Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第19001号 / 工博第4043号 / 新制||工||1622 / 31952 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 今堀 博, 教授 森 泰生 / 学位規則第4条第1項該当

<p>The modification of proteins by the covalent attachment of ubiquitin is a natural process that crucially regulates a wide range of eukaryotic signaling outcomes. This process has been understood as the linking of the C-terminus of ubiquitin to the lysine residue of a target protein via an isopeptide linkage, catalyzed by the coordinated effort by E1, E2, and E3 enzymes. Importantly, ubiquitination has only been observed to be a eukaryotic phenomenon. In recent years though, intracellular bacteria, including human pathogens, have been observed to possess ubiquitin-interacting proteins in their genomes. These proteins serve to subdue and manipulate their hosts’ ubiquitin signaling for their own benefit. While some of these proteins act within the eukaryotic context, more recent findings reveal the existence of prokaryotic enzymes that catalyze ubiquitination using mechanisms never before seen in nature. These remarkable processes utilize different cofactors and target different amino acid residues of both ubiquitin as well as substrate protein. The findings reported in this Thesis involve structural and biochemical studies on two new ubiquitinating proteins, the only two proteins known to catalyze ubiquitination outside of the canonical pathway. Both proteins are present in the genome of the intracellular human pathogen <i>Legionella pneumophila</i>: the SidE family, which catalyzes ubiquitination via a mechanism combining ADP-ribosylation and phosphodiesterase activities, and MavC, which utilizes a mechanism reminiscent of transglutaminases. Key insights provided in this document include the discovery that SidE enzymes can modify multiple ubiquitin moieties within a ubiquitin chain, and that modified ubiquitin chains are resistant to hydrolytic cleavage from many deubiquitinating enyzmes. Also, the development of a robust, continuous assay for SidE-catalyzed ubiquitination using a synthetic substrate is described. The catalytic action of MavC, which differs from both canonical E1/E2/E3 ubiquitination and SidE ubiquitination is also here elucidated. The crystal structure of MavC in complex with its ubiquitinated product is presented and provides an atomic view into the basis of substrate recognition. These findings bring to light a new dimension of host-pathogen interactions, where pathogenic ubiquitinating enzymes have appeared to arise from convergent evolution. The regulation of these pathogenic enzymes by other effectors is also discussed, as well as biochemical studies of these regulators. Further, these findings describe possible new drug discovery strategies, as well as possible techniques for discovering similar enzymes in organisms besides <i>Legionella</i>.</p>

In prokaryotes, protein glycosylation can be a determinant of pathogenicity as it plays a role in host adherence, invasion, and colonization. Impairment of glycosylation in some organisms, for example N-linked glycosylation in Campylobacter jejuni, leads to decreased pathogenicity; thus, opening new avenues for the development of antivirulence agents. A member of the protein glycosylation (pgl) gene locus in C. jejuni, PglC, is predicted single-pass transmembrane (TM) protein, that catalyzes the phosphoglycosyl transferase (PGT) reaction in the first membrane-committed step of the N-linked glycosylation pathway. The small size of PglC (201 aa) compared to homologous PGTs suggests it may represent the minimal catalytic unit for the monotopic PGT superfamily. Herein, the structure of C. concisus PglC including its putative TM domain has been solved to 2.74 Å resolution to reveal a novel protein fold with a unique alpha-helix-associated beta-hairpin (AHABh) motif and largely solvent-exposed structure. There is noted a parsimony of fold in the form of short-range motifs underpinning the structural basis for critical functions of PglC: membrane association and active-site geometry. Biochemical and bioinformatics studies support structural evidence suggesting the crystallographically-observed, kinked TM helix is re-entrant on the cytoplasmic face of the membrane rather than membrane spanning. Thus, PglC represents a first-in-class structure of a novel membrane interaction mode for monotopic membrane proteins. Additionally, the AHABh-motif and active-site helical geometry establishes co-facial positioning of the catalytic-dyad. Molecular docking of PglC substrates, undecaprenyl phosphate (UndP) and UDP-N,N-diacetylbacillosamine (UDP-diNAcBac), within the active-site reveals co-incident binding sites, consistent with the proposed ping-pong enzymatic mechanism. Loading of PglC into membrane-bilayer nanodiscs (ND) allows for the investigation of PglC structure and function within a native-like membrane environment by small-angle x-ray scattering (SAXS). Observation of PglC in ND via SAXS confirms the application of the method for studying small, integral, monotopic membrane proteins in a membrane environment. Moreover, development of a mathematical approach by which resident-protein: ND stoichiometry can be deduced from measured scattering intensity enables independent confirmation of loading stoichiometry. Overall, the membrane-interaction modality observed for PglC is the first structurally characterized example of a new membrane association mode for monotopic proteins with the membrane. These studies provide insight into the structural determinants of the chemical mechanism and substrate-binding for C. concisus PglC and for the extensive homologous monotopic PGT superfamily, thus allow homology modeling and enabling future inhibitor design. / 2019-06-12T00:00:00Z

Cluster analysis of a large body of data on 180 Oregon specimens of Eutamias minimus and E. amoenus suggest that overall length of skull, basal length of skull and length of palate are taxonomically critical. If their sum is less than 71.3 millimeters the animal is E. minimus. If the sum is greater than 72.7 millimeters the animal is E. amoenus. If the sum is between 71.3 and72.7 millimeters, other factors must be considered before the animal can be identified. Of the 180 specimens, there were 60 E. minimus, 114 E. amoenus, 2 hybrids, 2 unidentifiable and 2 mismatched skulls and skins.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19001号 / 工博第4043号 / 新制||工||1622(附属図書館) / 31952 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 今堀 博, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Characterization of Rubisco plays a crucial role when it comes to the development and understanding of carbon sequestration in plants. This project took place at BMC in Uppsala, in the Gunn lab, and aimed to structure three Rubisco structures and analyze these with regard to the assembly pathway of the biogenesis of Rubisco but also how fast the reaction of binding of atmospheric carbon dioxide takes place with regard to different isoforms of the small subunit. The structural regulations led to the conclusion that an additional step in the assembly pathway would be added when one side of Rubisco had the chaperone BSD2 bound while the other side of Rubisco had the small subunit bound.The different subunits are believed to effect the structure of the LSu. The result also indicate that when the SSu are binding to the LSu octomer the interactions between the BSD2 and the LSu changes. This indicats that the SSu could indirectly facilitate the binding of the SSu on the other side by affecting the interactions of the LSu and the BSD2. Therefore the cooperative binding of the different subunits would be interesting to further evaluate. The NtL8B4(S-T1)4 , which is the first model for this structure to be determined, and therefore extended the assembly pathway for the biogenesis of higher plants, had the CABP bound, indicating that this intermediate structure could be analytically competent. This hypothesis is only based on the analyses of the structural determination, therefore further studies are needed to determine whether this is legitimate. Teknisk-naturvetenskapliga fakulteten, Uppsala universitet. Utgivni

Heart disease is leading cause of death in the United States. High-density lipoprotein (HDL) levels are inversely correlated with the prevalence of coronary heart disease. The anti-atherogenic properties of HDL are associated with its role in the pathway of the reverse cholesterol transport, which removes cholesterol from peripheral cells for transport back to hepatocytes. The formation of HDL is facilitated by ATP cassette transporter ABCA1 and apolipoprotein A-I, the major protein of HDL particles. However, the underlying molecular mechanism behind the biogenesis of HDL is not well understood. To provide further understanding of this mechanism, we developed two ABCA1 expression systems in both Sf9 insect cells and FreestyleTM 293-F human cells for functional and structural studies. We designed all constructs of ABCA1 to contain a C-terminal rho1d4 tag that bound to an affinity matrix of rho1d4 antibodies for successful purification. To reconstitute ABCA1 in a detergent-free environment that models the native membrane, we developed three reconstitution systems for ABCA1: saposin A nanodiscs, peptidisc, and amphipol A8-35. Biochemical and structural studies were carried out to understand the mechanism behind ABCA1’s function. We demonstrated a potential direct interaction of ABCA1 and apolipoprotein A-I with a pull-down experiment. Two cryo-electron microscopy data collections were obtained of ABCA1 in a detergent environment in the presence of ATP with the goal of determining the structure of ABCA1’s active state. We produced a 12 Å reconstruction of ABCA1 from this first data collection. This low-resolution structure confirmed the general structure that currently exists for ABCA1. Processing the data helped us streamline and troubleshoot the electron microscopy workflow pipeline for future data collections. Unfortunately, the second data collection had astigmatism issues that prevented particle alignment during data analysis. However, these data collections provided considerable insight into the ideal sample freezing and grid preparation conditions that affect data collection and data processing. More transmembrane protein structures are being solved each year but there remain many obstacles and challenges in ABCA1 purification and grid preparation that affect the ability to perform functional studies and high-resolution structure determination. Our developmental work has helped move forward our biochemical understanding of ABCA1 to achieve these aims. The more that is learned about this important membrane protein the more likely it is that future consistent production of ABCA1 will be accomplished to answer the questions of how ABCA1 mediates the formation of HDL particles.

No description available.

Protein complexes mediate a diverse range of behavior in prokaryotic cells, yet the exact molecular mechanisms explaining how many of these complexes assemble and function remain unknown. This work focuses on understanding the molecular mechanisms of two different protein assemblies responsible for regulating virulence in the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa utilizes type IV pili (T4P) to adhere to, and move along, surfaces. Assembly of T4P is powered by a dedicated cytoplasmic ATPase, PilB. The structural study of PilB from a related system (chapter 2) resulted in the formulation of the first model describing the mechanism of force generation resulting from ATP hydrolysis, which explains how T4P are assembled. Chapter 3 focuses on the RetS/GacS interaction, which is responsible for globally regulating virulence in P. aeruginosa. A comprehensive structural study reveals a dynamics of a novel regulatory interaction and the discovery of a potentially universal transmembrane signaling mechanism. / Doctor of Philosophy / Bacteria have threatened human health since the beginning of recorded history. With the development of antibiotics in the early twentieth century, the threat posed by bacterial infection was greatly lessened. However, decades of antibiotic mismanagement has led to the evolution of bacteria which are no longer vulnerable to these antibiotics. In order to combat this rising threat of resistant bacteria, we require a deeper understanding of how bacteria function and cause disease. Proteins play a crucial role in the diseases caused by bacteria, either by directly damaging host cells or regulating the expression of these damaging factors. By increasing our knowledge of the roles played by protein during bacterial infections, it will be possible to create new antibiotics while minimizing the risk of resistance. The work presented here grants a deeper understanding into how proteins work together to allow bacteria to survive inside the human body.

One of the main goals in structural biology is to understand the folding mechanisms and three-dimensional structure of biomolecules. Many biomolecular systems adopt multiple structures as a function of their microenvironment, which makes them difficult to be characterized by traditional structural biology tools (e.g., NMR, X-ray crystallography). As an alternative, complementary tools that can capture and sample multiple conformations needed to be developed. In the present work, we pioneered the application of a new variant of ion mobility spectrometry, trapped ion mobility spectrometry (TIMS), which provides high mobility resolving power and the possibility to study kinetically trapped intermediates as a function of the starting solution (e.g., pH and organic content) and gas-phase conditions (e.g., collisional activation, molecular dopants, hydrogen/deuterium back-exchange). When coupled to mass spectrometry (TIMS-MS), action spectroscopy (IRMPD), molecular dynamics and biochemical approaches (e.g., fluorescence lifetime spectroscopy), a comprehensive description of the biomolecules dynamics and tridimensional structural can be obtained. These new set of tools were applied for the first time to the study of Flavin Adenine Dinucleotide (FAD), Nicotineamide Adenine Dinucleotide (NAD), globular protein cytochrome c (cyt c), the 31 knot YibK protein, 52 knot ubiquitin C terminal hydrolase (UCH) protein, and the 61 knot halo acid dehydrogenase (DehI) protein.