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

No description available.

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

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

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

Câncer se destaca no contexto de patologias por ser uma das doenças que mais acometem mortes por ano, sendo caracterizada como um conjunto de doenças multifatoriais que tem em comum o crescimento desordenado de células que invadem tecidos e órgãos, podendo espalhar-se para outras regiões do corpo, dando origem às metástases. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem carácter invasivo e migratório, além de se tornarem mais resistentes às drogas. Durante este processo, ocorrem inúmeras alterações celulares que modificam a estabilidade proteica e/ou promovem sua translocação subcelular, o transporte de proteínas para a membrana, alterações no citoesqueleto e incluindo o envio de proteínas para degradação pelo proteassoma. A desregulação de fatores de transcrição e modificação pós traducional de proteínas são fatores que podem levar à EMT. Após a eficiente indução da EMT in vitro utilizando o inibidor de histonas deacetilase (SAHA) em células de adenocarcinoma de mama MCF-7, foram realizadas análises proteômicas envolvimento os inibidores relacionados ao sistema ubiquitina proteassoma, MG132 e P5091. A modulação por inibição de USP7 resultou em variação da expressão de diversas proteínas biomarcadoras da EMT (SNAIL, ?-Catenina, CDK1) e proteínas envolvidas no ciclo celular (P53 e CDK1). O estudo proteômico permitiu a correlação do processo da EMT por SAHA com as vias de modificações pós traducionais relacionadas ao sistema ubiquitina proteassoma, e ainda propõe USP7 como alvo de estudos detalhados para EMT com potencial proposta terapêutica / Cancer stands out in the context of pathologies because it is one of the diseases that most affect deaths per year, being characterized as a set of multifactorial diseases that has in common the disordered growth of cells that invade tissues and organs, being able to spread to other regions of the body, giving rise to metastases. An important step in the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of the epithelial phenotype and acquisition of the mesenchymal phenotype by the tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. During this process, numerous cellular alterations occur that modify the protein stability and/or promote its subcellular translocation, the transport of proteins to the membrane, changes in the cytoskeleton and including the sending of proteins for degradation by the proteasome. Deregulation of transcription factors and posttranslational modification of proteins are factors that can lead to EMT. After an efficient induction of EMT using the histone deacetylase inhibitor (SAHA) in MCF-7 breast adenocarcinoma cells, proteomic analyzes were performed involving inhibitors related to the ubiquitin proteasome system, MG132 and P5091. Modulation by inhibition of USP7 resulted in varying expression of various EMT biomarker proteins (SNAIL, ?-Catenina, CDK1) and cell cycle (P53 e CDK1). The proteomic study allowed the correlation of the SAHA EMT process with the posttranslational modifications pathways related to the ubiquitin proteasome system and also proposes USP7 as the target of detailed studies for EMT with potential therapeutic proposal

A esquistossomose mansônica é a segunda maior endemia parasitária do mundo em termos de extensão das áreas endêmicas e do número de pessoas infectadas com 200 milhões de pessoas acometidas. Esta doença é causada pelo parasito trematódeo Schistosoma mansoni, o qual apresenta adequados mecanismos de resposta ao estresse envolvendo a regulação da expressão gênica e proteica, reparo ou substituição de moléculas danificadas, recuperação do balanço redox, controle do ciclo celular e apoptose. O sistema ubiquitina- proteassoma é importante para manter a homeostase proteica durante o estresse celular. Inibidores do proteassoma podem interferir em processos como crescimento, progressão do ciclo celular e replicação, e os seus efeitos vem sendo caracterizados em muitos parasitos. Nosso laboratório demonstrou que MG132 reduz o número de esquistossômulos, a carga parasitária e a ovoposição em camundongos infectados com S. mansoni. Neste trabalho, são descritos os efeitos in vitro do estresse oxidativo, choque térmico e estresse químico em vermes adultos de S. mansoni. Observou-se alteração no perfil de expressão proteica durante estresse oxidativo e térmico, sendo identificadas dezoito proteínas upreguladas nestas condições. Estas proteínas estão envolvidas em muitas vias intracelulares como dobramento de proteínas, proteólise, ligação a íons cálcio, regulação de proteínas e resposta a estresse. Além disso, o estresse oxidativo gerou mudanças em vermes adultos de S. mansoni em processos como produção de ovos, motilidade, morfologia do tegumento, viabilidade e pareamento dos vermes. O estresse químico induzido com Curcumina, IBMX e MG132 aumentou a produção de ROS intracelular e alterou o perfil de expressão de enzimas antioxidantes em S. mansoni. As enzimas SmGPx1 e SmPGx2 tiveram a expressão aumentada no estresse com Curcumina e IBMX, enquanto que SmSOD e SmTGR foram induzidas no estresse com Curcumina. As enzimas do proteassoma SmHul5 e SmUbp6 tiveram a expressão modulada durante o estresse oxidativo, choque térmico e estresse químico. Em adição, a análise de expressão no ciclo de vida de S. mansoni revelou que estes genes apresentam um nível alto de expressão em esporocistos, esquistossômulos e miracídios. Estes resultados sugerem que estas proteínas acessórias do proteassoma participam da resposta ao estresse e desenvolvimento do parasito. O nível de expressão de SmHul5 e SmUbp6 foi cerca 9 e 16 vezes menor em relação ao controle no estresse químico induzido com IBMX, respectivamente, sugerindo a desmontagem do proteassoma. Por outro lado, Curcumina, MG132, estresse oxidativo e choque térmico aumentaram o nível de expressão de SmHul5 e SmUbp6. Além disso, o nível de expressão da proteína de maturação do proteassoma (SmPOMP) aumentou no estresse com Curcumina, MG132 e estresse oxidativo, sugerindo a síntese de novas populações de proteassoma. Em relação ao estresse oxidativo, nós demonstramos o aumento no nível proteico de proteassoma 20S e da subunidade alfa-3 do proteassoma sugerindo que em S. mansoni as proteínas oxidadas são degradadas pelo proteassoma 20S. Além do mais, nós observamos que vermes adultos de S. mansoni parecem utilizar mecanismos de resposta similares para diferentes estresses. Nossos resultados demonstraram que o estresse oxidativo, choque térmico e estresse químico modificam o perfil de expressão de genes relacionados ao sistema ubiquitina-proteassoma e sugerem que o proteassoma é importante para as respostas celulares ao estresse neste parasito. / Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma) and affecting 200 million people worldwide. This disease continues to rank, following malaria, at the second position of the world\'s parasitic diseases in terms of the extent of endemic areas and the number of infected people. There are different types of stress and the organisms have many mechanisms to respond to these stressor agents. The responses involve the regulation of gene and protein expression and consist in events such as repair or substitution of damaged molecules, recovery of redox balance, cell cycle control and apoptosis. The proteasomal system is important to support the protein homeostasis during the cellular stress. Effect of proteasome inhibitors has been described in many protozoans, either inhibiting growth or cell cycle progression, or blocking replication. Our laboratory\'s results have shown that MG132 reduces the number of lung stage schistosomula, the worm burden and consequently decreases oviposition in S. mansoni-infected mice. Here, we describe the in vitro effects of oxidative stress, heat shock and chemical stress in S. mansoni adult worms. We report that the oxidative stress and heat shock cause drastic changes in the protein profile of S. mansoni adult worms, and we identified a total of eighteen upregulated proteins in these conditions. These proteins are involved with many intracellular pathways as protein folding, proteolysis, calcium ion binding, regulator proteins and stress response. In addition, oxidative stress induced with H2O2 generated significative changes in the adult worms concerning process such as egg production, motor activity, tegument morphology, viability and pairing of worms. Chemical stress induced with Curcumin, IBMX and MG132 increases ROS production and changes the gene expression profile of antioxidant enzymes of S. mansoni adult worms. The enzymes SmGPx1 and SmGPx2 were upregulated in Curcumin and IBMXinduced chemical stress, and both SmSOD and SmTGR were upregulated- Curcumin. The proteasomal enzymes SmHul5 and SmUbp6 had their gene expression modified during oxidative stress, heat shock and chemical stress. Besides of, expression analyses in the S. mansoni life cycle indicate that genes are different express in sporocyst, schistosomula and miracidia. These results suggest these accessory proteins proteasome participates of stress response and parasite development. The expression level of SmHul5 and SmUbp6 were 16 and 9 times less than the control in chemical stress induced by IBMX, and we suggest that these results are due to the proteasome disassembling. On the other hand, Curcumin, MG132, oxidative stress e heat shock increases the expression of SmHul5 and SmUbp6. Furthermore, the expression level of maturation proteasome protein (SmPOMP) increases in stress induced by Curcumin, MG132 and oxidative stress suggesting new proteasome synthesis. In addition, we demonstrate increase the both 20S level and alpha-3 subunit proteasome in the oxidative stress, suggesting that in S. mansoni oxidized protein formed due to oxidative damage are degrade by proteasome 20S. We observed that S. mansoni adult worms utilize similar mechanisms to respond different stresses. Ours results demonstrate that oxidative stress, heat shock and chemical stress modified the expression profile of genes related with the ubiquitinproteasome system and suggest that the proteasome is important to responses the cellular stresses in the parasite.

The work in this thesis is divided into two parts; the first is the synthesis of a ‘switch-on’ fluorophore to measure cell viability, and the second is the development of a fluorescent detection method for protein−peptide affinity assays applied in the identification of protein-protein inhibitors. Tetrazolium salts are often used in cytotoxicity assays as indicators of cell viability as they are reduced to deeply coloured formazans exclusively in healthy cells. However, measuring the absorbance of the formazan is prone to bias from other coloured species in the cell media, requires solubilisation and can be difficult to quantify. A preferable method of detection is direct fluorescence as it is easily quantified, more sensitive and would ideally remove the need to solubilise the insoluble dye. The aim of this project was to synthesise a tetrazolium salt that could be reduced to a soluble fluorescent formazan in healthy cells as an indicator of cell viability. A number of fluorescent formazans were synthesised by incorporation of a fluorophore. The corresponding tetrazolium salts were non-fluorescent and could be reduced to the formazan in vitro. Several formazans were synthesised to attempt to increase the emission wavelength and intensity to overcome cellular autofluorescence. Protein-protein interactions have been implicated in the pathogenesis of many human diseases but until recently were considered undruggable. However, peptides have emerged as ideal compounds for targeting the large and relatively featureless protein interfaces. Work focussed on the discovery of peptide inhibitors for the E3 ubiquitin ligase stationary-phase kinase associated protein (Skp2). Potential peptide inhibitors were identified using CelluSpot synthesis and array technology to screen peptide libraries. Qualitative analysis of the protein affinity assay results by enhanced chemiluminescent detection was found to be misleading, and so a quantifiable and more sensitive fluorescent detection method was developed.

The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.

Brain stem cardiovascular regulatory dysfunction during brain stem death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain stem death ensues. At the same time, the ubiquitin-proteasome system (UPS) is involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain stem death by engaging in both synthesis and degradation of NOS II in RVLM. In a clinically relevant experimental model of brain stem death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling or UCH-L1 potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-£eB or binding between NF-£eB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated I£eB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-£eB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated. We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain stem death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.