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Comparative Analysis of the Ubiquitin-Proteasome System in Homo Sapiens and Saccharomyces Cerevisiae

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Comparative Analysis of the Ubiquitin-Proteasome System in Homo Sapiens and Saccharomyces Cerevisiae Comparative Analysis of the Ubiquitin-proteasome system in Homo sapiens and Saccharomyces cerevisiae Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Hartmut Scheel aus Rheinbach Köln, 2005 Berichterstatter: Prof. Dr. R. Jürgen Dohmen Prof. Dr. Thomas Langer Dr. Kay Hofmann Tag der mündlichen Prüfung: 18.07.2005 Zusammenfassung I Zusammenfassung Das Ubiquitin-Proteasom System (UPS) stellt den wichtigsten Abbauweg für intrazelluläre Proteine in eukaryotischen Zellen dar. Das abzubauende Protein wird zunächst über eine Enzym-Kaskade mit einer kovalent gebundenen Ubiquitinkette markiert. Anschließend wird das konjugierte Substrat vom Proteasom erkannt und proteolytisch gespalten. Ubiquitin besitzt eine Reihe von Homologen, die ebenfalls posttranslational an Proteine gekoppelt werden können, wie z.B. SUMO und NEDD8. Die hierbei verwendeten Aktivierungs- und Konjugations-Kaskaden sind vollständig analog zu der des Ubiquitin- Systems. Es ist charakteristisch für das UPS, daß sich die Vielzahl der daran beteiligten Proteine aus nur wenigen Proteinfamilien rekrutiert, die durch gemeinsame, funktionale Homologiedomänen gekennzeichnet sind. Einige dieser funktionalen Domänen sind auch in den Modifikations-Systemen der Ubiquitin-Homologen zu finden, jedoch verfügen diese Systeme zusätzlich über spezifische Domänentypen. Homologiedomänen lassen sich als mathematische Modelle in Form von Domänen- deskriptoren (Profile) beschreiben. Diese Deskriptoren können wiederum dazu verwendet werden, mit Hilfe geeigneter Verfahren eine gegebene Proteinsequenz auf das Vorliegen von entsprechenden Homologiedomänen zu untersuchen. Da die im UPS involvierten Homologie- domänen fast ausschließlich auf dieses System und seine Analoga beschränkt sind, können domänen-spezifische Profile zur Katalogisierung der UPS-relevanten Proteine einer Spezies verwendet werden. Auf dieser Basis können dann die entsprechenden UPS-Repertoires verschiedener Spezies miteinander verglichen werden. In dieser Arbeit wurden basierend auf UPS-relevanten Homologiedomänen und unter Verwendung der Profilmethode solche Kataloge für den Menschen und die Hefe Saccharomyces cerevisiae erstellt. In Kombination mit phylogenetischen Methoden wurden die evolutionären Beziehungen zwischen den UPS-Komponenten dieser beiden Organismen untersucht und in geeigneten Fällen eine Orthologiebeziehung abgeleitet. Durch die Verwendung der hoch-sensitiven Profiltechnik und die Einbeziehung von genomischen Datenbanken wurden im Rahmen dieser Arbeit eine Reihe von Proteinen identifiziert, die bisher nicht mit dem UPS assoziiert worden waren. Zusätzlich konnten einige unerwartete Verwandtschaftsbeziehungen zwischen Proteinen des UPS abgeleitet werden. So konnte z.B. das lange gesuchte Hefe-Ortholog des 'Antizyms' der Ornithin- Decarboxylase identifiziert werden - eine wichtige Voraussetzung zur experimentellen Untersuchung des Ubiquitin- Zusammenfassung II unabhängigen Proteinabbaus durch das Proteasom. In einem weiteren Beispiel konnte gezeigt werden, daß Ataxin-3 aus Mensch eine Homologiedomäne mit funktioneller Ähnlichkeit zu den deubiquitylierenden Enzymen besitzt. Da Ataxin-3 bei Patienten mit spinocerebellarer Ataxie 3 (SCA3) mutiert ist, kann diese Entdeckung zur Aufklärung des Krankheitsmechanismus von SCA3 beitragen. In einer dritten exemplarischen Anwendung konnten weitreichende Vorhersagen für den strukturellen Aufbau des 19S-Proteasoms getroffen werden, insbesondere mit Bezug auf dessen 'lid' Subkomplex. Ein Vergleich der UPS-relevanten Proteinrepertoires der Hefe und des Menschen erlaubte Schlüsse über den evolutionären Ursprung einiger Komponenten des UPS. Insbesondere bei Proteinfamilien mit einer etablierten oder angenommenen Rolle in der Substraterkennung und -ubiquitylierung oder im reversen Prozess der Deubiquitylierung findet man beim Menschen eine starke Diversifizierung der Proteinfamilien, während die elementaren Funktionen des UPS durch annähernd vergleichbare Proteinsets ausgeführt werden. Trotz der teilweise erheblich größeren Proteinfamilien im Menschen, konnten nicht allen UPS- assoziierten Proteinen der Hefe humane Orthologe zugeordnet werden, was auf spezifische Prozesse innerhalb des UPS von S. cerevisiae hindeutet. Ingesamt überwiegen jedoch die Ähnlichkeiten der beiden Systeme und unterstreichen die Rolle von S. cerevisiae als Modellorganismus zur Aufklärung des UPS. Abstract III Abstract The UPS (ubiquitin-proteasome system) is the most important degradation pathway for intracellular proteins in the eukaryotic cell. In a first step, the protein to degrade (substrate) is tagged covalently with a Ubiquitin chain via an enzyme cascade. Subsequently, the Ubiquitin chain is recognized by the proteasome and the substrate is proteolytically cleaved. Ubiquitin has several homologues, which can be conjugated to proteins posttranslationally, e.g. SUMO or NEDD8. The enzymes used for activation and conjugation of the Ubiquitin homologues are completely analogous to the ones used in the UPS. A hallmark of the UPS is that most proteins involved belong to only a few protein families, which are characterized by common functional homology domains. Several of these homology domains are found in the modification systems of Ubiquitin homologues, but these systems appear to have specific homology domains on their own as well. Homology domains may be described as mathematical models in terms of domain descriptors (profile). These profiles together with appropriate search algorithms can be applied to screen a given protein sequence for the occurrence of the corresponding homology domains. As the homology domains involved in the UPS are almost exclusively found in proteins of this and analogous systems, profiles corresponding to these homology domains seem to be an appropriate means to catalogue proteins of the UPS of a given species. In this work catalogues of proteins with a known or putative role in the UPS or analogous systems were set up for human and Saccharomyces cerevisiae (here referred to as ‘yeast’) based on relevant homology domains and their corresponding profiles. In combination with phylogenetic methods the evolutionary relationships between the UPS components of these two organisms were analyzed and, if possible, orthologous relationships were derived. Using the highly sensitive profile technique and including genomic databases, several new proteins were identified that have not been associated with the UPS so far. Additionally, several unexpected relationships were revealed between proteins of the UPS. For example, the postulated yeast orthologue of the antizyme of the ornithine decarboxylase could be revealed, which may be important for the experimental analysis of Ubiquitin independent protein degradation by the proteasome. Another example is human ataxin-3, in which a homology domain was found with similarity to the catalytic site of deubiquitylating enzymes. As ataxin-3 is mutated in patients with a spinocerebellar ataxia 3 (SCA3), this discovery might have implications for the elucidation of the SCA3 disease mechanism. Furthermore, predictions on the structure of the ‘lid’ of the 19S regulatory particle could be formulated. Abstract IV A comparison of the UPS-relevant protein repertoires of yeast and human allowed conclusions on the evolutionary origin of UPS components. Especially protein families with an established or putative role in substrate recognition/ubiquitylation or in the reverse process of deubiquitylation exhibited a strong diversification in human. Simultaneously, elementary functions of the UPS are carried out by almost identical protein sets in both yeast and human. Despite the extensively expanded protein families in human, not all yeast proteins associated with the UPS could be assigned to human orthologues. This finding might indicate specific processes within the yeast UPS. To summarize, the similarities of both yeast and human UPS are significant and underline the role of S. Cerevisiae as a model organism used in analyzing the UPS. Abbreviations V Abbreviations ATPases associated with a variety of AAA MPN Mpr1, Pad1 N-terminal cellular activities anaphase-promoting complex APC MSA multiple sequence alignment (cyclosome) neural precursor expressed, BAG Bcl2-associated athanogene domain NEDD developmentally downregulated baculoviral IAP repeat-containing BIRC6 OAZ ODC antizyme protein 6 BLOSUM blocks substitution matrix ODC ornithine decarboxylase bp base pair ORF open reading frame Bric-a-brac (bab), Tramtrack (ttk), and BTB PAM per cent accepted mutation Broad-Complex (BR-C) cDNA complementary DNA PAZ poly-Ub associated Zn-finger carboxy terminus of hsp70-interacting CHIP PCI proteasome, COP9, initiation factor 3 protein Clp caseinolytic protease PCNA proliferating cell nuclear antigen CP core particle (20S proteasome) PIAS protein inhibitor of activated STAT CSN COP9 signalosome POMP proteasomal maturation protein coupling of Ub conjugation to ER CUE RING really interesting new gene degradation regulatory particle (19S proteasome Cvt Cytoplasm-to-vacuole targeting RP subcomplex) DNA desoxyribonucleic acid RPN regulatory particle non-ATPase DUB deubiquitylating enzyme RPT regulatory particle triple A ATPase E1 Ub activating enzyme SC, sc Saccharomyces cerevisiae E2 Ub conjugating enzyme SCF Skp1, cullin, F-box (E3 complex) E3 Ub ligating enzyme SGD Saccharomyces Genome Database E4 Ub chain elongation factor SUMO small Ub-like modifier eukaryotic translation
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