Ubiquitin is an essential 76 amino acid protein which can be conjugated to lysine residues on a variety of substrates via its C-terminal diglycine motif. This conjugation allows the protein to act as a molecular tag in a range of processes, including regulation of chromatin compaction, signalling cascades and DNA repair. In addition, ubiquitin moieties are capable of forming chains through the successive conjugation to lysine residues within ubiquitin itself. One of the most well characterized functions of ubiquitin is its role in protein quality control and degradation. Tetra-ubiquitin chains, most commonly through a lysine-48 linkage, are responsible for directing proteins to the 26S proteasome for degradation. This process is of importance both in the removal of miss-folded proteins, and in the regulated destruction of specific targets, such as the cyclins. The 90kDa AAA-ATPase Cdc48/p97/VCP is an essential protein that forms a hexameric complex, which interacts with a wide variety of ubiquitinated substrates. The specificity of Cdc48 is modulated by a series of different cofactors, which together allow Cdc48 to operate in several different contexts, from removal of misfolded proteins from the ER, to regulating securin stability. The role of two Cdc48 cofactors, Ubx4 and Ubx5, was studied in an attempt to dissect their function and to determine how they may modulate the function of Cdc48. Neither protein was found to be essential, as knockouts of either were found to be viable with no major defect in growth rate. The work also describes the findings of a yeast two-hybrid screen to identify potential substrates for both cofactors. Delivery of ubiquitinated proteins to the proteasome is mediated by shuttling factors, which are able to bind to both ubiquitin and the proteasome, and hence mediate the interaction between both. The shuttling factor Dph1 binds ubiquitin via a C-terminal UBA domain, while its N-terminal UBL domain mediates its interaction with the proteasome. This work identified a novel interaction between the Sti1 domains of Dph1 and the N-terminal region of a mitochondrial localized AAA-ATPase, homologous to the Saccaromyces cerevisiae protein Msp1. In addition, cell fractionation experiments revealed the presence of Dph1 at the mitochondria. This interaction provides hints that Mlp1 may be involved in the removal of ubiquitinated proteins from the mitochondria, and their delivery to the proteasome. The thesis begins to try and attempt to identify possible substrates of this proposed mitochondria associated degradation pathway, and looks for ways in which the hypothesis may be tested.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23462号 / 医博第4769号 / 新制||医||1053(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 長船 健二, 教授 渡邊 直樹, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Eukaryotic gene expression to proteins is a complex process that begins with transcription which is regulated by numerous regulatory factors and signals. Alterations in these regulatory factors that modulate gene expression are linked with a multitude of cellular pathologies including cancers. Thus, it is important to delineate these transcriptional regulatory mechanisms of gene expression. Therefore, a large number of studies have been aimed at understanding the mechanism of transcription at the level of initiation, elongation, and termination. In line with this, my dissertation work is focused towards elucidating novel regulatory mechanisms of transcription initiation and elongation. Our results illuminate genetically how TOR (target of rapamycin) signaling pathway regulates transcription initiation and hence, transcription, in response to nutrients. The process of transcription initiation at the promoter is followed by RNA polymerase II (Pol II) pausing at the promoter-proximal site for mRNA capping/quality control. Such promoter-proximal pausing of Pol II (paused Pol II) plays an important role in regulating transcription elongation. Our results unveil how paused Pol II is released to engage into productive elongation for mRNA synthesis. We show that the capping enzyme, Cet1, targets a transcription factor known as FACT (facilitates chromatin transcription) which subsequently recruits a transcription elongation factor, Paf1C (RNA polymerase II- associated factor 1 [Paf1] complex), to release the paused Pol II for productive transcription elongation for mRNA synthesis. During such transcription elongation, histones need to be evicted in front of Pol II and reassembled in the wake of Pol II, and this dynamic histone disassembly and reassembly are coordinated by a number of histone chaperones. The aforementioned transcription factor, FACT, is one such histone chaperone that plays a key role in histone reassembly during transcription elongation. Importantly, we find a new regulation of FACT, by the ubiquitin-proteasome system (UPS), and hence, histone dynamics at the coding sequence and transcription. Specifically, the Spt16 component of FACT is ubiquitinated by the E3 ubiquitin ligase San1, and subsequently degraded by the 26S proteasome in yeast. Such proteasomal regulation of Spt16 subunit of FACT regulates transcription, and impairment of this UPS regulation alters transcription, leading to cellular pathologies. Indeed, SPT16 has been found to be associated with a lot of cancers, and our results show that this proteasomal degradation of SPT16 is impaired in cancer cells. Further, upregulation of SPT16 is associated with alterations in transcription of genes linked to cancer. Subsequent to its synthesis, mRNA needs to be exported to cytoplasm for translation to proteins. Importantly, transcription elongation has been found to be coupled to mRNA export, and like elongation, mRNA export is also controlled by UPS. Our findings demonstrate the role of active transcription in the proteasomal degradation of a key mRNA export factor, Sub2, mediated via Mdm30 (an F-box protein), thus, enhancing our understanding of the UPS regulation of mRNA export. Taken together, my dissertation work elucidates novel regulatory mechanisms of gene expression in response to nutrients and UPS, with implications in cellular pathologies including cancers.

No description available.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term effects include onset of insulin resistance and cardiovascular diseases. Increased oxidative stress and dysregulation of the ubiquitin-proteasome system (UPS) are implicated in protease-inhibitor (PI)-mediated cardio-metabolic pathophysiology. We hypothesized that PI treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the UPS, thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for eight weeks vs. vehicle and sham controls. Subsequently, we evaluated metabolic parameters and heart function (ex vivo and in vivo methods) at baseline and following ischemia-reperfusion. PI-treated rats exhibited weight gain, increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. It also upregulated hepatic gene expression of acetyl-CoA carboxylase β and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. Further, PI-treated hearts displayed impaired UPS, increased superoxide dismutase (SOD) activity and unaltered superoxide levels, and elevated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) peptide levels. Perfusion data revealed contractile dysfunction at baseline and following ischemia-reperfusion, while post-ischemic hearts exhibited decreased ATPase specific activity vs. matched controls. Early changes initiated by PI treatment resemble the metabolic syndrome and reflect a pre-atherogenic profile. Moreover, the effects of PIs on cardiac contractile function may in part be triggered by impaired UPS activity together with strain on the mitochondrial energetic system. Our study alerts to cardio-metabolic side effects of PI treatment and raises the question of the most appropriate co-therapies for patients on chronic antiretroviral treatment. / AFRIKAANSE OPSOMMING: Alhoewel anti-retrovirale behandeling MIV-VIGS morbiditeit/mortaliteit verlaag, bestaan daar langtermyn effekte soos die aanvang van insulienweerstandigheid en kardiovaskulêre siektes. Verhoogde oksidatiewe stres en wanregulering van die ubikwitien-proteosoomsisteem (UPS) word geïmpliseer met protease-inhibeerder (PI) gemediëerde kardio-metaboliese patofisiologie. Ons hipotetiseer dat PI behandeling (Lopinavir/Ritonavir) miokardiale oksidatiewe stres verhoog, en gevolglik die UPS inhibeer waardeur dit kardiale funksie verander. Lopinavir/Ritonavir is in 1% etanol (draer) opgelos en in ‘n mini-osmotiese pomp ingespuit wat chirurgies in Wistar rottes ingeplant is vir agt weke vs. draer en valskontroles. Gevolglik het ons die metabolise parameters en hartfunksie (ex vivo en in vivo metodes) op basislyn en na afloop van ischemie-reperfusie ondersoek. PI-behandelde rotte het ‘n toename in massa getoon asook verhoogde serum LDL-cholesterol, hoër weefseltrigliseriede (hart, lewer), maar geen bewys van insulienweerstandigheid nie. Dit het ook hepatiese asetielko-ensiem A karboksilase β en 3-hidrokise-3-metielglutariel KoA reduktase geenuidrukking opwaarts gereguleer, wat sleutel reguleerders van vetsuuroksidasie en cholesterolsintese onderskeidelik is. Verder, het PI-behandelde harte ingeperkte UPS, verhoogde SOD aktiwiteit en onveranderde superoksiedvlakke vertoon, asook verhoogde peroksisoomproliferator-geaktiveerde reseptor-γ ko-aktiveerder 1-α (PGC-1α) peptiedvlakke. Perfusie data toon kontraktiele wanfunskionering gedurende basislyn en na afloop van ischemie-reperfussie, terwyl post-ischemiese harte verlaagde ATPase spesifieke aktiwiteit vs gepaarde kontrole vertoon. Vroeë veranderinge wat deur PI behandeling veroorsaak word, kom ooreen met die metabolise sindroom en reflekteer op ‘n pre-aterogeniese profiel. Bowendien kan die effekte van PIs op kardiale kontraktiele funksie deels veroorsaak word deur die ingeperkte UPS aktiwiteit tesame met die las op die mitochondriale energie sisteem. Ons studie waarsku teen kardio-metaboliese newe effekte met PI behandeling en rig die vraag; wat die mees gepaste ko-behandeling vir pasiënte op chroniese anti-retrovirale behandeling is.

The use of small molecules to probe the function of proteins is referred to as chemical genetics. The Proteolysis Targeting Chimera (PROTAC) is a chemical genetic tool that contains the ligand for a target protein of interest and the recognition motif for an E3 ubiquitin ligase attached by a linker. The PROTAC is capable of binding to and recruiting specific target proteins to the intracellular degradation system, the ubiquitin proteasome system (UPS). While the approach has had success it has not been optimized to be used on a broader scale. Optimization efforts focused on elucidating the ideal linker length between the ligand and the E3-ligase recognition motif, the preferred location for attachment of the linker to the two moieties, and the possibility for a dimeric PROTAC comprised of two ligands. An estrogen receptor (ER)-targeting PROTAC was chosen as a model for optimization attempts as the ER is known to have pathological significance in breast cancer. Optimization of the PROTAC technology will not only provide a novel tool to probe ER biology, but may also offer a novel approach to breast cancer therapies. The ER targeting PROTAC constitute the 17β-estradiol (E2), as the ligand for ER and a pentapeptide derived from HIF-1α as the E3-ligase recognition motif, joined by a linker. Following the successful synthesis and evaluation of a number of PROTACs, it was revealed that an optimum ER-targeting monomeric PROTAC (KC-3) has a spacer of 16 atoms between the E2 and HIF-1α pentapeptide. The spacer is attached at the C-7α position on E2 and at the N-terminus of the HIF-1α pentapeptide. It was also established that the PROTAC is capable of targeting the ER for degradation in a proteasome and E3- ligase dependent manner, which translated to a decrease in the proliferation of MCF-7 cells with an IC50 similar to that of tamoxifen. KC-3, in comparison with E2, displayed lower agonistic activity on an ER-regulated downstream target, the progesterone receptor (PR). A dimeric PROTAC more effectively binds and degrades the ER in a proteasome dependent manner, suggesting that the dimeric ligand approach may be applied to the design of other PROTACs.

Aspergillus fumigatus ist die häufigste Ursache für Lungeninfektionen in immunsuppri-mierten Patienten. Virulenzfaktoren sind häufig an Kontrollmechanismen für Entwick-lung gekoppelt, welche im verwandten Modellorganismus Aspergillus nidulans entdeckt wurden. Diese Arbeit präsentiert die Charakterisierung des F-box Proteins Fbx15 in A. fumigatus, welches einen starken Einfluss auf die Entwicklung in A. nidulans hat. Die Deletion von fbx15 resultierte in starken Wachstumsdefekten unter vielen Stress induzie-renden Bedingungen, welche klassische Virulenz Faktoren beinhalten, wie erhöhte Tem-peratur, oxidativer Stress und Aminosäuremangel, während das Wachstum unter Stan-dardbedingungen nicht beeinflusst war. Oxidativer Stress induziert eine transiente Erhöhung der fbx15 Expression, welche nach 40 Minuten zu einer dreifach erhöhten Pro-teinmenge führte. Fbx15 ist ein stabiles F-box Protein mit einer Halbwertszeit von 90 Minuten. Generell funktionieren F-box Proteine als Substratadapter für SCF-E3-Ubiquitin-Ligasen. Fbx15 liegt unter normalen Bedingungen phosphoryliert vor und in-teragiert mit der Skp1/A Untereinheit des SCF-Komplexes, vorzugsweise in kleineren Subpopulationen im Zytoplasma. Phosphoryliertes Fbx15 wird bevorzugt in SCF-Komplexe eingebaut. Oxidativer Stress führt zu einer schnellen Dephosphorylierung von Fbx15. Fbx15 Varianten, welche nicht phosphoryliert werden können, interagieren mit Skp1/A primär im Kern. Fbx15 rekrutiert drei Untereinheiten des COP9-Signalosoms und Proteine welche in Transkription, Translation, Signalübertragung, Morphologie oder Stoffwechsel involviert sind. Fbx15 bindet die Ssn6/F Untereinheit des konservierten Ssn6/SsnF-Tup1/RcoA Co-Repressors und wird für dessen Kernlokalisation benötigt. Dephosphoryliertes Fbx15 interagiert mit Ssn6/F im Kern und eine Fbx15-Ssn6/F be-dingte Genrepression wird für die Reduzierung der Gliotoxin-Biosynthese benötigt. fbx15 Deletionsstämme sind nicht in der Lage immunsupprimierte Mäuse in einem Model für invasive Aspergillose zu infizieren, was eine essentielle Funktion von Fbx15 für die Viru-lenz bestätigt. Diese Arbeit zeigt, dass Fbx15 nicht nur Teil von SCF-E3-Ubiquitin-Ligasen sein kann, sondern eine zweite neue molekulare Funktion aufweist, welche die physische Interaktion mit der Co-Repressor Untereinheit Ssn6/F und dessen Lokalisa-tionskontrolle beinhaltet. Diese duale Funktion resultiert in einer essentiellen Funktion von Fbx15 für die Kontrolle der oxidativen Stressantwort, des Sekundärmetabolismus und der Virulenz im opportunistischen Humanpathogen A. fumigatus.