Дисертації з теми "The N-end rule"

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.
Title of thesis missing on title page; supplied from the abstract, p. [1].
Includes bibliographical references (p. 107-115).
Protein degradation is a central mechanism in the regulation of gene expression and activity. Proteolysis regulates not only homeostatic activities, but also the cell's responses to stress. A recurring question underlying this regulatory process is the specificity of substrate selection by the proteolytic machinery. I designed an unbiased selection to isolate N-terminal degradation sequences in vivo, which led to a collection of N-end rule signals. The N-end rule describes how the identity of a protein's N-terminal residue determines its metabolic stability. In E. coli, CIpAP is the principal protease that degrades proteins bearing an N-terminal phenylalanine, tyrosine, tryptophan, or leucine residue. The CIpS adaptor, which displays homology to eukaryotic ubiquitin ligases that recognize N-end signals, is a recently discovered component of the bacterial N-end rule. Using the collection of N-end signals, I was able to demonstrate that ClpS enhances N-end degradation by ClpAP but is not required in vivo or in vitro. The collection of N-end signals also provided insight into the role of sequence context in the N-end rule. Specifically, acidic residues and the length of the N-end signal affect degradation rates in vitro. These defective N-end signals also allowed us to separately define recognition specificities of ClpS and ClpAP. Whereas ClpS bound poorly to acidic N-end signals, CIpAP was unable to degrade substrates with short N-end sequences. Although two decades of biochemical and cellular data support the importance of the Nterminal residue in N-end degradation, there has been no structural information explaining how a single residue is recognized as a degradation signal.
(cont.) To this end, we solved a cocrystal structure of CIpS in complex with an N-end peptide. CIpS uses an extensive hydrogen bonding network to dock the a-amino group and a cavity lined with hydrophobic residues to recognize the N-terminal residue. Furthermore, mutation of the hydrophobic cavity altered the specificity of CIpS toward N-terminal residues. Together these findings attribute molecular functions to CIpS and ClpAP in the bacterial N-end rule and define sequence rules for the N-end signal. Furthermore, this work provides the tools and background for investigating the mechanism of substrate delivery by ClpS to ClpAP.
by Kevin H. Wang.
Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. "June 2016." In title on title page [alpha] appears as lower case Greek letters.
Includes bibliographical references (pages 103-118).
by Benjamin J. Stein.
Ph. D.

Current protein sequencing methods include mass spectrometry and Edman degradation. We envision a novel high-throughput protein sequencing method using affinity adapters to recognize the N-terminal residue of a denatured peptide in an iterative process. This thesis takes a first step toward designing robust and selective affinity reagents. We outline our pipeline for designing selective protein adapters that recognize the N-terminal amino acid of a peptide independent of the following sequence. We based our design on a substrate recognition protein in the N-end rule pathway, ClpS. The bacterial N-recognin protein ClpS binds peptide substrates, termed N-degrons, that have a bulky hydrophobic amino acid (L/F/Y/W) at the N-terminus. Using full atom in silico models we designed hydrogen bonding and salt-bridge contacts in ClpS to novel N-degron substrates (N-end D/E/T), predicted the selectivity of these designs, and experimentally verified them. Of 11 designs, we purified nine that were soluble by SDS-PAGE, and obtained a peptide binding profile to 30 peptides with a modified ELISA assay. Most designs were non-specific or had no binding affinity. Four designs M53A, L112F, I45L, I45L_I45L_M53A had an increase in affinity to various substrates, but were not selective as they retained affinity to the native substrates (N-end L/F/Y/W). We performed molecular dynamics simulations on several proteins that were soluble or insoluble under standard expression conditions in E. coli, in order to learn parameters that were indicative of kinetic instability. Using a back-to-consensus approach, we identified a point mutant S104F that stabilizes the scaffold of ClpS as assayed by GFP fluorescence in a GFP-ClpS fusion protein. This thesis outlines the computational design pipeline we developed, which includes a RosettaScripts protocol, an in silico selectivity screen with AutoDock, and a kinetic stability confidence score from a molecular dynamics trajectory. Finally, we make suggestions toward designing selective affinity reagents for high-throughput N-end protein sequencing.

Caspase-7 is a member of a family of cysteine proteases that includes apoptotic initiators (caspases-8, -9 and -10) and executors (caspases-3, -6 and -7). During apoptosis, executioner caspases are cleaved by initiator caspases either by the extrinsic (death receptors) or by the intrinsic (mitochondrial) pathway of caspase activation. Caspase-7 is an obligate dimer in the cell and cleavage of the interdomain connector (IDC), which split the catalytic domain in two subunits, at either site 1 or site 2 allows the conversion of the enzyme from the zymogen (inactive) state to the active state through a conformation switch that leads to the creation of a substrate binding pocket and the catalytic site. During caspase-7 activation, a 23-residue N-terminal peptide is also cleaved. Consequently, caspase-7 displays different N-terminal residues from those of its zymogen. This can change the stability of caspase-7 according to the N-end rule, which relates the half-life of a protein with the residue presented at its N-terminus. This degradation pathway controls the ubiquitination of the protein based on the N-terminus. To replicate the different forms of caspase-7 produced during its activation process in a controlled manner, a TEV protease cleavage site [ENLYFQ[arrow down](S/A)] was strategically inserted to mimic the different possibilities of IDC cleavage: 1) cleavage at site 1 only, 2) cleavage at site 2 only, or 3) a double cleavage. This was done in order to obtain the N-terminal residues normally presented during the cleavage of caspase-7. These constructions have been also optimized to preserve the proteolytic activity of the enzyme with as little change as possible to the length of the IDC. These constructs were cleaved by TEV protease in vitro and in cellulo and allowed the activation of apoptosis. Furthermore, the cellular half-life of caspase-7 seems to be changed by its cleavage. In conclusion, we have developed an interesting tool for the study of caspase-7.

Flooding events are becoming more common throughout the world as a result of climate change, resulting in reduced crop yields. It was recently discovered that plants sense low oxygen (O2) (associated with flooding) through regulated proteolysis of the group VII Ethylene Response Factor transcription factors (ERFVIIs), via the Cys-Arg/N-end rule pathway of ubiquitin mediated proteolysis, which also senses another gas, nitric oxide (NO). The N-terminal (Nt) Cys of physiological (e.g. ERFVIIs) and artificial substrates was shown to be key for N-end rule function, and work in mammalian systems suggested that oxidation of Nt-Cys by O2 and NO was a required prerequisite for subsequent Nt arginylation by arginyl tRNA transferases (ATEs). However the exact mechanism of Nt-Cys oxidation has not been discovered. The primary aim of this thesis was to define the mechanism of in vivo Nt-Cys oxidation essential in determining the stability of Arg/N-end rule protein substrates, including ERFVIIs. In this study a novel approach was developed to investigate the oxidation of Nt-Cys in vivo using transgenic Arabidopsis expressing Cys-2 reporter proteins (derived from the transgenes 35S::MC-polyG-HA-GUS and Ubi1::MCGGAIL-GUS). Cys-2 of the reporter proteins is made Nt constitutively by co-translational Methionine Aminopeptidase (MAP) activity. Biochemical techniques were combined with analytical chemistry to investigate the in vivo oxidation of Nt-Cys using liquid chromatography mass spectrometry (LC-MS). In addition, synthetic peptides representing the Nt-sequence of the in vivoreporter protein were used to define the oxidative and nitrosylative modifications occurring at Nt-Cys in vitro. Findings of this study include the successful development of two new in vivo O2 sensor artificial N-end rule substrates, in the plant genetic model Arabidopsis thaliana. A further outcome revealed that the ERFVII RELATED TO APETALA 2.12 (RAP2.12) is stabilised in the shoot and root apical meristem to a greater extent than in other regions of seedlings in response to hypoxia, indicating that meristematic cells could be important oxygen sensory zones. Cys-2 reporter proteins were used in in vivo studies to attempt to identify the nature of the Nt residues following N-end rule action. Although Nt-peptides derived from affinity purified Cys-2 reporter protein were not identified by LC-MS, it was possible to demonstrate that ubiquitination is required on Cys-2 reporter proteins before 26S proteasome degradation. Combined results from Nt-Cys in vitro synthetic peptides and Cys-2 reporter proteins substrates of the Arg/N-end rule pathway, provide indirect evidence that post translational modifications (PTMs)not defined before occur in vivo. Using synthetic peptides it was possible to show evidence for Nt-Cys-sulfenamide formation after oxidation of Nt-Cys. This finding suggests that Cys-sulfenamide formation, a previously recognised reversible modification preventing irreversible oxidation to Cys-sulfonic acid, could occur at Nt-Cys of in vivoprotein substrates. An important finding of this study was the observednonreactionbetween the nitrosylated Nt-Cys and H2O or conversely oxidised Nt-Cys and NO. As Plant Cysteine Oxidases (PCOs) do not require NO to oxidise Nt-Cys, this result raises further uncertainty as to how NO is involved in the oxidation of Nt-Cys, suggesting that NO may be involved in the enzymatic activation of PCOs or the arginylation of Nt-Cys by ATEs. The findings of this study did not identify the Nt-peptide of the Cys-2 reporter protein, and hence the Nt-Cys oxidation state of an in vivo substrate of the Arg/N-end rule pathway remains unidentified. Despite this the result that Nt-Cys can be oxidised to Cys-sulfenamide is novel and an important discovery to the field of reactive Cys biology and chemistry.

L'éthylène comme la stratification au froid et le GA3, stimule la germination des graines d'Arabidopsis thaliana (Col-0) ayant une dormance primaire, à 25 °C et à l'obscurité. L'élimination de la dormance au froid n'exige pas ETR1, EIN4 et EIN2, alors que l'effet du GA3 nécessite ETR1. L'effet stimulateur de l'éthylène est associé à une réduction de l'expression des gènes impliqués dans les voies de signalisation des GAs (DELLAs) et de l'ABA (ABI5). Les graines du mutant prt6 affectées dans la voie "N-end rule" de la protéolyse, sont insensibles à l'éthylène, montrant que PRT6 intervient dans l'action de l'éthylène, et cette insensibilité résulte aussi d'une interrelation avec la balance ABA/GAs. D'autre part, les facteurs de transcription de la réponse à l'éthylène du groupe VII, (ERF VIIs), substrats de la voie "N-end rule", interviennent dans cette insensibilité. La sensibilité à l'éthylène de prt6rap2.2rap2.3rap2.12 et l'insensibilité de prt6hre1hre2, permet de montrer que les 3 RAPs et les 2 HREs interviennent respectivement en aval et en amont de PRT6. L'éthylène induit une diminution de l'expression des 3 RAPs dans Col, mais maintient ou induit celle-ci dans prt6. De plus, l'expession de HRE2 augmente dans prt6, mais diminue dans prt6rap2.2rap2.3rap2.12, suggérant que les 3 RAPs peuvent réguler l'expression de HRE2. De plus, l'éthylène modifie différentiellement le protéome de Col et de prt6 avec respectivement 587 et 30 protéines significatives. L'analyse des classes fonctionnelles a permis d'identifier la réponse à hypoxie comme processus biologique spécifique de prt6, mais leur insensibilité à l'éthylène est indépendante des ROS et de l'intensité respiratoire
Ethylene as chilling and GA3, was able to improve the germination of primary dormant seeds of Arabidopsis thaliana (Col-0) at 25 °C in darkness. Chilling did not require EIN4, ETR1 and EIN2 involved in ethylene signaling to break seed dormancy while GA required ETR1.The improving effect of ethylene in seed germination is EIN4 independent, and is associated with a decrease in ABA/GA ratio and a down-regulation of DELLAs and ABI5 genes related to GA and ABA signaling, respectively. The mutant affected in the proteolytic N-end rule pathway, prt6, was insensitive to ethylene in seed germination evidenced that PRT6 was involved in dormancy release by ethylene, and this insensitivity was related to a crosstalk with ABA/GAs. The substrates of the N-end rule pathway, ERFVIIs (HRE1, HRE2, RAP2.2, RAP2.3, and RAP2.12), might result in the insensitivity with an increased germination in prt6rap2.2rap2.3rap2.12 rather than in prt6hre1hre2, which also indicated that the 3 RAPs acted downstream of PRT6, while the 2 HREs acted upstream of PRT6. Ethylene reduced the transcript expression of the 3 RAPs in Col-0, but the 3 RAPs were maintained or induced by ethylene in prt6. Besides, HRE2 was up-regulated in prt6 seeds, but it was down-regulated in prt6rap2.2rap2.3rap2.12, suggesting that the 3 RAPs might stimulate the expression of HRE2. Ethylene differently changed the seed proteome of Col and prt6 with 587 and 30 significant proteins, respectively. The functional class scoring analysis identified one biological process, response to hypoxia, which was distinct in prt6, however the insensitivity of prt6 to ethylene was independent of ROS production or respiration intensity

Le mûrissement des tomates est un processus finement régulé avec de nombreux changements dynamiques qui concernent les voies du métabolisme primaire et secondaire, la respiration ou encore la signalisation hormonale. Comme les autres fruits climactérique, la tomate connait une transition développementale du stade « pré-maturation » à la phase de maturation qui est associée à une augmentation de la respiration au début de la maturation et à une augmentation de la production autocatalytique d'éthylène. Les facteur de réponse à l'éthylène, de la sous-clade E (ERF.E), ont un profil d'expression fortement associé au mûrissement du fruits de tomate. Les protéines ERF.E de la tomate sont notamment caractérisées par la présence d'un motif N-terminal conservé, qui a été identifié comme cible de la voie N-end rule en condition d’hypoxie chez leurs orthologues d'Arabidopsis. Cette étude dévoile pour la première le rôle positif de ERF.E1 dans la maturation des fruits de la tomate. La surexpression d'ERF.E1 favorise l'initiation du mûrissement, et est accompagnée d'une production avancée d'éthylène et d'une déplétion précoce de l'oxygène dans le gel, par rapport aux lignées WT et KO-erf.e1. ERF.E1 régule positivement les activités promotrices des gènes clés de la maturation, RIN et NOR, ainsi que les gènes de biosynthèse de l'éthylène du système 2. De plus, les résultats ont montré qu’en condition d’hypoxie, ERF.E1 est régulé aux niveaux transcriptionnel et post-traductionnel. En effet, les conditions hypoxiques induisent l’expression d’ERF.E1 et stabilise la protéine ERF.E1. Cette thèse se concentre principalement sur le rôle d'ERF.E1 dans l'initiation de la maturation, grâce à la caractérisation des lignées altéré dans l’expression de ERF.E1 et à l’étude des réseaux d'interaction protéiques potentiels d'ERF.E1
Tomato fruit ripening is a finely regulated process with many well-known dynamic changes, from metabolites, color, and respiration rate to hormone level. As a climacteric fruit, tomato fruit experiences a developmental transition characterized by a burst of respiration at the onset of ripening and a rise in autocatalytic ethylene production. Ethylene Response Factors (ERFs) are end mediators of ethylene responses and members of subclade E (ERF.E) of this transcription factor family display remarkable ripening-associated expression patterns in tomato fruit. Notably, tomato ERF.E proteins are characterized by the presence of a consensus N-terminal motif, which has been identified in their Arabidopsis orthologs as the target of the N-end rule pathway under hypoxia. The first outcome of our study is to reveal that initiation of tomato fruit ripening is preceded by a dramatic drop in oxygen levels in locular gel, thus giving a solid physiological marker of ripening initiation. The study also unveils the function of ERF.E1 in tomato fruit ripening showing that overexpression of ERF.E1 promotes the initiation of ripening, by advancing both ethylene production and oxygen depletion event in the gel, compared with the WT and KO-erf.e1 lines. ERF.E1 positively regulates the promoter activities of key ripening genes, RIN and NOR, as well as the ethylene biosynthesis genes involved in System 2 ethylene production. In addition, the data show that ERF.E1 is regulated by hypoxia at both transcriptional and post-translational levels. Hypoxia conditions induce transcription of ERF.E1 and promote ERF.E1 protein stability. Overall, while the study unraveled the role of ERF.E1 in ripening initiation based on the analysis of ERF.E1 impaired lines are altered at the early ripening stages it also uncovered potential proteins interacting with the ERF.E1 network components

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2011.
"June 2011." Cataloged from PDF version of thesis.
Includes bibliographical references (p. 158-165).
Regulated protein degradation is crucial in the regulation of many physiological processes as well as in protein quality control. In all organisms, ranging from bacteria to mammals, ATP-dependent proteases carry out regulated protein degradation in order to maintain homeostasis as well as to respond to stress. ATP-dependent proteases are responsible for the degradation of a broad array of substrates and for that reason, a high degree of substrate specificity is required in order to target only desired proteins for destruction. Adaptor proteins can provide ATP-dependent proteases an extra-layer of specificity by binding and delivering a specific class of substrates, therefore regulating the activity of the protease. Understanding how adaptor proteins work in combination with their partner protease will provide a better understanding on how specificity is achieved by these proteolytic machines. In this thesis, different aspects of the bacterial N-end rule degradation pathway are examined. The N-end rule is a highly conserved degradation pathway that relates protein stability to the identity of its N-terminal residue in both prokaryotes and eukaryotes. For example, in bacteria, Tyr, Phe, Trp, and Leu serve as degradation signals when located at the N-terminus of a protein. In E. coli, the bacterial adaptor CIpS recognizes these signals and delivers these substrates to the AAA+ protease CIpAP. Here, we present the first crystal structure of a bacterial N-end rule adaptor, CIpS, bound to a peptide mimic of an N-end rule substrate. This structure pioneered the understanding behind the basis of N-end rule recognition by CIpS. The CIpS structure reveals the adaptor recognizes the peptide a-amino group via hydrogen bonding and shows that the peptide's N-terminal side chain is buried in a deep hydrophobic cleft that preexists on the surface of CIpS. We also present here the crystal structures of CIpS alone and engaged with peptides containing the rest of the primary N-end degrons (N-terminal phenylalanine, leucine, and tryptophan). These structures, together with the first structure of CIpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule residues. Moreover, we show that mutation of critical CIpS contact residues impairs substrate delivery to and degradation by the AAA+ protease CIpAP. In addition to the structural studies, the biochemical studies presented here provide a better understanding on how CIpS and CIpA work together for efficient N-end rule substrate delivery. Here, we show that substrate binding is enhanced substantially when CIpS binds ClpA6. Reciprocally, N-end-rule substrates increase CIpS affinity for CIpA6. Some of the features required for enhanced binding include the substrate N-end residue and the substrate first peptide bond. It also requires multiple features of CIpS, including a side chain that contacts the substrate a-amino group and two regions of a flexible N-terminal extension (NTE). We also show that enhancement in affinity requires the N domain and AAA+ rings of CIpA to be connected by a sufficiently long linker. One major novel finding uncovered in this thesis is that the CIpS NTE can be engaged by the CIpA translocation pore, but CIpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the CIpAP proteolytic machine.
by Giselle Roman Hernandez.
Ph.D.

The PROTEOLYSIS 6 (PRT6) branch of the N-end rule pathway is a well-characterized negative regulator of flooding and low oxygen tolerance in plants. This study investigated the role of this pathway in adaptation to salinity stress in Arabidopsis and maize via physiological and molecular characterization of Arabidopsis prt6-1 and maize prt6 MU insertion mutants, respectively. Our study demonstrated that the loss of function mutation of prt6 in Arabidopsis activated hormonal and transcriptional responses associated with adaptation to salinity stress, enhancing high salt tolerance at seed germination, seedling, and adult plant stages. Our data also indicated that salinity tolerance conferred by the prt6 mutation is attributed to increased mRNA abundance of key transcriptional factors in ABA-dependent (AREB/ABFs) and independent (DREBs) pathways, together with the dominant expression of downstream dehydrins. Furthermore, this study revealed that the prt6 mutation enhances ethylene and brassinosteroid responses, resulting in restricted Na+ accumulation in roots and shoots as well as increased expression of dehydrin genes such as RD29A and RD29B. Maize prt6 mutant plants, contrary to our observation in Arabidopsis, showed lower seed germination, primary root elongation, and shoot biomass growth along with increased malondialdehyde (MDA) accumulation under high salt. Moreover, maize prt6 mutants exhibited reduced grain yield and yield-related components under high salt. These results indicate that PRT6 functions as a negative regulator for salinity tolerance in Arabidopsis, whereas this gene plays a positive role in salinity tolerance in maize. In wheat, we compared two genotypes with contrasting nitrogen-use-efficiency (NUE), VA08MAS-369 and VA07W-415, to dissect physiological and molecular mechanisms underlying NUE regulation. Our agronomic data revealed that line 369 maintained yield and yield-related parameters and exhibited greater NUE indexes relative to line 415 under N deficient conditions. Furthermore, our analyses suggested that the significantly higher nitrogen use efficiency (NUE) in line 369 could be attributed to the greater N uptake efficiency in this genotype. In fact, line 369 was able to maintain the development of root systems under N limitation. Consistently, genes encoding high-affinity nitrate transporters such as TaNRT2.1 and TaNRT2.2 were expressed more abundantly in the roots of line 369 than line 415 at limited N. Overall, the results of this study characterized physiological and molecular phenotypes associated with high N uptake efficiency in line 369. This is useful information for the development of new wheat accessions with improved NUE.
Doctor of Philosophy
In coastal areas, sea-level rise increases the chances of saltwater intrusion into cultivable lands, making a hostile environment for crop growth and production by imposing flooding and salinity stresses simultaneously. Identification of central regulators that regulate the adaptation to both flooding and salinity is a critical step for the development of new crop genotypes with enhanced tolerance to these stresses. Previous studies have characterized the function of the PROTEOLYSIS 6 (PRT6) gene in adaptation to flooding stress in plants. This study assessed whether this gene is involved in adaptation to salinity stress in Arabidopsis and maize by evaluating the growth and survival of their respective prt6 mutants under high salt. Consistent with the flooding tolerance data, our study showed that the PRT6 gene also functions as a negative regulator of salinity stress tolerance in Arabidopsis. The prt6 mutation in Arabidopsis activated the key transcriptional and hormone response pathways associated with adaptation to both salinity/osmotic stress and sodium toxicity, expressed as enhanced tolerance to excess salt at seed germination, seedling, and adult plant stages. In maize, disruption of the PRT6 gene decreased seed germination, primary root elongation, and shoot biomass growth under high salt, which is opposite to our observations in Arabidopsis. Additionally, the maize mutant plants encountered more oxidative stress, as demonstrated by the higher accumulation of malondialdehyde (MDA) under high salt. Moreover, maize prt6 mutants exhibited reduced grain yield under high salt. Overall, these results indicate that disruption of the PRT6 gene confers increased tolerance to high salt in Arabidopsis, whereas it conversely reduced salinity tolerance in maize. In wheat, we compared two genotypes with distinct nitrogen use efficiency (NUE), VA08MAS-369 and VA07W-415, to determine critical traits involved in NUE regulation. Our study showed that grain yield and yield-related parameters were significantly higher in line 369 than line 415 under low N. Moreover, high NUE in line 369 was attributed to efficient N uptake in this genotype under limited N. Our root architecture analysis demonstrated that line 369 was able to maintain root depth, volume, and thickness even under N limitation. Consistently, line 369 highly induced expression of genes associated with nitrogen transport at low N. Altogether, this study identified key traits involved in high NUE in wheat, facilitating the breeding of new wheat genotypes with enhanced NUE.

El óxido nítrico (NO) es una molécula gaseosa altamente reactiva que regula el crecimiento y el desarrollo de las plantas así como sus respuestas de defensa. El NO se produce principalmente a partir de nitrito por las nitrato reductasas (NRs) en balance con las nitrito reductasas (NiRs), y es percibido a través de un mecanismo en el que está involucrada la proteólisis dirigida por la secuencia aminoterminal del grupo VII de los factores de transcripción ERF (ERFVIIs). El NO ejerce especialmente su función señalizadora al causar modificaciones postraduccionales en las proteínas y alterar su función, estructura y/o estabilidad. Por estos medios y en colaboración con distintas rutas de señalización fitohormonales, el NO es capaz de regular un amplio abanico de procesos celulares en plantas, incluyendo aquellos relacionados con la adquisición de tolerancia a la congelación. Utilizando Arabidopsis thaliana como planta modelo, en este trabajo se descubrió que el NO puede regular su propia biosíntesis, puesto que las enzimas NRs y NiRs fueron reguladas por tres factores principales: señalización inducida por nitrato y controlada por la función del factor de transcripción NIN-like protein 7 (NLP7), la proteólisis dirigida por la secuencia aminoterminal, y la degradación mediada por el proteasoma, probablemente ocasionada por modificaciones postraduccionales relacionadas con el NO. Adicionalmente, se descubrió que el factor de transcripción ERFVII RAP2.3 regula negativamente tanto la biosíntesis de NO como las respuestas que desencadena a través de un mecanismo similar a un reóstato en el que están involucradas ramas específicas relacionadas con el NO de las rutas de señalización de jasmonato y ácido abscísico. Por otro lado, una caracterización metabolómica y transcriptómica combinada de plantas mutantes nia1,2noa1-2 deficientes en NO y plantas fumigadas con NO permitió desentrañar una serie de mecanismos que están controlados por NO. En primer lugar, la percepción de NO en los hipocotilos requeriría varias hormonas para ser completada, como fue confirmado por los rastreos de acortamiento de hipocotilo por NO con mutantes relacionados con hormonas y la colección TRANSPLANTA de líneas transgénicas que expresan condicionalmente factores de transcripción de Arabidopsis. En segundo lugar, dosis elevadas de NO causan una reprogramación masiva aunque transitoria de los metabolismos primario y secundario, incluyendo la alteración del estado redox celular, la alteración de la permeabilidad de estructuras lipídicas y el recambio de proteínas y ácidos nucleicos. Por último, se descubrió que el NO previene el desarrollo de la tolerancia a congelación bajo condiciones no estresantes de temperatura, mientras que resulta esencial para la aclimatación a frío desencadenada por bajas temperaturas que conduce a una tolerancia mejorada a congelación. El NO conseguiría esta modulación afinada de la activación de respuestas relacionadas con frío al coordinar la acumulación de diferentes metabolitos y hormonas. En conjunto, este trabajo arroja luz sobre los mecanismos mediante los cuales, al interactuar con varias rutas señalizadoras y metabólicas, el NO puede regular distintos procesos clave de la fisiología vegetal.
L'òxid nítric (NO) és una molècula gasosa altament reactiva que regula el creixement i desenvolupament de les plantes així com les seves respostes de defensa. El NO es produeix principalment a partir de nitrit per les nitrat reductases (NRs) en balanç amb les nitrit reductases (NiRs), i és percebut a traves d'un mecanisme que inclou la proteòlisi dirigida per la seqüència aminoterminal del grup VII dels factors de transcripció ERF (ERFVII). El NO exerceix la seva funció senyalitzadora majoritàriament al provocar modificacions postraduccionals en les proteïnes i alterar la seva funció, estructura i/o estabilitat. Mitjançant aquestes modificacions i en col·laboració amb distintes rutes de senyalització fitohormonals, el NO es capaç de regular un ampli espectre de processos cel·lulars en plantes, inclosos aquells relacionats amb l'adquisició de tolerància a la congelació. Emprant Arabidopsis thaliana com a planta model, en aquest treball es va descobrir que el NO regula la seva pròpia biosíntesi, donat que els enzims NRs i NiRs foren regulades per tres factors principals: senyalització induïda per nitrat i controlada per la funció del factor de transcripció NIN-like protein 7 (NLP7), la proteòlisi dirigida per la seqüència aminoterminal, i la degradació mitjançant el proteasoma, probablement a causa de modificacions postraduccionals relacionades amb el NO. A més, es va descobrir que el factor de transcripció ERFVII RAP2.3 regula negativament tant la biosíntesi de NO com les respostes que desencadena aquest a través d'un mecanisme similar a un reòstat en el que estan involucrades branques específiques de les rutes de senyalització de jasmonat i àcid abscísic relacionades amb el NO. Per altre costat, una caracterització metabolòmica i transcriptòmica combinada de plantes mutants nia1,2noa1-2 deficients en NO i plantes fumigades amb NO va permetre desentranyar una sèrie de mecanismes que estan controlats per NO. En primer lloc, la percepció de NO en els hipocòtils requeriria de varies hormones, com fou confirmat pels rastrejos d'acurtament d'hipocòtil per NO amb mutants relacionats amb hormones i la col·lecció TRANSPLANTA de línies transgèniques d'expressió condicional de factors de transcripció d'Arabidopsis. En segon lloc, dosis elevades de NO causen una reprogramació massiva, encara que transitòria, dels metabolismes primari i secundari, incloent l'alteració de l'estat redox cel·lular, canvis en la permeabilitat de estructures lipídiques i el recanvi de proteïnes i àcids nucleics. Per últim, es va descobrir que el NO prevé el desenvolupament de la tolerància a congelació en condicions no estressants de temperatura, mentre que resulta essencial per a l'aclimatació a fred induïda per baixes temperatures que condueix a una tolerància millorada a congelació. El NO aconseguiria aquesta modulació minuciosa de l'activació de les respostes relacionades amb fred al coordinar l'acumulació de diferents metabòlits i hormones. En conjunt, aquest treball clarifica els mecanismes pels quals el NO pot regular distints processos clau de la fisiologia vegetal al interactuar amb varies rutes senyalitzadores i metabòliques.
Nitric oxide (NO) is a highly reactive gaseous molecule that regulates plant growth and development as well as defense responses. NO is mainly produced from nitrite by nitrate reductases (NRs) in balance with nitrite reductases (NiRs), and is sensed through a mechanism involving the N-end rule pathway-mediated proteolysis of the group VII of ERF transcription factors (ERFVIIs). NO especially exerts its signaling function by triggering post-translational modifications in proteins and altering their function, structure and/or stability. By these means and in collaboration with different phytohormone signaling pathways, NO is capable of regulating a wide array of cell processes in plants, including those related to the acquirement of freezing tolerance. By using Arabidopsis thaliana as model plant, during the development of this work it was found that NO can regulate its own biosynthesis, as NRs and NiR enzymes were regulated by three main factors: nitrate-induced signaling controlled by the function of the NIN-like protein 7 (NLP7) transcription factor, N-end rule proteolytic pathway, and proteasome-mediated degradation, likely triggered by NO-related post-translational modifications. In addition, the ERFVII transcription factor RAP2.3 was found to negatively regulate both the NO biosynthesis and their triggered responses through a rheostat-like mechanism that involves specific NO-related branches of jasmonate and abscisic acid signaling pathways. On the other hand, a combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1-2 mutant plants and NO-fumigated plants allowed to unravel a number of mechanisms that are controlled by NO. First, NO perception in hypocotyls would require various hormones to be fulfilled as it was confirmed by NO-triggered hypocotyl shortening screenings with hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis transcription factors. Second, high NO doses caused a massive but transient reprogramming of primary and secondary metabolism, including alteration of the cellular redox status, alteration of the permeability of lipidic structures or turnover of proteins and nucleic acids. Lastly, NO was found to prevent the development of freezing tolerance under non-stress temperature conditions, while being essential for the low temperature stress-triggered cold acclimation that leads to enhanced freezing tolerance. NO would achieve this fine-tuned modulation of the activation of the cold-related responses by coordinating the accumulation of different metabolites and hormones. Altogether, this work sheds light on the mechanisms by which, by interacting with various signaling and metabolic pathways, NO can regulate several key processes of plant physiology.
Costa Broseta, Á. (2018). Regulation of the nitric oxide synthesis and signaling by posttranslational modifications and N-end rule pathway-mediated proteolysis in Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114825
TESIS

The N-end rule pathway of targeted proteolysis is regulated by Group VII ethylene response factors (ERFVII’s). The aim of this research work was to analyse the relationship between substrates (ERFVII’s) of the N-end rule pathway and genes, which have promoters containing a double ‘GCCGCC’ Ethylene-Responsive Element Binding Protein (EBP) cis-element. Several genes were identified containing double EBP elements. Cloning and transformation of the promoters from five of these genes (PYL, ERD4, AT1G14810, AT3G13440 and AT5G44420) carrying two copies of the GCC-boxes present in the 5' UTR (5’ untranslated region) or promoter region was conducted into Arabidopsis wild-type (Col-0) and prt6-1 mutant plants. Expression driven by these promoters in the leaves and flowers of transgenic plants was analysed through GUS staining to reveal promoter activities. Enhanced promoter activity was seen in prt6-1 lines (mutated in the E3-ligase of the N-end rule pathway) in comparison to Col-0. Further, cDNA of leaves and flowers of Col-0 and prt6-1 were analysed by q-RT-PCR (quantitative real-time PCR) for expression of PYL7, ERD4, AT1G14810, AT3G13440; t-test analysis showed a significant difference (p-value<0.05) only in leaves of Col-0 and prt6-1 for PYL7. Analysis of the genetic relationship between N-end rule pathway and genes containing GCC-boxes was also performed by analysing double mutant combinations of prt6-1 and mutants of genes containing the EBP elements (pyl7prt6-1, erd4prt6-1 and abi5prt6-1) and Col-0 under different concentration of salt to determine the effect of stress due to salinity on the regulation of genes. At 125mM salt concentration significant difference was identified in highest number of mutant lines in comparison to Col-0. An analysis of the in-vivo binding of the ERFVII RAP2.3 to the promoter of GCC-boxes containing genes was performed through Chromatin Immuno-precipitation assay (ChIP). The t-test analysis on qChIP-PCR data indicated significant difference between IgG and HA-IPs for both ABI5 and PYL7 performed on normoxic 35S:MA-RAP2.3-HA in Col-0 line. Further, in-vivo localization of ERFVII’s HRE1 and RAP2.2 conditional stability was analysed using promERFVII’s:MC/MA-ERFVII’s-YFP constructs in Col-0 and prt6-1. This thesis suggests that genes PYL7, ERD4, AT1G14810, AT3G13440 that have double ‘GCCGCC’ EBP elements are downstream targets of the N-end rule pathway. Further analysis via ChIP suggests that RAP2.3 interacts with the ‘GCCGCC’ binding site of promoters in the ABI5 and PYL7 genes, however further work is needed to confirm this. Additionally, sub-cellular localization of promERFVII’s:MC/MA-ERFVII’s-YFP studies suggest the location of HRE1 and RAP2.2 in nuclei of early stage root tips studied on 4-days old etiolated seedlings.

Die Tetrapyrrolbiosynthese (TBS) führt zu wichtigen Endprodukten wie Häm und Chlorophyll. Das gemeinsame Vorstufenmolekül aller Tetrapyrrole ist die 5-Aminolävulinsäure (ALA), die in Pflanzen über den C5-Weg aus Glutamat synthetisiert wird. Das erste spezifische Enzym der ALA-Synthese und somit auch der TBS ist die Glutamyl-tRNA Reduktase (GluTR). Sie unterliegt als Schlüsselenzym einer strengen Regulation. Aufgrund der unterschiedlichen Expression der HEMA-Gene in Arabidopsis wird ein differenzieller Beitrag der GluTR-Isoformen zu den Endprodukten der TBS vermutet. Analysen von knockout-Mutanten gaben Aufschluss darüber, inwiefern die Isoformen den Verlust des jeweils anderen kompensieren können. Die knockout-Mutante von HEMA1 zeigte einen blassgrünen Phänotyp, war nicht mehr in der Lage photoautotroph zu wachsen und demonstrierte eine essentielle Rolle der GluTR1 gegenüber GluTR2, wohingegen hema2-Mutanten einen wildtypartigen Phänotyp aufzeigten. Die Bedeutung der N-terminalen GluTR-Domäne in der posttranslationalen Regulation der ALA-Synthese wurde durch BiFC-Analysen und Komplementationsversuche aufgeklärt. BiFC-Analysen zeigten eine Interaktion der N-terminalen Domäne der GluTR1 mit Proteinen der Clp Proteasen und dem GluTR-Bindeprotein (GBP). Veränderte GluTR-Stabilitäten in gbp-Mutanten lassen eine schützende Funktion des GBP gegenüber dem Abbau des Proteins postulieren. Die Expression einer N-terminal verkürzten GluTR1 komplementierte hema1-Mutanten vollständig. Die in diesen Pflanzen und in clp-Mutanten beobachteten erhöhten GluTR1-Proteinstabilitäten im Dunkeln lassen einen Abbau der GluTR durch Clp Proteasen vermuten, bei dem der N-Terminus des Enzyms für die Substraterkennung notwendig zu sein scheint. Die Detektion von erhöhten Pchlid-Mengen als Folge der erhöhten Proteinstabilität in Linien, die die verkürzte GluTR1 exprimierten, demonstriert erstmals die Bedeutung einer kontrollierten Proteolyse der GluTR in der Regulation der ALA-Synthese.
In plants 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrrols and formed from glutamate via the C5 pathway. Glutamyl-tRNA reductase (GluTR) is the initial enzyme of ALA synthesis and thus tetrapyrrole biosynthesis (TBS). The most important control point of the TBS is the synthesis of ALA and GluTR is the key enzyme, that is tightly regulated. Due to the different expression of HEMA genes in Arabidopsis, a differential contribution to endproducts of the TBS is proposed for GluTR isoforms. Analysis of knockout mutants gave some indications of how the isoforms can compensate each other. I introduced a new knockout mutant of HEMA1 that was pale-green and not able to grow photoautotrophically, indicating that the remaining GluTR2 does not sufficiently compensate ALA synthesis for the extensive needs of chlorophyll. In contrast, hema2 mutants were wild-type-like. The function of the N-terminal region of GluTR1 in posttranslational regulation has been analyzed by BiFC analysis and complementation experiments of hema1. BiFC analysis showed an interaction of the N-terminal region of GluTR1 with the GluTR binding protein (GBP) and with proteins of the Clp proteases. Mutants of GBP revealed a decreased GluTR1 stability during the dark period, indicating a protective role of GBP against proteolysis of GluTR1 in darkness. The expression of a GluTR1 lacking the N-terminal amino acid residues successfully complemented hema1. These plants as well as clp mutants revealed an increased GluTR1 stability in darkness, suggesting a degradation of the protein through Clp proteases. Thereby, the N-terminal region of GluTR1 seems to be necessary for the recognition by Clp proteins. The observed high amount of truncated GluTR1 in transformed hema1 mutants was caused by the increased GluTR1 stability and lead to an accumulation of Pchlide in prolonged dark periods, demonstrating the importance of a controlled proteolysis of GluTR in the regulation of ALA synthesis.

At the end of G1 phase, cells have to decide between continue proliferation or remain in a quiescent state (G0). This decision point, known as “Start” in yeast and “Restriction Point” in metazoans, marks irreversibly the commitment to the completion of a new cell cycle, and is regulated mainly by the activity of the G1 CDK and the induction of the G1-to-S transcriptional program. The MBF transcription factor complex (functional homolog of pRB-E2F in metazoans) drives the G1-to-S transcriptional wave in the fission yeast Schizosaccharomyces pombe. We have previously described how the co-repressors Nrm1 and Yox1 bind to MBF complex at the end of S phase, inhibiting the MBF activity. However, the mechanisms involved in the activation of MBF at the onset of an unperturbed cell cycle have remained elusive. Here, we show that Nrm1 is the responsible for the activation of the MBF-dependent transcription through a two-step mechanism. Its phosphorylation by CDK1 and its posterior degradation by APCSte9 induce the irreversible MBF activation until the end of S phase. We have also studied the role of chromatin remodelers in the control of the G1-to-S transcriptional program. In this sense, we have found that chromatin-remodeling complexes SWI/SNF and RSC are recruited to MBF-regulated genes, having a clear impact in the activation of the G1-to-S transcriptional wave. Furthermore, we have created a short-lived fluorescent reporter to measure small and transient changes in the MBF activity in vivo by flow cytometry, to further identify new MBF regulators.
Al final de la fase G1, les cèl·lules han de decidir entre continuar proliferant o romandre en un estat de quiescència (G0). Aquest punt de decisió, conegut com “Start” en llevats o “Restriction Point” en metazous, compromet irreversiblement a les cèl·lules a completar el següent cicle cel·lular, i està principalment regulat per l’activitat CDK de G1 i per la inducció del programa transcripcional de G1/S. El complex MBF (homòleg funcional de pRB-E2F en metazous) es el factor de transcripció encarregat de la inducció de l’onada transcripcional de G1/S en el llevat de fissió Schizosaccharomyces pombe. Anteriorment, vam descriure com els repressors Nrm1 i Yox1 s’uneixen al complex MBF al final de la fase S per inhibir la seva activitat. Fins ara, els mecanismes implicats en l’activació de MBF a l’inici d’un cicle cel·lular no pertorbat s’han mantingut desconeguts. En aquest treball, hem vist que Nrm1 es el responsable de l’activació transcripcional depenent de MBF mitjançant un mecanisme de dos passos. La seva fosforilació per CDK1 i la seva posterior degradació per APCSte9 donen lloc a l’activació irreversible de MBF fins al final de la fase S. També hem estudiant el paper dels remodeladors de cromatina en el control del programa transcripcional de G1/S. En aquest sentit, hem trobat que els complexes remodeladors de la cromatina SWI/SNF i RSC són reclutats als gens regulats per MBF i tenen un clar impacte en l’activació transcripcional de G1/S. A més, hem creat un reporter fluorescent de vida curta per mesurar canvis petits i transitoris de l’activitat MBF in vivo mitjançant citometria de flux, per a poder identificar nous reguladors de MBF.

Les légumineuses sont connues pour leurs capacités à établir une relation symbiotique avec des bactéries du sol fixatrices de l'azote atmosphérique. Cette interaction aboutit à la formation d'un nouvel organe au niveau des racines, la nodosité, au sein duquel le symbiote convertit l'azote atmosphérique (N2) en ammoniac, qui peut être directement consommé par les plantes. A l’intérieur de cette nodosité, la concentration en oxygène (O2) est maintenue à un très faible niveau car la réaction de réduction du N2 par l’enzyme bactérienne nitrogénase est inhibée par des traces d’oxygène. Un mécanisme de perception directe de l'O2 impliquant des membres de la famille des facteurs de transcription « Ethylene Responsive Factors » (ERFs) du groupe VII a récemment été découvert chez Arabidopsis thaliana. Ces facteurs de transcription (FT) possèdent une extrémité N-terminale caractéristique avec un résidu de cystéine à la seconde position. Dans des conditions normales d'O2, les FT sont conduit à la dégradation suivant une voie spécifique du protéasome. En condition de stress hypoxique, les TFs sont stabilisés et peuvent activer l’expression des gènes de réponse à l'hypoxie. Il a été démontré que la présence d’O2 et de NO était nécessaire pour déstabiliser ces protéines, et qu'une réduction de la disponibilité de l'un ou l'autre des gaz est suffisante pour protéger le résidu cystéine N-terminale de l'oxydation. L’objectif de cette thèse a été d'étudier le rôle de la famille ERF-VII dans la perception et l'adaptation au manque d'O2 chez M. truncatula. Des travaux ont aussi été menés pour déterminer l’importance du NO dans le fonctionnement en microoxie de la nodosité. Quatre gènes codant pour des facteurs de transcription de la famille ERF-VII ont été identifiés dans le génome de M. truncatula. La caractérisation de cette famille au niveau transcriptionnel a révélé que seul MtERF-B2.2 était induit par le stress hypoxique et au cours du développement des nodosités. Les trois autres, MtERF-B1.1, MtERF-B1.11 et MtERF-B2.3, sont constitutivement exprimés dans les feuilles, les racines et les nodosités. Pour étudier la stabilité de la protéine MtERF-B2.1, l’orthologue de RAP2.12 principal ERF-VII décrit dans la perception de l’O2 chez Arabidopsis, en fonction de la disponibilité de O2/NO, nous avons réalisé une protéine de fusion entre l’extrémité N-terminale de notre protéine et la protéine rapporteur luciférase. Les résultats obtenus sur des protoplastes d'Arabidopsis montrent l’implication la partie N-terminale de MtERF-B2.1 dans la régulation de la stabilité de la protéine, mais en contradiction avec les résultats obtenus en plantes composites de M. truncatula. La fonction de MtERF-B2.1 et MtERF-B2.11 a également été étudiée dans le cadre de la réponse au stress hypoxique et au cours du processus de nodulation en utilisant une stratégie d'interférence ARN. Des racines transgéniques dérégulées sur l’expression de MtERF-B2.1 et MtERF-B2.11 ont montré un défaut d’activation de plusieurs gènes de réponses à l'hypoxie tels que l’alcool déshydrogénase (ADH1) ou la pyruvate décarboxylase (PDC1). Ces racines transgéniques ARNi-MtERF-B2.1/B2.11 sont également affectées dans l'interaction symbiotique avec une réduction significative de la capacité de nodulation et de l'activité de fixation de l'azote dans les nodules matures. En conclusion, ces travaux révèlent que le mécanisme de détection d'O2 est médié par les ERF-VII dans les nodosités de M. truncatula et que ce mécanisme, associé aux cibles moléculaires régulées en aval, participe au développement de cet organe et au maintien de la capacité de fixatrice de celui-ci. De plus, les résultats indiquent que MtERF-B2.1/B2.11 sont des régulateurs positifs du métabolisme anaérobie et que les gènes associés au cycle hémoglobine-NO sont susceptibles d'activer d'autres voies de génération d'ATP
Legume crops are known for their capacities to establish a symbiotic relationship with nitrogen fixing soil bacteria. This mutualism culminates in the formation of a new plant organ, the root nodule, in which the symbiont converts atmospheric nitrogen (N2) into ammonia, which can be directly consumed by plants. In nodules, bacterial nitrogenase enzyme is inhibited by traces of oxygen (O2) so different mechanisms maintain this organ at low O2 level. At the same time, nodules need to maintain a high ATP level to support the nitrogenase activity, which is highly energy demanding. Thus, a balance between a tight protection from O2 and an efficient energy production, referred as the “O2 paradox” of N2-fixing legume nodules, has to be reached. In Arabidopsis thaliana, a direct oxygen sensing mechanism has recently been discovered involving members of the ethylene responsive factors (ERFs) group VII. These transcription factors (TFs) possess a characteristic N-terminal amino acid with a cysteine residue at the second position that, under normal O2 conditions, leads to protein degradation following a specific pathway called the N-end rule pathway. Furthermore, it was shown that both O2 and nitric oxide (NO) are required to destabilize the ERFs VII and that a reduction in the availability of either gas is sufficient to stabilize these proteins. Therefore, the goal of this thesis was to investigated the role of ERF-VII family in O2 sensing and adaptation to hypoxia in M. truncatula, model plant for legumes, and to understand how NO interacts with O2 in hypoxic signalization in the microoxic environment that characterizes the nodule. We identified four genes belonging to the ERF-VII TF family in the M. truncatula genome, which present a strong similarity with ERF-VII of Arabidopsis. The characterization of this family at the transcriptional level revealed that only MtERF-B2.2 is up-regulated by hypoxia stress and during nodule development. The three others, MtERF-B1.1, MtERF-B1.11 and MtERF-B2.3 are found constitutively expressed in leaves, roots and nodules. To investigated the protein stability of MtERF-B2.1, the closest orthologous to AtRAP2.12 described as O2-sensors in Arabidopsis, in function of O2/NO availability, we realized a fusion protein with the luciferase reporter protein. Our results on Arabidopsis protoplasts indicated that the N-terminal part of MtERF-B2.1 drives its O2-dependent degradation by the N-end rule pathway. The function of MtERF-B2.1 and MtERF-B2.11 was also investigated both in response to hypoxia stress and during the nodulation process using an RNA interference strategy. Silencing of MtERFB2.1 and MtERF-2.11 showed a significant lower activation of several core hypoxia-responsive genes such as ADH1, PDC1, nsHb1 and AlaAT. These double knock-down transgenic roots were also affected in symbiotic interaction with a significant reduction of the nodulation capacity and nitrogen fixation activity in mature nodules. Overall, the results reveal that O2 sensing mechanism is mediated by ERF-VIIs in M. truncatula roots and nodules and that this mechanism, together with downstream targets, is involved in the organ development and ability to efficiently fix nitrogen. Furthermore, results indicated that MtERF-B2.1/B2.11 are positive regulator of the anaerobic metabolism and the Hb-NO cycle– related genes likely in order to activate alternative ATP generation pathways

Les légumineuses sont connues pour leurs capacités à établir une relation symbiotique avec des bactéries du sol fixatrices de l'azote atmosphérique. Cette interaction aboutit à la formation d'un nouvel organe au niveau des racines, la nodosité, au sein duquel le symbiote convertit l'azote atmosphérique (N2) en ammoniac, qui peut être directement consommé par les plantes. A l’intérieur de cette nodosité, la concentration en oxygène (O2) est maintenue à un très faible niveau car la réaction de réduction du N2 par l’enzyme bactérienne nitrogénase est inhibée par des traces d’oxygène. Un mécanisme de perception directe de l'O2 impliquant des membres de la famille des facteurs de transcription « Ethylene Responsive Factors » (ERFs) du groupe VII a récemment été découvert chez Arabidopsis thaliana. Ces facteurs de transcription (FT) possèdent une extrémité N-terminale caractéristique avec un résidu de cystéine à la seconde position. Dans des conditions normales d'O2, les FT sont conduit à la dégradation suivant une voie spécifique du protéasome. En condition de stress hypoxique, les TFs sont stabilisés et peuvent activer l’expression des gènes de réponse à l'hypoxie. Il a été démontré que la présence d’O2 et de NO était nécessaire pour déstabiliser ces protéines, et qu'une réduction de la disponibilité de l'un ou l'autre des gaz est suffisante pour protéger le résidu cystéine N-terminale de l'oxydation. L’objectif de cette thèse a été d'étudier le rôle de la famille ERF-VII dans la perception et l'adaptation au manque d'O2 chez M. truncatula. Des travaux ont aussi été menés pour déterminer l’importance du NO dans le fonctionnement en microoxie de la nodosité. Quatre gènes codant pour des facteurs de transcription de la famille ERF-VII ont été identifiés dans le génome de M. truncatula. La caractérisation de cette famille au niveau transcriptionnel a révélé que seul MtERF-B2.2 était induit par le stress hypoxique et au cours du développement des nodosités. Les trois autres, MtERF-B1.1, MtERF-B1.11 et MtERF-B2.3, sont constitutivement exprimés dans les feuilles, les racines et les nodosités. Pour étudier la stabilité de la protéine MtERF-B2.1, l’orthologue de RAP2.12 principal ERF-VII décrit dans la perception de l’O2 chez Arabidopsis, en fonction de la disponibilité de O2/NO, nous avons réalisé une protéine de fusion entre l’extrémité N-terminale de notre protéine et la protéine rapporteur luciférase. Les résultats obtenus sur des protoplastes d'Arabidopsis montrent l’implication la partie N-terminale de MtERF-B2.1 dans la régulation de la stabilité de la protéine, mais en contradiction avec les résultats obtenus en plantes composites de M. truncatula. La fonction de MtERF-B2.1 et MtERF-B2.11 a également été étudiée dans le cadre de la réponse au stress hypoxique et au cours du processus de nodulation en utilisant une stratégie d'interférence ARN. Des racines transgéniques dérégulées sur l’expression de MtERF-B2.1 et MtERF-B2.11 ont montré un défaut d’activation de plusieurs gènes de réponses à l'hypoxie tels que l’alcool déshydrogénase (ADH1) ou la pyruvate décarboxylase (PDC1). Ces racines transgéniques ARNi-MtERF-B2.1/B2.11 sont également affectées dans l'interaction symbiotique avec une réduction significative de la capacité de nodulation et de l'activité de fixation de l'azote dans les nodules matures. En conclusion, ces travaux révèlent que le mécanisme de détection d'O2 est médié par les ERF-VII dans les nodosités de M. truncatula et que ce mécanisme, associé aux cibles moléculaires régulées en aval, participe au développement de cet organe et au maintien de la capacité de fixatrice de celui-ci. De plus, les résultats indiquent que MtERF-B2.1/B2.11 sont des régulateurs positifs du métabolisme anaérobie et que les gènes associés au cycle hémoglobine-NO sont susceptibles d'activer d'autres voies de génération d'ATP
Legume crops are known for their capacities to establish a symbiotic relationship with nitrogen fixing soil bacteria. This mutualism culminates in the formation of a new plant organ, the root nodule, in which the symbiont converts atmospheric nitrogen (N2) into ammonia, which can be directly consumed by plants. In nodules, bacterial nitrogenase enzyme is inhibited by traces of oxygen (O2) so different mechanisms maintain this organ at low O2 level. At the same time, nodules need to maintain a high ATP level to support the nitrogenase activity, which is highly energy demanding. Thus, a balance between a tight protection from O2 and an efficient energy production, referred as the “O2 paradox” of N2-fixing legume nodules, has to be reached. In Arabidopsis thaliana, a direct oxygen sensing mechanism has recently been discovered involving members of the ethylene responsive factors (ERFs) group VII. These transcription factors (TFs) possess a characteristic N-terminal amino acid with a cysteine residue at the second position that, under normal O2 conditions, leads to protein degradation following a specific pathway called the N-end rule pathway. Furthermore, it was shown that both O2 and nitric oxide (NO) are required to destabilize the ERFs VII and that a reduction in the availability of either gas is sufficient to stabilize these proteins. Therefore, the goal of this thesis was to investigated the role of ERF-VII family in O2 sensing and adaptation to hypoxia in M. truncatula, model plant for legumes, and to understand how NO interacts with O2 in hypoxic signalization in the microoxic environment that characterizes the nodule. We identified four genes belonging to the ERF-VII TF family in the M. truncatula genome, which present a strong similarity with ERF-VII of Arabidopsis. The characterization of this family at the transcriptional level revealed that only MtERF-B2.2 is up-regulated by hypoxia stress and during nodule development. The three others, MtERF-B1.1, MtERF-B1.11 and MtERF-B2.3 are found constitutively expressed in leaves, roots and nodules. To investigated the protein stability of MtERF-B2.1, the closest orthologous to AtRAP2.12 described as O2-sensors in Arabidopsis, in function of O2/NO availability, we realized a fusion protein with the luciferase reporter protein. Our results on Arabidopsis protoplasts indicated that the N-terminal part of MtERF-B2.1 drives its O2-dependent degradation by the N-end rule pathway. The function of MtERF-B2.1 and MtERF-B2.11 was also investigated both in response to hypoxia stress and during the nodulation process using an RNA interference strategy. Silencing of MtERFB2.1 and MtERF-2.11 showed a significant lower activation of several core hypoxia-responsive genes such as ADH1, PDC1, nsHb1 and AlaAT. These double knock-down transgenic roots were also affected in symbiotic interaction with a significant reduction of the nodulation capacity and nitrogen fixation activity in mature nodules. Overall, the results reveal that O2 sensing mechanism is mediated by ERF-VIIs in M. truncatula roots and nodules and that this mechanism, together with downstream targets, is involved in the organ development and ability to efficiently fix nitrogen. Furthermore, results indicated that MtERF-B2.1/B2.11 are positive regulator of the anaerobic metabolism and the Hb-NO cycle– related genes likely in order to activate alternative ATP generation pathways

Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS). In eukaryotes, the N-end rule pathway is a part of the UPS. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. E3 Ub ligases of the N-end rule pathway are called N-recognins. They bind to primary destabilizing N-terminal residues of N-end rule substrates. The N-end rule pathway comprises two major branches, the Arg/N-end rule pathway and the Ac/N-end rule pathway.

The Arg/N-end rule branch involves the N-terminal arginylation of protein substrates and also the targeting of specific unmodified N-terminal residues by E3 N-recognins. The S. cerevisiae Arg/N-end rule pathway contains a single N-recognin, Ubr1. The Ub-fusion degradation (UFD) pathway is also a part of the UPS. This pathway recognizes a "nonremovable" N-terminal Ub moiety of a Ub fusion as a primary degron. My collaborator, Cheol-Sang Hwang, and I demonstrated that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. We showed that the Ubr1-Ufd4 complex targets the S. cerevisiae Mgt1 DNA repair enzyme through a degron near its N-terminus, in addition to mediating the Arg/N-end rule pathway and a part of the UFD pathway as well. We also further characterized the physical interaction between Ubr1 and Ufd4.

I also report the discovery of the other branch of the N-end rule pathway, the Ac/N-end rule pathway, which recognizes N-terminally acetylated residues as N-degrons, termed Ac/N-degrons. We showed that Ac/N-degrons are recognized by the Doa10 E3 Ub ligase and apparently by other E3s as well. Given the prevalence of Ac/N-degrons, as nearly 90% of human proteins are Nt-acetylated, we also demonstrated the physiological role of Ac/N-degrons in protein quality, including the regulation of input stoichiometries of subunits in oligomeric proteins.

Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a minute or so. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS), in conjunction with molecular chaperones, autophagy, and lysosomal proteolysis. The N-end rule pathway, the first specific pathway of UPS to be discovered, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Physiological functions of the N-end rule pathway are strikingly broad and continue to be discovered. In bacteria and in eukaryotic organelles mitochondria and chloroplasts all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. What is the main biological function of this metabolically costly, transient, and not strictly essential modification of N-terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the formyl groups of N-terminal Met in Nt formylated bacterial proteins may signify a proteolytic role of Nt-formylation. My colleagues and I addressed this hypothesis experimentally, as described in Chapter 3 of this thesis.

Among the multitude of biological functions of the mammalian Arg/N-end rule pathway are its roles in the brain, including the regulation of synaptic transmission and the regulation of brain’s G-protein circuits. This regulation is mediated, in part, by the its Ate1-mediated arginylation branch of the Arg/N-end rule pathway. One role of the Ate1 arginyltransferase (R-transferase) is to mediate the conditional degradation of three G-protein down-regulators, Rgs4, Rgs5, and Rgs16. Ate1^-/- mice, which lack the Ate1 R-transferase, exhibit a variety of abnormal phenotypes. Chapter 4 describes our studies of neurological abnormalities in Ate1^-/- mice (and also in mice that express Ate1 conditionally, upon the addition of doxycycline), with an emphasis on the propensity of these mice to epileptic seizures.

The molecular mechanisms of apoptosis are evolutionarily-conserved with caspases being the chief executioners of this process. Though key regulators of apoptosis, including caspases, inhibitor of apoptosis (IAP) proteins, and IAP antagonists exist in both mammals and flies, there are reportedly mechanistic differences in the way the apoptotic process is executed. One of the differences pertains to the importance of mitochondrial permeabilization for caspase activation. Herein, we demonstrate that dOmi, a Drosophila homologue of the serine protease Omi/HtrA2, is a developmentallyregulated mitochondrial intermembrane space protein that undergoes processive cleavage in situ to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the pro-apoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. Thus, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis, but also results in the release of a bona fide pro-apoptotic protein. DIAP1, in addition to being regulated by dOmi, is also regulated by RINGdependent autoubiquitination and by the N-end rule degradation (NERD) pathway. Despite decreasing the cellular levels of DIAP1, the NERD pathway enhances its antiapoptotic function through an unknown mechanism(s). Herein, we show for the first time that the NERD pathway facilitates trans-ubiquitination and degradation of IAP antagonist bound to DIAP1. Indeed, Grim is trans-ubiquitinated in an Ubr1-dependent manner and requires its interaction specifically with the BIR1 domain of DIAP1. These results demonstrate that similar to RING domain-dependent ubiquitination, the NERD pathway regulates not only the levels of DIAP1, but also of the levels of IAP antagonists bound to it.

Regulation of the in vivo half-lives of intracellular proteins is an important cellular process. Many intracellular proteins are short-lived, owing to their regulated and processive degradation by the Ubiquitin (Ub)-Proteasome System (UPS). In eukaryotes, the N-end rule pathway is one specific pathway within the UPS. The N-end rule pathway relates the identity of the N-terminal residue of a protein, or a protein fragment, to its in vivo half-life. Substrates of the N-end rule pathway are recognized by the presence of degradation signals, termed N-degrons. Recognition components of the N-end rule pathway are E3 ubiquitin ligases that are capable of binding to N-degrons. The N-end rule pathway consists of two distinct branches: the Arg/N-end rule pathway and the Ac/N-end rule pathway.

In the present studies, we demonstrate a complementary targeting of the rat serotonin N-acetyltransferase (AANAT), an important mediator of circadian physiology, by both branches of the N-end rule pathway. The co-targeting results from incomplete N-terminal (Nt-) acetylation of a Met-Ф motif at the N-terminus of AANAT in vivo. In the same study, we demonstrate that human AANAT is substantially longer-lived than its rat counterpart, owing to differences in their N-terminal sequences. This molecular genetic investigation of the degradation of a physiological N-end rule substrate followed an analogous earlier study, in which we reported that a clinically-relevant (blood pressure-increasing) Q2L mutant of human RGS2 (termed ML-RGS2), a regulator of G proteins, could likewise be co-targeted by both branches of the N-end rule pathway. Together, AANAT and RGS2 are the first identified and characterized physiological substrates of the Ac/N-end rule pathway in mammals.

We also report on the development and use of in vitro N-terminal arginylation (Nt-arginylation) assays using CelluSpots peptide arrays, in conjunction with pulse-chase assays in rabbit reticulocyte extract, for the systematic investigation of the effects of N-terminus-proximal sequence context on the Nt-arginylation activity of the Ate1 arginyltransferase, a component of the Arg/N-end rule pathway. These experiments help to define the sequence requirements for efficient Nt-arginylation by Ate1. Finally, we demonstrate that Rec8, a subunit of the cohesin protein complex during meiosis, is a natural short-lived substrate of the mammalian Arg/N-end rule pathway.

Les fichiers accompagnant le document sont en format Microsoft Excel 2010.
Les modèles classiques de signalisation cellulaire eucaryotes sont généralement organisés en voies linéaires et hiérarchiques, impliquant un ensemble de facteurs restreint. Ces facteurs forment un circuit isolé qui transmet une information externe vers sa destination, d’où une réponse cellulaire sera alors engendrée. Or, ces modèles sont justement le fruit d’approches expérimentales réductionnistes qui ne permettent pas d’intégrer aisément la contribution de facteurs multiples, ni de faire une évaluation quantitative de l’apport des composantes du système. Le développement de techniques d’investigation plus holistiques, telles la génomique fonctionnelle et la protéomique, permettent d’examiner de manière systématique et quantitative l’apport d’ensembles larges de facteurs et de les mettre en relation avec d’autres systèmes cellulaires. Il y aurait donc lieu de réévaluer le modèle de voie de signalisation linéaire au profit d’un modèle de réseau de signalisation multiparamétrique, comportant plusieurs branches d’entrée et sortie de signal interagissant avec d’autres systèmes cellulaires. Cet ouvrage porte sur la voie RAS/MAPK, l’un des principaux axes de signalisation associé à la prolifération et la différenciation cellulaires. Le sujet y est d’abord abordé sous l’angle d’une perspective historique, en mettant l’emphase sur les contributions des études de génétique classique chez les organismes modèles D. melanogaster et C. elegans. Il fait ensuite état du développement du criblage par ARNi pan-génomique dans ces deux modèles en le comparant aux approches de criblage génétique classique. Le corps de l’ouvrage décrit ensuite les résultats expérimentaux d’une campagne de criblage par ARNi visant à dresser une carte globale des régulateurs de la voie chez la drosophile. Trois groupes de régulateurs identifiés dans ce crible ont été caractérisés de manière plus détaillée. Dans un premier article, nous démontrons que les composantes du complexe EJC ont un impact sur l’épissage de mapk; une découverte doublement intéressante puisque l’EJC était jusqu’alors associé qu’à la régulation post-épissage des ARNm. Une seconde publication fait état de l’ensemble des résultats du crible ARNi, mettant l’emphase sur un ensemble de facteurs d’épissage qui modulent également mapk. Nous y montrons que l’impact de ces facteurs sur l’épissage alternatif est différent de celui de l’EJC, suggérant ainsi deux modes de régulation distincts. Finalement, dans un troisième manuscrit, nous nous attardons au rôle d’Usp47, une déubiquitinase qui, contrairement aux autres facteurs identifiés dans le crible, régule l’expression de MAPK de manière post-traductionnelle. Nous y détaillons une stratégie de criblage d’interaction génétique par ARNi visant à identifier des facteurs reliés fonctionnellement à Usp47. Ce second crible a permis l’identification de trois facteurs reliés au « N-end rule », un mécanisme de dégradation des protéines caractérisé par la reconnaissance des résidus N-terminaux de protéines ou peptides. Il existait jusqu’alors très peu de données quant à la régulation de l’expression des composantes de la voie MAPK, ce qui rend la description d’un large réseau de régulateurs agissant sur l’expression de MAPK d’autant plus insoupçonnée. L’absence d’un réseau équivalent rattaché aux autres composantes de la voie laisse supposer que MAPK serait un noeud servant de point d’entrée à ce type de régulation dans le système RAS/MAPK. De plus, nos travaux témoignent de la capacité de la génomique fonctionnelle à mettre en relation différents systèmes cellulaires de manière plus globale et à quantifier les liens établis entre eux.
The classical model of eukaryotic cellular signalling generally involves hierarchically organized linear pathways involving a restricted set of elements. These generally function together as an insulated circuit, transmitting information from the outside to the intracellular compartment involved in eliciting a response. These models, often the fruit of reductionist experimental approaches, do not allow for the integration of multiple inputs nor for a gradation of responses. The recent emergence of more holistic investigation techniques has brought about the re-evaluation of these classical models in favor of multiparametric signalling networks. This thesis focuses on the RAS/MAPK pathway, one of the cell’s main proliferation and differentiation signalling conduits, beginning with a historical perspective covering the contributions of model organism genetics to the current pathway model. This provides context for the description of a whole-genome RNAi screen experiment that we carried out to obtain a global view of regulators in Drosophila. Three groups of factors emerging from this screen were then examined in more detail. A first article shows that the exon junction complex (EJC) plays a role in mapk alternative splicing, an observation that is unexpected given that this complex was not previously known to act on splicing. A second paper details the genome wide screening campaign and focuses on a large set of splicing factors that also regulate mapk, albeit in a distinct manner than the EJC’s. Finally, a manuscript in a third segment examines Usp47 function and finds it to control MAPK levels post-translationally. An RNAi-based genetic interaction screen is then used to identify factors functionally related to Usp47 capable of counteracting its impact on MAPK levels. Three such factors identified through this technique are linked to the N-end rule protein degradation pathway. Regulation of core pathway component expression is a poorly described process, which makes the identification of a large set of factors regulating MAPK expression all the more unusual. Moreover, the absence of such regulation linked to other pathway components suggests that MAPK may act as a node incorporating inputs of this type into RAS/MAPK signaling dynamics.

The perpetration of theft by employees in a nursing home is the subject of this study. The investigation focuses mainly on the theft of clients’/residents’ possessions by those people who are supposed to care for them. Previous research indicated a relationship between ethical climate and the behavioural outputs of employees in an organization. This research, which is mainly qualitative in nature, endeavours to study the assumed relation between ethical climate and theft by employees in a specific location, namely at Vergenoeg vir Seniors, an old age home in Pretoria. With the aid of a standardized questionnaire the nature of the ethical climate in the home is clarified. Thereafter the results are evaluated and investigated by utilizing (i) a focus group interview and (ii) six in- depth individual interviews. Certain substantial and formal tendencies which present themselves, are discussed fully. For example, it is detected that “rules and regulations” play a paramount role as regards ethical climate, whereas “friendship and team work” rate very low. Ethical climate however does affect employee theft. Therefore it is recommended that this aspect should be managed with care and should also enhance the climate type of “social responsibility”. Other recommendations involve the need for team building and proper job descriptions, as well as dealing with the perception of most older people that theft practices differ according to race. Finally, some suggestions for further study are made.
Dissertation (MCom (Human Resources Management))--University of Pretoria, 2005.
Human Resource Management
unrestricted