京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22665号 / 農博第2420号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5296(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The type VII secretion system is a protein export pathway linked to diverse phenotypes in both Actinobacteria and Firmicutes. The Actinobacterial subtype of the T7SS, referred to as T7SSa, has been shown to play a critical role in various aspects of Mycobacterial life including virulence, conjugation, and metal homeostasis. The T7SSb of Firmicutes bacteria on the other hand has similarly been shown to influence virulence but by the direct growth inhibition of competitor bacteria. Structure-function analyses of the T7SSa apparatus as well as various effectors and chaperones have begun to build a more mechanistic understanding of how T7SSa functions. In contrast, we know little of how the T7SSb functions despite its noted importance to both pathogens and environmental bacteria such as Bacillus, Staphylococcus, Enterococcus, and Streptococcus. During my thesis work, I have addressed several gaps in our understanding of T7SSb function. The three major questions that I have studied are: (1) how do T7SSb immunity proteins inhibit the toxicity of their cognate toxins, (2) how does the T7SSb export effectors through the thick Gram-positive cell wall, and (3) what is the role of chaperone proteins in facilitating T7SSb effector export? / Thesis / Doctor of Philosophy (PhD) / Bacteria require space and various nutrients to survive and grow and must therefore compete against other bacteria for access to these resources. To gain advantage over their competitors, many bacteria have developed molecular weapons that target and kill other closely related bacteria. Some of these weapons take the form of protein secretion machines that export antibacterial toxins. Gram-positive bacteria use the type VIIb secretion system (T7SSb) to inhibit the growth of other Gram-positive bacteria. In this work, I explore several aspects of T7SSb including: (1) how toxins are inhibited by immunity proteins, (2) how toxins are secreted through the cell envelope, and (3) how toxins are recognized by the secretion apparatus. The goal of this work is to better understand how T7SSb functions at the molecular level.

Clostridium perfringens is a Gram-positive bacterium that secretes a biofilm matrix material. The goal of these experiments was to identify pilin mutants that are needed for secretion of the biofilm matrix and develop a functional model for a type II secretion system (T2SS) in C. perfringens. Protein tagging, western blot, and slot blot experiments were done to quantify protein secretion. After performing experiments using a CPE0515-FLAG construct, it was concluded from immunoblot densitometry data that, except for the pilA1 deletion mutant, none of the 18 tested pilin mutants had a statistically significant difference from the wild type (WT) with regard to protein secretion. From slot blot densitometry assays, it was concluded that the pilA1 and CPE2280 mutants showed statistically significant lower values than the WT but the pilA2 and CPE1841 mutants had values that were higher than the wild type. Testing the construct containing only CPE0514 and CPE0515-FLAG showed that CPE0516 and CPE0517 are not needed for secretion of the protein CPE0515. HA-tagged CPE0516 qualitative immunoblots showed that, unlike CPE0515, oligomerization of CPE0516 is not occurring, and that this protein likely forms a heat stable dimer. Overall, the data did not allow us to construct a T2SS model, since there were not enough proteins revealed to be involved to create a complete Type II secretion system. / Master of Science / The methods by which C. perfringens can persist and survive in environmental conditions is something that would be useful to learn more about. One of the methods that many bacteria use to survive is by creating a biofilm matrix material, which provides protection for the bacteria from environmental stresses. In this study, the goal was to determine which specific proteins are needed for the secretion of the biofilm matrix material. Using molecular biology techniques, the proteins thought to be involved in biofilm formation quantified. The results showed that while two proteins ultimately appeared to be needed for secretion, there were not enough proteins involved to create a complete model for a functional secretion system in C. perfringens.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22495号 / 農博第2399号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5275(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 木岡 紀幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Part One. X-ray crystallography has benefited from the synthetic introduction of selenium to different positions within nucleic acids by easing the solving of the phase problem. Interestingly, its addition to the 2' position of the ribose ring also significantly enhances crystal formation. This phenomenon was investigated to describe the effect of selenium-based and other 2' modifications to the ribose ring of nucleosides in solution, as well as the incorporation of the selenium-modified nucleotides into a helical structure. This work correlates the difference in conformation propensity between the selenium containing nucleosides and oligomers towards a rationale behind the enhanced crystal forming behavior. Part Two. Recombinant protein production is a critical tool in laboratories and industries, and inducing extracellular transport of these products to the culture medium shows potential for improving cases where the yields are not sufficient in quality or quantity. This review incorporates current practices and systems with future perspectives.

Les protéines destinées à la sécrétion ou adressées à la membrane transitent par le réticulum endoplasmique (RE) où elles acquièrent leur conformation native et subissent des modifications post-traductionnelles comme la formation de ponts disulfures. Dans ce compartiment, la formation de ponts disulfures repose sur l’activité de l’oxydase Ero1 et de la Protein Disulfure Isomerase (PDI). Ero1 catalyse la formation de ponts disulfures et les transmet à la PDI qui à son tour oxyde les substrats. L’isomérisation ou la réduction terminale des ponts disulfures non-natifs repose sur un système de réduction dans le RE encore non élucidé. Des études suggèrent l’importance du glutathion réduit (GSH) dans ce système de réduction. Le GSH est un tripeptide redox exclusivement synthétisé dans le cytosol. Notre étude s’attache à (i) décrire les flux de glutathion entre RE et cytosol et (ii) identifier les acteurs de ce transport (iii) comprendre l’impact d’une modification de l’homéostasie redox du glutathion sur la physiologie du RE.Nous avons établi un système permettant d’étudier les flux de glutathion entre cytosol et RE. Afin de démasquer ces flux intracellulaires, nous avons utilisé une souche de S. cerevisiae surexprimant le transporteur plasmatique du glutathion, HGT1. Ce système permet de modifier rapidement et drastiquement la concentration cytosolique de glutathion. Les flux intracellulaires engendrés sont ensuite suivis grâce à des sondes redox spécifiques du glutathion adressées dans le RE ou le cytoplasme.(i) Nos résultats suggèrent que le GSH et le GSSG sont importés dans le RE depuis le cytosol. Le GSH est transporté selon un gradient de concentration via un système de transport de diffusion facilité. Ces flux sont également observés lors de stress stimulant la synthèse de GSH (stress thermique, arsenite…).(ii) Le transport de GSH dans le lumen est assuré par le translocon Sec61, et une régulation de cet import par la chaperone luminale Kar2 est observée.(iii) une réduction rapide de l’état redox du glutathion dans le RE conduit à une mort cellulaire programmée non apoptotique, également observée lors d’autre stress RE (traitement tunicamycine). / The endoplasmic reticulum (ER) is the first intracellular compartment of the protein secretion pathway. Protein maturation in this compartment involves protein folding and post-traductionnal modification including formation of disulfide bonds. The formation of disulfide bonds is operated by a highly conserved redox relay made of the thiol oxidase Ero1 and the protein disulfide isomerase (PDI). Ero1p catalyzes disulfide bond formation and relays them by thiol-disulfide exchange to PDI, which in turn oxidizes substrates. Isomerization and terminal reduction of non-native disulfide bonds both rely on a reduction system that remains to be formally identified. Studies however suggest the importance of reduced glutathione in this reducing system. GSH is small redox tripeptide exclusively synthesized in the cytosol. In this study we (i) describe the main parameters of glutathione traffic across the ER membrane (ii) identify the main actors involved in the transport and (iii) analyze the physiological impact of a modification of the ER glutathione redox state.We established a system to monitor the fluxes of glutathione from the cytosol to the ER in S. cerevisiae. To artificially increase fluxes of glutathione, we used a cell over-expressing the GSH plasma membrane transporter HGT1, which when grown in presence of glutathione import high levels of this compound. Consequently, we monitored the intracellular relocation of imported GSH by following GSH fluxes using two specific redox probes. Our data indicate that:(i) GSH is transported into the ER by facilitated diffusion along a concentration gradient. GSSG can also be imported into the ER. Similarly, stress conditions that stimulate GSH synthesis, such as heat shoc, arsenite treatment, also triggered a GSH import in the ER.(ii) GSH import in the ER is achieved by the translocon Sec61, and is regulated by the lumenal chaperone Kar2.(iii) A rapid reduction of glutathione ER redox state leads to the activation of a non-apoptotic programmed cell death pathway, usually observed during high ER stress.

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Komagataella pastoris, formerly known as Pichia pastoris, and hereafter referred to as Pichia, is a methylotrophic yeast widely employed as a recombinant protein factory for biotechnical and industrial purposes. P. pastoris boasts the ability to thrive at high cell densities, executes numerous post-translational modifications, and exhibits minimal secretion of endogenous proteins, thus greatly facilitating the expression and purification of recombinant proteins. Despite these advantages, Pichia still presents certain challenges as an expression system. Occasionally, recombinant proteins are retained within the cell and subject to degradation. Furthermore, Pichia falls short in matching the production capabilities of more widely used systems like Escherichia coli in terms of sheer numbers of recombinant proteins generated. When incubating a strain of Pichia expressing reporter protein enhanced green fluorescent protein (eGFP) (yJC100:pDT300) with fetal bovine serum (FBS), western blot analysis revealed a novel, higher molecular band in addition to the expected band, suggesting that FBS was altering recombinant protein expression. The alterations to this higher molecular weight variant were confirmed to happen intracellularly, but the molecular mechanisms behind it remain unclear. To elucidate the intracellular molecular mechanisms behind the production of the novel recombinant protein variant, our lab utilized site-directed mutagenesis and mass spectrometry. Through site-directed mutagenesis, we were able to localize the alteration facilitated by FBS to the C-terminus of eGFP, demonstrating that post-translational modifications incurred by FBS incubation occur at the C-terminus. Analysis of intracellular lysate of yJC100:pDT300 revealed proteomic alteration caused by treating P. pastoris with fetal bovine serum, presenting possible key players in FBS’s interaction with yJC100:pDT300. LC-MS was also used to analyze the extracellular media of FBS-treated yJC100:pDT300, revealing peptide discrepancies in the C-terminus of eGFP between FBS-treated and FBS-untreated samples. These results confirm that FBS’s interaction with yJC100:pDT300 significantly affects secretion through interaction with eGFP’s C-terminus. Our research serves to characterize the FBS’s interaction with yJC100:pDT300 and set precedence for future work in further characterization and optimization of the novel mechanism occurring due to FBS’s presence in growth media.

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA. The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (∆psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ∆psi, the delay likely represents some preparatory step that results in a transport competent translocon. In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system’s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery’s ability to transport substrates determined by the protein’s molecular weight, as well as by its dimensions. In total, these results suggest a dynamic translocon that undergoes functionally significant, ∆psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape.

Ovarian cancer (OC) has the highest mortality among gynecological cancers. The high mortality is associated with the lack of an accurate screening tool to detect disease in early stage. As a result the majority of OCs are diagnosed in late stage. Further, the molecular events responsible for malignant transformation in the ovary remain poorly understood. Consequently, delineating key molecular players driving OC could help elucidate potential diagnostic, prognostic and therapeutic targets. Receptor for hyaluronan-mediated motility (RHAMM) belongs to a group of hyaladherins, which share a common ability to bind to hyaluronan (HA). Intracellularly, RHAMM is involved in microtubule spindle assembly contributing to cell cycle progression. On the cell surface, loosely tethered RHAMM forms a complex with cluster differentiation 44 and HA to activate cell signaling pathways that promote cellular migration, invasion and proliferation. Since RHAMM is overexpressed in a number of cancer types and it is often associated with an aggressive cancer phenotype, I sought to determine if RHAMM similarly contributes to OC. I found that RHAMM is overexpressed in clinical specimens of OC by immuno-histochemistry and although both primary and metastatic OCs stain equally for RHAMM, RHAMM staining was most intense among clinically aggressive OC histologic subtypes. Further, using an in vitro model system, I was able to show that OC cells express and secrete RHAMM. Abrogation of RHAMM using silencing RNA technology inhibited OC cell migration and invasion suggesting that RHAMM may contribute, at least in part, to the metastatic propensity of OC. Since RHAMM lacks an export signal peptide sequence and has not been reported to employ alternate mechanisms for extracellular secretion, I utilized computational analyses to predict post-translational glycosylation events as a novel mode for RHAMM secretion. N- glycosylation inhibitors abrogated RHAMM secretion by OC cells in vitro validating my prediction and identify a novel and potentially unconventional mode for RHAMM secretion. Lastly, since RHAMM is secreted by OC cells, I sought to determine whether RHAMM could be detected in bodily fluids. In a pilot study, I found that urinary levels of RHAMM are elevated in OC patients as measured by enzyme-linked immunosorbant assays. Decreased urinary RHAMM levels noted following cytoreductive surgery support OC as the source of elevated urinary RHAMM levels. Finally, while obesity was associated with high urinary RHAMM levels in OC patients, combined measurements of urinary RHAMM and serum CA125 improved prediction of OC. Taken together, the studies described herein suggest that RHAMM contributes to OC and that further studies are warranted to further elucidate the clinical role of RHAMM in OC.