The Role of the GATA Transcription Factor Gaf1 in Nutrient Responses and Cellular Ageing

The discovery of the biological bases of ageing continues to be one of the most fascinating challenges in modern science. Current efforts have narrowed the complexity of such task by focusing on mechanisms used by the cell to couple its physiology with environmental stimuli as they are often involved in the regulation of ageing. The Target of Rapamycin (TOR) have been proved to be a rheostat of nutritional status orchestrating cellular growth and homeostasis mainly through the regulation of transcriptional responses that remain to be understood. Recent studies unveiled novel functions of the evolutionarily conserved GATA transcription factor Gaf1 in nutrient sensing pathways and potentially in cellular ageing by regulating transcription downstream of TOR signalling. To elucidate these questions, the robust model organism Schizosaccharomyces pombe was used in this study due to its relevant similarity with higher eukaryotes and thoroughly described genetics. The experimental settings involved a combination of in silico analyses, fitness assessments, revivability assays, transcriptomics, mutagenesis, chemical-genetics, and interactome to further characterise functions of Gaf1. This study also contributed to the identification of candidate genes that promote longevity and mediate the resistance of mutant cells depleted of gaf1 gene to the TOR-kinase inhibitor torin1. The results indicate that upon TOR complex 1 (TORC1) inhibition, Gaf1 represses genes that induce protein translation (anabolism) and upregulates genes required for survival (catabolism) under adverse nutritional conditions downstream of TORC1

CHARACTERIZATION OF MITOTIC REGULATORS ACM1 AND CDC14

Mitotic exit and cytokinesis are driven by a complex set of processes that serve t

Regulation of polyamine biosynthesis in Saccharomyces cerevisiae

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme. The synthesis of mammalian antizyme was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. In this work, a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian antizyme was identified. Synthesis of yeast antizyme (Oaz1) involves polyamine-regulated frameshifting. New elements, termed OFRE (OAZ1 frameshifting repressor element) and OPRE (OAZ1 polyamine responsive element) that are necessary for the polyamines to regulate frameshifting were mapped in the OAZ1 mRNA. Degradation of yeast ODC by the proteasome depends on Oaz1. Oaz1 mediates the degradation by binding to ODC thereby exposing a degradation signal at the N-terminus of ODC. Using the novel transplantable yeast ODC degradation signal (ODS) identified in this work a new possible role of the shuttle factor Rad23 in ODC degradation was identified. In addition, ODS is shown to interact with multiple 19S lid subunits in the proteasome. Using this novel model system for polyamine regulation another level of its control was discovered. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is efficiently inhibited by polyamines. I propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation. In a second part of the work, peptide aptamers were isolated that inhibit the ubiquitin-dependent turnover of test substrates of the proteasome. These aptamers appear to either inhibit ubiquitylation or the proteasome and thereby lead to a stabilization of test substrates. I Propose that ODS due to its ubiquitin-independent mode of degradation can be used as a tool in aptamer screens that are aimed at identifying additional peptide inhibitors of the proteasome with potential clinical relevance

The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

This review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. Antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. The bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. The inhibition of ornithine decarboxylase by antizyme can be relieved to different degrees by DNA or by a variety of synthetic nucleic acid polymers, attributed to a specific interaction between nucleic acid and antizyme. Recently, this interplay between bacterial antizyme and nucleic acid was determined by discerning an additional function to antizyme that proved to be the atoC gene product, encoding the response regulator of the bacterial two-component system AtoS-AtoC. The gene located just upstream of atoC encodes the sensor kinase, named AtoS, that modulates AtoC activity. AtoC regulates expression of atoDAEB operon which is involved in short-chain fatty acid metabolism. Antizyme is thus referred to as AtoC, functioning both as a post-translational and transcriptional regulator. Also, the AtoS-AtoC signal transduction system in E. coli has a positive regulatory role on poly-(R)-3-hydroxybutyrate biosynthesis. The properties and gene structural similarities of antizymes from different organisms were compared. It was revealed that conserved domains are present mostly in the C-domain of all antizymes. BLAST analysis of the E. coli antizyme protein (AtoC) showed similarities around 69–58% among proteobacteria, g-proteobacteria, enterobacteria and the thermophilic bacterium Thermus thermophilus. A working hypothesis is proposed for the metabolic role of antizyme (AtoC) describing the significant biological implications of this protein molecule. Whether antizymes exist to other enzymes in different tissues, meeting the criteria discussed in the text remains to be elucidated

Funktionelle Charakterisierung des Komplexes aus Ornithin-Decarboxylase, Antizym und Antizym-Inhibitor sowie Identifikation einer Antizym-Inhibitor-Spleißvariante durch Bindung an FK506

Das von (Licitra and Liu, 1996) entwickelte Hefe-3-Hybrid-System ermöglicht die in vivo Identifikation von Ligand-Rezeptor-Interaktionen. Es ist eine Weiterentwicklung des klassischen Hefe-2-Hybrid-Systems und beinhaltet den Einsatz eines synthetischen Hybridmoleküls. Der feste Bestandteil dieses Hybridmoleküls ist das Hormon Dexamethason, das über die Hormon-bindende Domäne des Glukokorticoidrezeptors in der Hefezelle verankert wird und kovalent mit einem Molekül verbunden ist, für das der Interaktionspartner gesucht wird. Ziel dieser Arbeit war es, das Hefe-3-Hybrid-System im Labor zu etablieren und das System zur Identifikation neuer Interaktionspartner für das Immunsuppressivum FK506 einzusetzen. Um das Hefe-3-Hybrid-System sensitiver zu gestalten, wurde zunächst der ABC-Transporter PDR5 deletiert, der an dem Export von Steroidhormonen beteiligt ist. Dabei wurde gezeigt, daß der Pdr5-Transporter auch chemisch modifizierte Steroidhormone wie beispielsweise Dexamethason-Linker transportiert. Darüberhinaus wurden zwei Aminosäurepositionen innerhalb der Hormon-bindenden Domäne des Glukokorticoidrezeptors identifiziert, die entscheidend zur die Aktivierbarkeit des Rezeptors beitragen. Im Rahmen des durchgeführten 3-Hybrid-Screens wurde zusätzlich zu dem bekannten FK506- Bindeprotein, FKBP12, eine unbekannte, C-terminal verkürzte Spleißvariante von Antizym- Inhibitor als neuer Interaktor für FK506 identifiziert. Die Interaktion war FK506-spezifisch, da keine Interaktion mit anderen an Dexamethason gekoppelten Liganden nachzuweisen war. Die im 3-Hybrid-Screen isolierte Spleißvariante wurde zusätzlich über RT-PCR amplifiziert, wobei noch eine weitere Spleißform von Antizym-Inhibitor identifiziert werden konnte. Es wurde gezeigt, daß beide Formen ubiquitär exprimiert werden und Homologe in der Maus existieren. Antizym-Inhibitor ist an der Regulation der Polyaminbiosynthese beteiligt. Polyamine spielen für das Zellwachstum, die Zelldifferenzierung und die Proteinbiosynthese eine essentielle Rolle. Die Aktivität von Antizym-Inhibitor wird anhand seiner Fähigkeit bestimmt, Ornithin- Decarboxylase (ODC, EC 4.1.1.17) aus dem inhibitorischen Komplex mit Antizym freizusetzen. Die freigesetzte ODC-Aktivität gibt somit Auskunft über die Antizym-Inhibitor- Aktivität. Bei den Antizymen handelt es sich um eine Proteinfamilie, die aus vier Mitgliedern besteht, deren Interaktion mit Antizym-Inhibitor bislang nur für Antizym 1 beschrieben ist. Für die Charakterisierung der Antizym-Inhibitor-Spleißvarianten, wurde ein nichtradioaktiver Aktivitätsassay entwickelt, der auf der funktionellen Expression von humaner ODC, Antizym und Antizym-Inhibitor in der Hefe Saccharomyces cerevisiae beruht. Dabei wurde gezeigt, daß humane ODC die Deletion der Hefe-ODC (∆spe1) komplementiert und so das Hefezellwachstum auf Polyamin-freiem Medium ermöglicht. Dieser Assay erwies sich auch als geeignet für ein Hochdurchsatzverfahren (HTS) zur Identifikation von ODCInhibitoren. Die Koexpression von Antizym führte zu einer Wachstumshemmung, die auf einem Polyaminmangel beruht und durch Zugabe von Putrescin, oder durch die zusätzliche Koexpression von Antizym-Inhibitor wieder aufgehoben werden konnte. Darüber hinaus wurde ein 3-Hybrid-Assay für den Proteinkomplex aus ODC, Antizym und Antizym-Inhibitor entwickelt. Hiermit wurde untersucht, inwieweit Antizym-Inhibitor die Heterodimerisierung zwischen ODC und Antizym unterbindet. Aufgrund dieser Ergebnisse konnte erstmals gezeigt werden, daß Antizym-Inhibitor in der Lage ist, an alle Proteine der Antizym-Familie zu binden und ODC aus dem Komplex mit Antizym 1, 2, 3 und 4 verdrängt. Bislang war dies nur für Antizym 1 beschrieben. Desweiteren wurde für das noch nicht charakterisierte Mitglied der Antizym-Familie, Antizym 4, gezeigt, daß auch dieses an ODC bindet und die Aktivität der ODC hemmt. Die C-terminal verkürzte Spleißvariante konnte zwar an die Antizyme 1, 2 und 3 binden, war jedoch nicht der Lage, ODC aus dem Komplex freizusetzen. Um die Antizym-Bindungsdomäne von Antizym-Inhibitor weiter zu charakterisieren, wurden Fragmente hergestellt mit deren Hilfe die Antizym-Bindungsdomäne auf einen Bereich von Leucin45 bis Serin300 eingegrenzt werden konnte. Die Antizym-Bindungsdomäne überlappt dabei mit der FK506-Bindungsdomäne, die vollständige Form von Antizym-Inhibitor bindet interessanterweise nicht an FK506. Zusammengefaßt zeigt dies, daß der C-Terminus von Antizym-Inhibitor von großer Bedeutung für die Konformation des Proteins ist und einen wichtigen Beitrag zur Stabilisierung der Antizym / Antizym-Inhibitor-Interaktion leistet. Die Relevanz der in der Hefe gewonnenen Daten wurde abschließend mit HEK 293-Zellextrakten überprüft. Die Komplexbildung der transient exprimierten Proteine ODC, Antizym und Antizym-Inhibitor konnte durch Immunpräzipitation nachgewiesen werden. Damit wurde gezeigt, daß die Hefe-Daten auf höhere Organismen übertragen werden können

Transcriptional analysis of the histone acetylation and deacetylation in Candida albicans

[EN] The kingdom of fungi includes many important species from different points of view as are the ecological, economic or doctor. The fungal species by decomposing organic matter contributes to continued nutrient cycling within ecosystems, most vascular plants could not grow without the symbiotic fungi associated with their roots. Some fungi provide food directly to the man and others are used to impart certain characteristics to products consumed by humans, such as bread. Of particular medical relevance the ability of some species of this kingdom to produce and secrete antibiotics to the environment

Doctor of Philosophy

dissertationThe nonsense mediated mRNA decay (NMD) pathway is a conserved posttranscriptional mRNA decay pathway that functions to destabilize a variety of naturally occurring target mRNAs. The NMD pathway functions in all eukaryotes and regulates a significant portion of the transcriptome. It is thought that this regulation is critical as inhibition of NMD leads to physiological and developmental defects in all organisms and in the case of more complex organisms, lethality. It is predicted that overexpression of NMD pathway target genes leads to these defects in NMD mutants. Despite the critical nature of this pathway, little is know about how NMD functions in a developmental and physiological context, including which target genes are most critically regulated by NMD and how the overexpression of these targets may mediate the NMD mutant phenotype. To address this knowledge gap, we first use two genome-wide techniques to identify and characterize the kinds of transcripts targeted by NMD in the context of an intact metazoan, Drosophila melanogaster. We then examine more closely the function of one of these target genes, Gadd45, and find that overexpression of this target in NMD mutants may explain important aspects of the NMD mutant phenotype

Respostas celulares aos erros de tradução do genoma

Doutoramento em BioquímicaLow level protein synthesis errors can have profound effects on normal cell physiology and disease development, namely neurodegeneration, cancer and aging. The biology of errors introduced into proteins during mRNA translation, herein referred as mistranslation, is not yet fully understood. In order to shed new light into this biological phenomenon, we have engineered constitutive codon misreading in S. cerevisiae, using a mutant tRNA that misreads leucine CUG codons as serine, representing a 240 fold increase in mRNA translational error relative to typical physiological error (0.0001%). Our studies show that mistranslation induces autophagic activity, increases accumulation of insoluble proteins, production of reactive oxygen species, and morphological disruption of the mitochondrial network. Mistranslation also up-regulates the expression of the longevity gene PNC1, which is a regulator of Sir2p deacetylase activity. We show here that both PNC1 and SIR2 are involved in the regulation of autophagy induced by mistranslation, but not by starvation-induced autophagy. Mistranslation leads to P-body but not stress-granule assembly, down-regulates the expression of ribosomal protein genes and increases slightly the selective degradation of ribosomes (ribophagy). The study also indicates that yeast cells are much more resistant to mistranslation than expected and highlights the importance of autophagy in the cellular response to mistranslation. Morpho-functional alterations of the mitochondrial network are the most visible phenotype of mistranslation. Since most of the basic cellular processes are conserved between yeast and humans, this study reinforces the importance of yeast as a model system to study mistranslation and suggests that oxidative stress and accumulation of misfolded proteins arising from aberrant protein synthesis are important causes of the cellular degeneration observed in human diseases associated to mRNA mistranslation.Erros no processo da síntese proteica podem ter profundos efeitos na fisiologia celular e no desenvolvimento de doenças, nomeadamente doenças neurodegenerativas, cancro e envelhecimento. A introdução de erros durante a síntese de proteínas e, em particular durante o processo da tradução, é designado por “mistranslation” que é um processo pouco estudado e mal compreendido. Neste projecto, construímos leveduras que, sistemática e constitutivamente, treslêem o codão de leucina CUG como serina, o que corresponde a um aumento de erro de 240 vezes relativamente à taxa de erro basal da síntese proteica (0.001%). Os resultados obtidos demonstram que os erros de tradução induzem a actividade autofágica, acumulação de proteínas insolúveis, produção de espécies reactivas de oxigénio, disrupção funcional e morfológica das mitocôndrias, não ocorrendo, no entanto, destruição selectiva destas. A expressão do gene PNC1, associado ao aumento da longevidade e regulador da actividade da deacetilase Sir2p, é fortemente aumentada em resposta aos erros da tradução. Os genes PNC1 e SIR2 estão envolvidos no controlo da autofagia induzida pelos erros de tradução mas não em situações de stress nutricional. O aumento dos erros de tradução leva à formação de P-bodies, mas não induz a formação de grânulos de stress e reduz a expressão de genes que codificam proteínas ribosomais em vez de se verificar destruição selectiva de ribosomas - ribofagia. Este estudo mostra que as células de levedura são muito mais resistentes aos erros na tradução do que o esperado. Os resultados mostram um papel fundamental da autofagia na resposta celular aos erros de tradução e indicam que estes têm um forte impacto em alterações morfo-funcionais das mitocondrias, sendo este um dos fenótipos mais marcantes nestas células. Considerando que a maior parte dos mecanismos celulares são conservados entre leveduras e células humanas, este estudo mostra que a levedura é um excelente modelo para estudar a resposta celular aos erros de tradução e sugere que o stress oxidativo, a acumulação de espécies reactivas de oxigénio e a acumulação de proteínas insolúveis podem ser a causa da degeneração celular observada em múltiplas doenças humanas associadas a defeitos na síntese proteica

RNA Directed Gene Regulation in Toxoplasma gondii

Toxoplasma gondii is an intracellular obligate parasite of phylum Apicomplexa. Toxoplasma is able to infect any nucleated cell including up to one third of the world’s population. Within its hosts, the asexual life cycle of Toxoplasma consists of two distinct forms; a rapidly growing form called tachyzoite and a latent cyst encapsulated form called bradyzoite. Although tachyzoites can be removed by the host’s immune system, parasites can convert to bradyzoites thereby evading the host’s immune response. If the host’s immune system becomes weakened, bradyzoites are able to reconvert to tachyzoites. Taken together, these observations suggest Toxoplasma contains intricate gene regulation mechanisms that could be shared by other intracellular parasites. MicroRNAs (miRNAs) are crucial genetic effectors involved in numerous gene regulation mechanisms in eukaryotes. Although a post-transcriptional gene silencing can be observed in Toxoplasma the roles and functions of Tg-miRNAs are still elusive. In this work an engineered dual luciferase reporter system was used to examine and standardize the ability of long and short double-stranded RNA to control gene expression in Toxoplasma. The effects of endogenous Tg-miRNAs were also evaluated based on (i) their abundance and (ii) the number of binding sites within a transcript. Tg-miRNA effectiveness can be altered by use of miRNA mimics and inhibitors. Also a genetic knockdown system that exploits and directs Tg-miRNAs for loss-of-function analysis of essential and non-essential genes was developed. To further our understanding of miRNA induced gene silencing on parasite cell biology, a model gene target was selected for the most abundant Tg-miR-60a. The locus of the Ubiquitin-like protease 1 (Ulp1) contains the highest number of predicted Tg-miR-60a binding sites. The antisense RNA expresses a likely precursor of Tg-miR-60a suggesting TgUlp1 expression could be self-regulating. Ulp1 activity is required for the first and last step in the SUMOylation pathway. TgUlp1 is able to compliment the function of yeast Ulp1 and exhibits cleavage activity in vitro. Knockout of TgUlp1 is detrimental to parasite egress and survival. Since a miR-60a inhibitor or antisense RNA can alter TgUlp1 expression and activity, a TgUlp1-SF line was created to study the effects of miR-60a and its effects on total protein SUMOylation to demonstrate the importance of proper gene regulation by miRNAs. Furthermore, Toxoplasma Argonaute, the core protein of RNA-induced gene silencing that is required for a gene silencing effect, is expressed as two isoforms from the same locus, suggesting alternative translational starts sites. Overall, this dissertation reveals the importance of proper gene regulation directed by non-coding RNAs in Toxoplasma that could lead to the development of anti-Toxoplasma therapeutic