京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20223号 / 医博第4182号 / 新制||医||1019(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 中川 一路, 教授 岩田 想, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The Grr1 (Glucose Repression Resistant) protein in Saccharomyces cerevisiae is an F-box protein for the E3 ubiquitin ligase protein complex known as the SCFGrr1 (Skp, Cullin, F-box). F-box proteins serve as substrate receptors for this complex and in this capacity Grr1 serves to promote the ubiquitylation and subsequent proteasomal degradation of a number of intracellular protein substrates. Substrates of SCFGrr1 include the G1-S phase cyclins, Cln1 and Cln2, the Cdc42 effectors and cell polarity proteins, Gic1 and Gic2, the FCH-bar domain protein, Hof1, required for cytokinesis, the meiosis activating serine/threonine protein kinase, Ime2, the transcriptional regulators of glucose transporters, Mth1 and Std1, and the mitochondrial retrograde response inhibitor Mks1. Stabilization of these substrates lead to pleiotrophic phenotypic defects in grr1Δ strains including resistance to glucose repression, accumulation of grr1Δ cells in G2 and M phase of the cell cycle, sensitivity to osmotic stress, and resistance to divalent cations. However, many of these phenotypes are not reflected at the gene expression level. We conducted a quantitative genomic vii and proteomic comparison of 914 loci in a grr1Δ and wild-type strain grown to early log-phase in glucose media. These loci encompassed 16.7% of the Saccharomyces proteome of which 22.3% exhibited discordance between gene and protein expression. GO process enrichment analysis revealed that discordant loci were enriched in the processes of “trafficking”, “mitosis”, and “carbon/energy” metabolism. Here we show that these instances of discordance are biologically relevant and in fact reflect phenotypes of grr1Δ strains not evident at the transcriptional level. Additionally, through combined biochemical and network analysis of discordant loci among “carbon and energy metabolism” we were able to not only construct a model for central carbon metabolism in grr1Δ strains but also were able to elucidate a novel molecular event that may serve to regulate glucose repression of genes needed for respiration in response to changes in glucose concentration.

Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23919号 / 工博第5006号 / 新制||工||1781(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 今堀 博, 教授 田中 庸裕, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Maturation of spinal motoneurons in rodents is characterized by a period of cell loss in the embryo, but researchers have claimed that some cell death occurs postnatally. This form of cell death is called apoptosis and involves active participation of the cell. Apoptotic cells have certain recognizable morphological and molecular features. I have used a monoclonal antibody against ubiquitin, (a putative marker of apoptotic cells), to do immunochemistry on mouse spinal cords at various postnatal ages till early adulthood. Staining is seen in large amotoneurons in the ventral horn. Staining is intense till P28, and faint thereafter. Substantial proportions of motoneurons stain till P21, followed by a sharp decline in the number of immunopositive cells. None of the cells exhibit signs of apoptosis.

Ubiquitinierungen sind dynamische, posttranslationale Proteinmarkierungen, die eine Vielzahl zellulärer Reaktionen hervorrufen. Die strukturell unterschiedlichen Signale werden von einer Ubiquitinierungsmaschinerie, bestehend aus E1-, E2- und E3-Enzymen, aufgebaut. Die Synthese von Polyubiquitin wird durch ubiquitinbindende Domänen (UBD) innerhalb der enzymatischen Kaskade stimuliert. Das E2-Enzym Ubc7 katalysiert zusammen mit dessen Kofaktor Cue1 die Polymerisierung von Ubiquitineinheiten und kennzeichnet Substratproteine mit Lysin 48 (K48)-ver¬knüpf¬ten Ubiquitinketten für den Endoplasmatische Retikulum-assoziierten Proteinabbau (ER-associated protein degradation, ERAD). In dieser Arbeit konnte mittels in vitro rekonstitu¬ierter Ubiquitinierungsreaktionen die Funktionsweise der ubiquitinbindenden CUE-Domäne von Cue1 während der Synthese von Polyubiquitin aufgeklärt werden. Verlängerungs¬reaktionen von Ubiquitinketten konnten durch Fluoreszenzmessungen verfolgt und die CUE-Domäne als Substratrezeptor von Ubc7 beschrieben werden. Anscheinend erhöht die Ubiquitin¬bindung durch Cue1 die lokale Konzentration von Ubc7 an den Ketten und positio¬niert das E2-Enzym effizient für die Übertragung der gebundenen Ubiquiti-neinheit. Die Reaktionen werden durch eine Bindungspräferenz der Cue1-CUE-Domäne für K48-ver¬knüpfte Ubiquitinmoleküle zusätzlich beschleunigt. Es ist bekannt, dass UBDs Ubiquitin¬signale entschlüsseln. Die Charakterisierung der CUE-Domäne beschreibt eine Notwendigkeit der Bindung von Ubiquitin bereits während der Entstehung von Polyubiquitin. Neben den E3-Ubiquitinligasen existieren Deubiquitinasen (DUB), die an der Reifung und dem Abbau von Ubiquitinsignalen beteiligt sind. Die proteasomalen DUBs Ubp6 und Rpn11 zeigen basale Aktivitäten in Isolation, die eingebunden in den 26S-Komplex moduliert werden. Fluoreszenz-basierte Untersuchungen von Kettenabbaureaktionen lassen erste Schlüsse über die Spezifitäten und die Abbaumechanismen der Enzyme zu. / Polyubiquitination is an essential process modulating protein function in eukaryotic cells. Only recently ubiquitin binding activity has emerged as an important factor in ubiquitin chain assembly. Cue1 is a crucial component of yeast endoplasmic reticulum associated protein degradation complexes which recruits and activates the E2 ubiquitin conjugating enzyme Ubc7. Our NMR solution structure reveals an unconventional CUE domain of Cue1 that substantially stimulates ubiquitin chain elongation by Ubc7.Results from NMR analysis combined with interaction studies and in vitro ubiquitination reactions imply that binding of CUE to a ubiquitin moiety adjacent to the acceptor ubiquitin is a prerequisite for rapid chain elongation. By this mode of action, the CUE domain counteracts the inability of associated Ubc7, to progressively elongate ubiquitin chains. Elongation of K48-linked ubiquitin chains is additionally accelerated since the CUE domain preferentially binds chains of K48-linkage. Our data support a model, where dynamic binding of ubiquitin chains assist to position Ubc7 for rapid elongation of K48-linked chains. Thus, the CUE domain acts as acceleration factor of elongation. Our study provides detailed mechanistic insight into how a ubiquitin binding domain governs polyubiquitin chain formation.

Ubiquitinierung ist eine essentielle posttranslationale Proteinmodifikation (PTM), die vielfältige Prozesse in eukaryotischen Zellen steuert. Ubiquitin wird zu unterschiedlichen polymeren Ketten zusammengesetzt, wobei E2-Ubiquitin-konjugierende Enzyme häufig eine entscheidende Rolle spielen. Im Rahmen meiner Promotion habe ich die molekularen Grundlagen der Ub Kettensynthese durch die E2-Enzyme Ubc1 und Ubc7 untersucht. Dies geschah mithilfe von in vitro Ubiquitinierungs-Reaktionen, biochemischen und strukturellen Untersuchungen sowie zellbiologischen Experimenten. Ich konnte zeigen, dass zugehörige Ubiquitin-Binde-Domänen (UBDs) die Funktion der E2-Enzyme maßgeblich regulieren. Als einziges unter elf E2-Enzymen in S. cerevisiae enthält Ubc1 eine Ub-bindende UBA Domäne, deren Funktion bisher unklar blieb. Ubc1 modifiziert ausschließlich Lysin 48 (K48) in Ub und wurde mit Proteinqualitätskontrolle sowie der Regulation des Zellzyklus in Verbindung gebracht. Meine Ergebnisse zeigen, dass Ubc1 mithilfe seiner UBA-Domäne vorzugsweise mit K63-verknüpftem Polyubiquitin interagiert, wodurch K48/K63 verzweigte Ub-Ketten entstehen. Basierend auf vorhandenen Strukturinformationen und meinen eigenen röntgenkristallographischen Untersuchungen zeige ich eine Modellstruktur für die Reaktion auf. Meine Ergebnisse stellen eine wesentliche Untersuchungsgrundlage für verzweigten Ub-Ketten dar, über deren Vorkommen und Funktion bisher wenig bekannt ist. Ubc7 assembliert mithilfe seines Kofaktors Cue1 K48-verknüpfte Ub-Ketten im Rahmen des Endoplasmatisches-Retikulum-assoziierten Proteinabbaus (ERAD). In einem kollaborativen Projekt haben wir die Ub-bindende CUE-Domäne in Cue1, die hierfür eine Schlüsselrolle spielt, untersucht. Sie ermöglicht die Ausrichtung des E2-Enzyms an der distalen Spitze der Ub-Kette für eine schnelle Kettenverlängerung, besitzt einzigartige auf den Prozess angepasste Bindungseigenschaften und ihre Beeinträchtigung stört den Abbau des ERAD-Substrates Ubc6. / Ubiquitination is an essential posttranslational protein modification (PTM) that regulates widespread intracellular processes in eukaryotic cells. Ubiquitin (Ub) can be assembled into polymeric chains through its seven internal lysine residues and the N-terminus enabling the formation of a complex "Ubiquitin Code". Factors that guide the molecular machinery which produces this code remain poorly understood. In this study, I demonstrate that ubiquitin binding domains (UBDs) associated with the E2 enzymes Ubc1 and Ubc7 substantially contribute to the assembly of particular Ub chains. Uniquely among the eleven E2 enzymes of S. cerevisiae Ubc1 contains a ubiquitin binding UBA domain. Ubc1 exclusively modifies lysine 48 (K48) in Ub and has been implicated in protein quality control and cell cycle progression. However, the function of its UBA domain remained elusive. I identified Ubc1 to preferentially target specific Ub molecules in K63-linked polyubiquitin via its UBA domain. This activity results in the assembly of K48/K63 branched Ub chains. Based on existing structural information and my own X-ray crystallographic experiments, I propose a structure for the transition state of branched chain assembly by Ubc1. My findings provide a basis for the study of this unusual Ub chain type. Ubc7 has previously been shown to be activated by its co-factor Cue1 to assemble Ub chains linked through lysine 48 (K48) in the context of endoplasmic reticulum associated protein degradation (ERAD). In collaboration with Dr. Maximilian von Delbrück and Dr. Andreas Kniss, we identified the ubiquitin binding CUE domain in Cue1 to play a key role in aligning Ubc7 with the distal tip of a K48-linked Ub chain for rapid chain elongation. Furthermore, we showed how binding of Ub by the CUE domain is well adapted towards the chain elongation process and how its disruption impairs degradation of the ERAD substrate Ubc6.

No description available.

In bacteria, mechanosensitive ion channels are essential for the cellular response to hypoosomitic shock, relieving the build up of membrane pressure. This thesis focuses on the Mechanosensitive Channel of Small Conductance (MscS) for which three conflicting gating models exist. These models were generated from structural studies of the closed and open conformations of MscS using three different experimental techniques. Pulsed Electron Double Resonance (PELDOR) spectroscopy was applied to MscS in the detergent solubilised state and the membrane-like bilayer. The distances between selectively introduced unpaired electrons in MscS were measured. PELDOR data in the detergent solubilised state were only consistent with the crystal structure depicting an open conformation of MscS, indicating that the transmembrane helices were unperturbed during crystallisation in detergent. MscS was reconstituted into membrane bilayer mimics, bicelles and nanodiscs, and PELDOR data in these environments suggested that both closed and open conformations determined by X-ray crystallography are stabilised in the membrane bilayer. The second part of this thesis involved stabilisation of an enzyme complex of the ubiquitin (Ub) pathway for structural analysis. This pathway is a eukaryotic signalling system involving post-translational modification of target protein amino groups with Ub. The variety of modification provided by Ub and its interplay with other Small Ubiquitin-like Modifier (SUMO) signalling proteins controls an array of cellular responses. The pathway functions to activate the Ub C-terminus, forming highly reactive thioester conjugates between Ub and the active site cysteine of a series of enzymes: E1, E2 and in some cases E3. This thesis explores the use of an isopeptide linkage to stabilise an E2~Ub conjugate in complex with an E3 enzyme. Sample conditions were optimised for the future use of PELDOR spectroscopy to structurally analyse the E2~Ub alone and in complex with E3.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy