis and egg activation. TPEN chelates Ca2+ with lower affinity, although in the Endoplasmic Reticulum (ER), where Ca2+ concentrations are high, TPEN may sequester Ca2+ preventing release into the cytoplasm. Initial exposure of TPEN to MII eggs failed to cause spontaneous intracellular Ca2+ release from the ER. Interestingly, in the case of GV oocytes, addition of TPEN caused Ca2+ influx. This influx could be blocked via the addition of 2-APB, a plasma membrane Ca2+ channel blocker. To determine the possible role of TPEN on chelation of ER Ca2+, MII and GV cells were incubated in TPEN and ER Ca2+ released was by exposure to Cyclopiazonic Acid (CPA), a sarco/endoplasmic reticulum (SERCA) pump inhibitor, or Ionomycin (IO), a Ca2+ ionophore. In MII oocytes, the amplitude of the rises caused by CPA and IO, in TPEN-treated oocytes, was smaller than controls and experienced a delay in return to baseline. In GV oocytes, TPEN enhanced rather than reduced Ca2+ responses to CPA and IO. Given its inability to fully chelate ER Ca2+, the use of TPEN as a tool to study Ca2+ homeostasis in mouse oocytes needs additional investigation.

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.

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)

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