Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20), which showed a halving in the risk of disease progression in a randomised phase II study. However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, I studied the effect of macrophages, abundant in mesothelioma, on the tumour cytotoxicity of ADI-PEG20. A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified and validated in ADI-PEG20-treated ASS1 negative MPM cell lines. In vivo induction of pro-inflammatory cytokines was also seen in ADI-PEG20-treated patient plasma. Notably, in vitro co-culture experiments demonstrated a significant increase in ASS1 negative MPM cell viability upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumour cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumour cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumour volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 alone. Collectively, the data indicate that as a result of metabolic 'cross-talk' between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. My studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references (leaves 179-187). Also available online.

The discovery of nitric oxide (NO) as the endothelial-derived relaxing factor has led to significant research on NO and the proteins involved in its function, generation, location and regulation. Synthesis of NO by blood vessel endothelial cells results from the enzymatic oxidation of arginine by endothelial nitric oxide synthase (eNOS) resulting in the formation of equimolar amounts of NO and citrulline. Citrulline is sequentially recycled to arginine by successive reactions involving the enzymes argininosuccinate synthase (AS) and argininosuccinate lyase (AL), respectively. eNOS activity has been shown to be regulated by post-translational modifications including dynamic phosphorylation on multiple serine/threonine and tyrosine residues and dynamic O-linked beta-N-acetylglucosamine (O-GlcNAc) modifications on serine/threonine residues.Previous studies showed that even though intracellular endothelial arginine levels range from 0.1 to 0.8 mM and the Km of eNOS for arginine is 3 uM, the addition of exogenous arginine caused an increase in NO production. To explain this "arginine paradox" we hypothesize that there is a separate and distinct cellular source of arginine substrate directed to NO production and that this source is maintained through the regeneration of arginine via a citrulline-NO cycle. The presented research has provided the following evidence in support of this hypothesis: Citrulline stimulates NO production in an arginine-rich medium, without an increase in intracellular arginine. The enzymes of the citrulline-NO cycle, eNOS, AS and AL, co-fractionate with caveolin-1 in an endothelial cell caveolar membrane fraction. In vitro interaction assays demonstrate protein-protein interactions between fusion tagged AS or AL with eNOS or caveolin-1. Simultaneous monitoring of apparent citrulline and NO production demonstrates an efficient and essential coupling of the reactions of the citrulline-NO cycle. Glucosamine treatment of endothelial cells results in increased NO production in the basal state and decreased NO production in the stimulated state.Our findings demonstrate the enzymes of the citrulline-NO cycle, eNOS, AS and AL, are functionally associated, the reactions are efficiently coupled and enzyme activities are changed by post-translational modifications based on nutrient levels. These alterations ensure a constant and distinct source of arginine which is available for NO production to ensure vascular health.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 130-144). Also available online.

Abstract Enzymes catalyze chemical changes in biological systems. Therefore, to understand the chemistry of living systems, it is important to understand the enzyme structure and the chemistry of the enzyme's functional groups which are involved in catalysis. In this study, structure and function relationships of two enzymes, (1) α-methylacyl-CoA racemase from Mycobacterium tuberculosis (MCR) and (2) argininosuccinate lyase from Escherichia coli (eASL) have been studied using X-ray crystallography. The main focus of this study has been understanding the structure-function relationship of MCR. The eASL has been crystallized from a highly concentrated sample of purified recombinant α-methylacyl-CoA racemase in which it occurred as a minor impurity. The structure of eASL has been solved using molecular replacement at 2.44 Å resolution. The enzyme is a tetramer, but in this crystal form there is a dimer in the asymmetric unit. Each active site is constructed from loops of three different subunits. One of these catalytic loops, near residue Ser277 and Ser278, has been disordered in the previous structures of active lyases, but is very well ordered in this structure in one of the subunits due to the presence of two phosphate ions in the respective active site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex and therefore this structure has provided new information on the reaction mechanism of this class of enzymes. α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of α-methyl-branched CoA esters. An Amacr homologue from the eubacteria Mycobacterium tuberculosis, referred to as MCR, was taken as a model protein. MCR was purified, crystallized and the structure of unliganded protein was determined at 1.8 Å resolution using the MIRAS procedure. The structure shows that the enzyme is an interlocked dimer. To understand the reaction mechanism and the mode of substrate binding, several crystallographic binding studies were done using both wild type MCR and mutant H126A MCR crystals. In particular, the structures of the wild type MCR-complexes with (R, S)-ibuprofenoyl-CoA (1.85 Å), (R)-2-methylmyristoyl-CoA (1.6 Å) and (S)-2-methylmyristoyl-CoA (1.7 Å) were important in this respect. These crystal structures show that Asp156 and His126 are the two catalytic residues which are involved in proton donation and abstraction, respectively; when the (S)-enantiomeric substrate is bound in the active site and vice versa when the (R)-enantiomeric substrate is bound. The tight geometry of the active site also shows that His126 and Asp156 are involved in stabilizing the transition state. These crystal structures show that in the active site of MCR, there is one binding pocket for the CoA part and there are two different binding pockets (R-pocket and S-pocket) connected by a hydrophobic methionine rich surface for binding the fatty acyl part of the substrate. After substrate binding, proton abstraction takes place which produces a planar intermediate. Then, donation of a proton to the other side of the planar intermediate changes the configuration at the chiral center. During the stereochemical interconversion of the two enantiomers, the acyl group moves between R-pocket and S-pocket by sliding over the hydrophobic surface connecting these two pockets.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20661号 / 医博第4271号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 柳田 素子, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays a role, and how phosphorylation affects AS function in endothelial cells. We developed a phospho-specific antibody directed against pS328 AS and assayed for increases or decreases in phosphorylation relative to physiological factors. We found that AS phosphorylation at S328 occurred when endothelial cells were stimulated with physiological factors that stimulate nitric oxide production through calcium-dependent stimulation of eNOS. Furthermore, by utilizing kinase inhibitors and kinase knockdown experiments, we showed that phosphorylation at S328 significantly decreased when PKCá was knocked down, suggesting that S328 phosphorylation of AS is involved in PKCá signaling. In addition, by confocal microscopy, immunoprecipitation, and membrane fractionation, we showed that phosphorylation at S328 of AS promotes its co-localization with eNOS in the perinuclear region. These findings describe a novel pathway involving AS regulation of nitric oxide production, and may serve as a novel drug target in the restoration of vascular nitric oxide homeostasis.

O BPP-10c é um decapeptídeo bioativo, rico em resíduos de prolina e é expresso em uma proteína precursora no cérebro e na glândula de veneno da Bothrops jararaca. Recentemente demonstramos que o BPP-10c tem um potente e sustentado efeito anti-hipertensivo em ratos espontaneamente hipertensos (SHR), sem, no entanto, causar qualquer efeito em ratos normotensos, por um mecanismo farmacológico independente da inibição da enzima conversora de angiotensina (ECA), levando à hipótese de que outro mecanismo poderia estar envolvido na atividade do peptídeo. Neste trabalho, usamos cromatografia de afinidade para isolar e identificar as proteínas renais com afinidade pelo BPP-10c e demonstramos que a argininosuccinato sintase (AsS) é a principal proteína a se ligar ao peptídeo. Além disso, mostramos que essa interação promove um aumento na atividade catalítica da enzima, de forma dose-dependente. A AsS é reconhecida como uma peça chave na regulação do ciclo da citrulina-óxido nítrico (NO), e sua ação é passo limitante na síntese de NO. A interação funcional do BPP-10c com a AsS foi evidenciada pelos seguintes efeitos promovidos pelo peptídeo: i) estimulação da produção de NO por células HUVEC e da produção de arginina por células HEK 293, ii) aumento da concentração plasmática de arginina em SHR. Corroborando esses achados, mostramos a reversão dos efeitos do peptídeo, inclusive sobre a pressão arterial em SHR, quando o MDLA, um inibidor específico da AsS, foi co-administrado. Em conjunto, os resultados apresentados neste trabalho sugerem que a AsS é fundamental para o efeito anti-hipertensivo do BPP-10c. Tais resultados nos levaram a propor a AsS como um novo alvo terapêutico, e o BPP-10c como molécula-líder para a geração de medicamentos para tratamento de doenças relacionadas à hipertensão arterial / BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological mechanism independent of angiotensin converting enzyme inhibition; therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-nitric oxide (NO) cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of BPP-10c and AsS was evidenced by the following effects promoted by the peptide: i) increase of NO production in human umbilical vein endothelial cell culture, and of arginine in human embryonic kidney cells; ii) increase of arginine plasma concentration in SHR. Moreover, MDLA, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of BPP-10c in SHR. These results led us to suggest AsS as a new therapeutically useful target for the development of activators, such as BPP- 10c, useful to treat hypertension related diseases

O BPP-10c é um decapeptídeo bioativo, rico em resíduos de prolina e é expresso em uma proteína precursora no cérebro e na glândula de veneno da Bothrops jararaca. Recentemente demonstramos que o BPP-10c tem um potente e sustentado efeito anti-hipertensivo em ratos espontaneamente hipertensos (SHR), sem, no entanto, causar qualquer efeito em ratos normotensos, por um mecanismo farmacológico independente da inibição da enzima conversora de angiotensina (ECA), levando à hipótese de que outro mecanismo poderia estar envolvido na atividade do peptídeo. Neste trabalho, usamos cromatografia de afinidade para isolar e identificar as proteínas renais com afinidade pelo BPP-10c e demonstramos que a argininosuccinato sintase (AsS) é a principal proteína a se ligar ao peptídeo. Além disso, mostramos que essa interação promove um aumento na atividade catalítica da enzima, de forma dose-dependente. A AsS é reconhecida como uma peça chave na regulação do ciclo da citrulina-óxido nítrico (NO), e sua ação é passo limitante na síntese de NO. A interação funcional do BPP-10c com a AsS foi evidenciada pelos seguintes efeitos promovidos pelo peptídeo: i) estimulação da produção de NO por células HUVEC e da produção de arginina por células HEK 293, ii) aumento da concentração plasmática de arginina em SHR. Corroborando esses achados, mostramos a reversão dos efeitos do peptídeo, inclusive sobre a pressão arterial em SHR, quando o MDLA, um inibidor específico da AsS, foi co-administrado. Em conjunto, os resultados apresentados neste trabalho sugerem que a AsS é fundamental para o efeito anti-hipertensivo do BPP-10c. Tais resultados nos levaram a propor a AsS como um novo alvo terapêutico, e o BPP-10c como molécula-líder para a geração de medicamentos para tratamento de doenças relacionadas à hipertensão arterial / BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological mechanism independent of angiotensin converting enzyme inhibition; therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-nitric oxide (NO) cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of BPP-10c and AsS was evidenced by the following effects promoted by the peptide: i) increase of NO production in human umbilical vein endothelial cell culture, and of arginine in human embryonic kidney cells; ii) increase of arginine plasma concentration in SHR. Moreover, MDLA, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of BPP-10c in SHR. These results led us to suggest AsS as a new therapeutically useful target for the development of activators, such as BPP- 10c, useful to treat hypertension related diseases