München, Techn. Universiẗat, Diss., 2004.

Bochum, Universiẗat, Diss., 2001.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

No description available.

Ubiquitin is a multifunctional 76 amino acid protein which plays critical roles in many aspects of cellular metabolism. Ubiquitin protein structure and gene structure are highly conserved among eukaryotes. In C. elegans the major source of ubiquitin RNA was shown to be the polyubiquitin locus, UbiA. UbiA was shown to be transcribed as a polycistronic mRNA which contained eleven tandem repeats of ubiquitin sequence and possessed a two amino acid carboxy terminal extension on the final repeat. Mature UbiA mRNA was demonstrated to acquire a 22 nucleotide leader sequence via a trans splicing reaction involving a 100 nucleotide splice leader RNA derived from a different chromosome. UbiA was also shown to be unique among known polyubiquitin genes in containing four cis spliced introns within its coding sequence. Thus UbiA was shown to be one of a small class of genes found in higher eukaryotes whose hnRNA undergoes both cis and trans splicing. The expression of the UbiA gene was studied under various heat shock conditions, and was monitored during larval molting and throughout the major stages of development. These studies indicated that the expression of the UbiA gene was not inducible by acute or chronic heat shock, and did not appear to be under nutritional or developmental regulation. A second ubiquitin gene, UbiB, was cloned from C. elegans and a related nematode species C. briggsae. This gene was comprised of (at least) one ubiquitin unit followed by a basic 52 amino acid tail sequence. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Magister Scientiae - MSc / The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (“ubiquitinated”) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called “ubiquitin-like modifiers”, which consist of a single domain that structurally resembles ubiquitin, and “ubiquitin-domain” proteins, which are multi-domain proteins, which include domains that resemble ubiquitin. This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained. Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb. / South Africa

Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in concert with E2 enzymes to attach molecules of ubiquitin to protein substrates. There are seven CRLs in mammalian cells (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) that are highly homologous in sequence and structure. CRLs possess a highly conserved C- terminal domain that interacts with E2 enzymes, and a more variable N- terminal domain which recruits substrates through distinct substrate adapter molecules. Despite the structural similarity, these CRLs recognize distinct substrates and carry out unique functions in cells. In order to characterize the functional domains of cullins that are responsible for their unique activity, we generated cullin chimeras for expression and analysis in mammalian cells. These chimeras are Cul3 mutants in which the C- terminal domain or N- terminal domain of Cul3 has been replaced by that of Cul1 or Cul2, respectively. These chimeras were cloned into a mammalian expression vector for the purpose of experimentation in cultured cells. The chimeric cullin constructs provided a valuable tool for investigating how different functional domains of CRLs contribute to their specific functions in cells. In this study, we first investigated if the chimeras that we engineered were able to interact with their respective substrate adapters. We performed co- immunoprecipitation experiments in which we tested the ability of wild type, chimeric, or mutant cullin proteins to bind to three different substrate adapter proteins. We found that the chimera possessing the C- terminus of Cul1 and the N- terminus of Cul3 retains the ability to interact with the BTB substrate adapters Ctb57 and KLHL3. We also found that the chimera that possesses the C- terminus of Cul3 and the N- terminus of Cul1 was unable to interact with BTB proteins. Lastly, we found that the Cul1 adapter Skp1 was able to bind to Cul1, but did not bind to Cul3 or either chimera. We concluded that the chimera possessing the N- terminus of Cul3 likely retains the functional binding abilities of Cul3 at the N- terminus and would therefore be useful for conducting experiments. In this study, we also used the cullin chimeras to investigate the binding interactions between E2 enzymes and cullin RING ligases. We performed co- immunoprecipitation assays to examine the interactions between E2 enzymes and wild type, mutant or chimeric cullin proteins. We found that E2 enzyme UbE2E1 selectively binds to Cul3 and not to Cull. Notably, the BTB binding region at the N- terminus of Cul3 is required for binding to UbE2E1. Furthermore, we found that UbE2E1 also binds to Cul3 substrate adapter protein Ctb57. These experiments revealed a novel interaction between and E2 enzyme and the N- terminus of Cul3, as well as with a Cul3 substrate adapter protein. In conclusion, the chimeras generated in this study have provided valuable information regarding what regions of CRLs are important for interactions with other proteins, and will continue to be a useful tool for investigating CRL structure and function.

Protein quality control systems are critical for cellular proteostasis and survival under stress conditions. The ubiquitin proteasome system (UPS) plays a pivotal role in proteostasis by eliminating misfolded and damaged proteins. However, exposure to the environmental toxin arsenite results in the accumulation of polyubiquitylated proteins, indicating an overload of the UPS. Arsenite stress induces the rapid formation of stress granules (SGs), which are cytoplasmic assemblies of mRNPs stalled in translation initiation. The mammalian proteins ZFAND2A/B (also known as AIRAP and AIRAPL, respectively) bind to the 26S proteasome, and ZFAND2A has been shown to adapt proteasome activity to arsenite stress. They belong to a small subfamily of AN1 type zinc finger containing proteins that also comprises the unexplored mammalian member ZFAND1 and its yeast homolog Cuz1. In this thesis, the cellular function of Cuz1 and ZFAND1 was investigated. Cuz1/ZFAND1 was found to interact with the ubiquitin-selective, chaperone-like ATPase Cdc48/p97 and with the 26S proteasome. The interaction between Cuz1/ZFAND1 and Cdc48/p97 requires a predicted ubiquitin-like domain of Cuz1/ZFAND1. In vivo, this interaction was strongly dependent on acute arsenite stress, suggesting that it is a part of the cellular arsenite stress response. Lack of Cuz1/ZFAND1 caused a defect in the clearance of arsenite induced SG clearance. ZFAND1 recruits both, the 26S proteasome and p97, to arsenite-induced SGs for their normal clearance. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products and become aberrant. These aberrant SGs persist after arsenite removal and undergo degradation via autophagy. ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder, inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS). / Systeme zur Sicherung der Proteinqualität sind von essentieller Bedeutung für die zelluläre Proteostase und das Überleben unter Stressbedingungen. Dabei spielt das Ubiquitin-Proteasom-System (UPS) eine entscheidende Rolle: Es beseitigt fehlgefaltete und beschädigte Proteine. Sind Zellen dem Umweltgift Arsenit ausgesetzt, kommt es zu einer Akkumulation von polyubiquitinierten Proteinen, was auf eine Überlastung des UPS hinweist. Dieser durch Arsenit verursachte Stress bewirkt eine schnelle Bildung von Stressgranula (SGs), einer cytoplasmatischen Ansammlung von mRNPs, die in der Initiation der Translation blockiert sind. Die Säuger Proteine ZFAND2A/B (auch bekannt als AIRAP und AIRAPL) binden an das 26S Proteasom. Zusätzlich wurde gezeigt, dass ZFAND2A die Aktivität des Proteasoms an durch Arsenit verursachten Stress, anpasst. Diese Proteine gehören zu einer kleinen Unterfamilie von Zinkfinger von AN1-type enthaltenden Proteinen, zu der auch das bislang nicht erforschte Säuge Protein ZFAND1 und sein Hefehomolog Cuz1 gehören. In dieser Arbeit wurde die zelluläre Funktion von Cuz1 und ZFAND1 untersucht. Es zeigte sich, dass Cuz1/ZFAND1 mit dem 26S Proteasom und der Ubiquitin-selektiven, Chaperon-ähnlichen ATPase Cdc48/p97 interagiert. Die Interaktion zwischen Cuz1/ZFAND1 und Cdc48/p97 benötigt eine vorhergesagte Ubiquitin-ähnliche Domäne von Cuz1/ZFAND1. Diese Interaktion ist in vivo stark von akutem Arsenitstress abhängig, was darauf hindeutet, dass sie Teil der zellulären Stressantwort gegen Arsenit ist. Fehlt Cuz1/ZFAND1, so kommt es zu einer Störung bei der Beseitigung der von Arsenit verursachten SGs. Normalerweise rekrutiert ZFAND1 sowohl das 26S Proteasom als auch p97 zu diesen SGs, um sie zu entfernen. Wenn ZFAND1 jedoch fehlt, sind auch p97 und das 26S Proteasom nicht an den SGs lokalisiert. Dadurch sammeln sich dort defekte ribosomale Produkte an, und die SGs werden abnormal. Auch nach Entfernung des Arsenitstresses bestehen diese abnormalen SGs fort und werden schließlich über Autophagie abgebaut. Bei der Beseitigung der SGs ist das Fehlen von ZFAND1 epistatisch zu der Expression einer pathogenen p97-Mutante, was darauf hinweirt, dass ZFAND1 bei der degenerativen Multisystemerkrankung Einschlusskörper-Myopathie assoziiert mit Pagets Erkrankung der Knochen und frontotemporaler Demenz und Amyotrophe Lateralsklerose (IBMPFD/ALS) eine Rolle spielt.

Der WNT-Signalweg ist ein hochkonservierter Signalweg, dessen zentraler intrazellulärer Regulationsschritt die Proteinstabilität des Proteins β-Catenin ist. Deregulierende Mutationen in diesem sind frühe Ereignisse bei der Entstehung von Darmtumoren. Ist der Abbau von β-Catenin gestört, so ist unabhängig von äußerer Kontrolle der Signalweg konstitutiv aktiviert und liefert ein Wachstumssignal. Untersuchungen haben aber gezeigt, dass beim Vorliegen solcher Mutationen immer noch eine – unzureichende – Ubiquitinylierung und ein Abbau von β-Catenin stattfindet. Ziel dieser Studie war Deubiquitinasen (DUBs) zu finden, die durch ihre Aktivität den Abbau von β-Catenin verhindern. Mithilfe eines siRNA Screens in der Vorarbeit konnten DUBs als Kandidaten für einen CRISPR Ansatz ausgewählt werden. APC Wildtyp HEK293T Zellen und Darmkrebszellen wurden mit lentiviralen CRISPR/Cas9 Vektoren infiziert, in welche sgRNAs gegen exonische Sequenzen von DUBs geklont waren. Einzelne Zellklone von USP10 CRISPR Zellen wurden weiter untersucht. In Western Blots und Immunofluoreszenz zeigte sich bei den USP10 CRISPR Zellen eine verminderte Expression von USP10 und damit einhergehend β-Catenin. Proteinstabilitätsversuche mit MG132 und Cycloheximid zeigten einen erhöhten Abbau von β-Catenin in HEK293T USP10 CRISPR Zellen, vor allem nach Stimulierung des WNT-Signalwegs durch LiCl. In Aktivierungsassays (Luciferase und TOP-GFP FACS) des WNT-Signalwegs zeigte sich in HEK293T Zellen nach Behandlung mit LiCl eine geringere Aktivierung in den USP10 CRISPR Zellen. In einem Wachstumsassay zeigten HT29 USP10 CRISPR ein geringeres Wachstum als Kontrollzellen. Während in einer histologischen Färbung von Mausgewebe eine erhöhte Expression von USP10 nachweisbar war, zeigten sich in einer TMA Färbung kein eindeutiger Unterschied zwischen gesundem Gewebe und Tumorgewebe. Die Studie identifiziert USP10 als eine mögliche DUB für β-Catenin und potenzielles Ziel für eine Beeinflussung des mutierten WNT-Signalwegs in Darmkrebszellen. / The WNT- signaling pathway is a highly preserved pathway. Its central intracellular regulation measure is the protein stability of the protein β-Catenin. Mutations that cause a deregulation in said pathway occur early within the development of intestinal tumors. If the breakdown of β-Catenin is impaired, the WNT- signaling pathway will get activated constitutively, providing growth signals, regardless of outer controls. Investigations have shown, that if such mutations exist there will still be a (insufficient) ubiquitinylation and a breakdown of β-Catenin. It was the aim of this research to identify deubiquitinases (DUBs) whose activity prevent the breakdown of β-Catenin. It was possible to select DUBs as potential candidates for a CRISPR approach by using sIRNA Screens. APC Wildtype HEK293T cells and intestinal tumor cells where infected by lentiviral CRISPR/Cas9 vectors which contained sgRNAs clones that worked against exonic sequences of DUBs. Individual cell clones of USP10 CRISPR cells where further investigated. Within western blots and immunofluorescence USP10 CRISPR cells showed a reduced expression of USP10 and thus, β- Catenin. Protein stability trials using MG132 and cycloheximide showed an increased breakdown of β-Catenin in HEK293T USP10 CRISPR cells, especially in response to stimulation of the WNT- signaling pathway using LiCl. Within activation assays (Luciferase and TOP-GFP FACS) of the WNT- signaling pathway, a reduced activation in USP10 CRISPR cells after treatment with LiCl was shown within HEK293T cells. Within a growth assay HT29 USP10 CRISPR showed a reduced growth when compared to control cells. While there was evidence of an increased expression of USP10 within the histological coloration of mouse tissue, the TMA coloration did not show a significant difference between healthy tissue and tumor tissue. This study identified USP10 as a possible DUB for β- Catenin and as a possible target for the manipulation of the mutations that cause a deregulation within the WNT- signaling pathway in intestinal tumor cells.

The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment. De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis. SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis. SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation. It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth. A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation. In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients. / Die Reprogrammierung metabolischer Stoffwechselwege ist ein Kennzeichen von Krebs: Tumorzellen sind abhängig von der Versorgung mit Metaboliten und Bausteinen, um ihren wachsenden Bedarf als hoch proliferierende Zellen zu decken. Vor allem die de novo Stoffwechselsynthesewege sich hochreguliert, wenn die Zellen des wachsenden Tumors nicht mehr in der Lage sind, ihr erforderliches metabolisches Niveau mithilfe der Aufnahme aus der Umgebung zu erfüllen. De novo Synthesewege sind oft unter der Kontrolle von zentralen Transkriptionsfaktoren die die Genexpression von Enzymen, die im Syntheseprozess beteiligt sind, regulieren. Die vorherrschenden Regulatoren, für die de novo Fettsäuresynthese und der Cholesterogenese sind die Steroid-regulatorisches-Element-bindende Proteine (SREBPs). Während SREBP1 bevorzugt die Expression von Enzymen die an der Fettsäuresynthese beteiligt sind kontrolliert, reguliert SREBP2 die Transkription von Enzymen des Mevalonat Stoffwechselwegs, sowie Prozesse unterhalb, namentlich die Cholesterol-, Isoprenoid- und die die Synthese von Bausteinen für die Ubiquinonsynthese. Die Aktivität von SREBP ist streng reguliert auf verschiedenen Ebenen: Die post-translationale Modifikation mittels Ubiquitinierung reduziert die Stabilität von aktiven SREBPs. Das Anhängen von K48-verlinkten Ubiquitinketten markiert die Transkriptionsfaktoren für den proteasomalen Abbau. In Tumorzellen sind hohe Niveaus von aktiven SREBPs essentiell für die Induktion der entsprechenden metabolischen Stoffwechselwege. Die erhöhte Stabilität und Aktivität von SREBPs wurden im Rahmen dieser Arbeit untersucht. SREBPs werden von der E3-Ligase Fbw7 ubiquitiniert, was zur Proteolyse der Transkriptionsfaktoren führt. In dieser Arbeit wurde gezeigt, dass das entgegenwirkende Deubiquitinierungsenzym USP28 die Ubiquitinketten von SREBPs entfernt und deren proteasomalen Abbau verhindert. Diese Forschungsarbeit zeigt weiterhin, dass die Stabilisierung von SREBP2 durch USP28 eine wichtige Rolle im Kontext von Epithelkarzinomen spielt. Erhöhte USP28 Niveaus werden mit einem schlechten Überleben von Patienten in der Krebs-Untergruppe der Plattenepithelkarzinomen verbunden. Es konnte gezeigt werden, dass reduzierte USP28 Niveaus, in Zelllinien und in vivo, niedrigere SREBP2-Aktivität und eine Herunterregulierung des Mevalonat Stoffwechselwegs ergeben. Diese Manipulation führte zu reduzierter Proliferation und Tumorwachstum. Ein direkter Vergleich von Adenokarzinomen und Plattenepithelkarzinomen in Lungenkrebspatienten zeigte zudem eine Hochregulierung von USP28 ebenso wie SREBP2 und dessen Zielgenen. Der gezielte Einsatz von Inhibitoren gegen die USP28-SREBP2 regulatorische Achse in Plattenepithelzellen reduzierte die Lebensfähigkeit und Proliferation der Zellen. Abschließend berichtet diese Forschungsarbeit von der Bedeutung des durch die USP28-SREBP2 Achse regulierten Mevalonat Stoffwechselwegs bei der Tumorinitiation und dem Fortschreiten von Plattenepithelkarzinomen. Die kombinatorische Behandlung mit USP28- und Inhibitoren der HMGCR, dem Schlüsselenzym des Mevalonat Stoffwechselwegs, mithilfe von Statinen eröffnet die Möglichkeit für eine gezielte therapeutische Behandlung von Patienten mit Plattenepithelkarzinomen.