Dissertations / Theses: 'DDAH1'

1

Dowsett, Laura Bethany. "The role of the NOS-ADMA-DDAH1 pathway in adipocytes and obesity." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/24709.

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Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) which is metabolised by two isoforms of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH has been shown to increase ADMA concentrations both in vitro and in vivo. Clinically, high concentrations of ADMA have been associated with a range of diseases, particularly cardiovascular disease and more recently obesity. The role of the NO-ADMA-DDAH pathway has not yet been studied in adipose tissue. This thesis sets out to investigate the effects of pathological ADMA exposure firstly on adipocytes in vitro and secondly, on whole adipose physiology in vivo. In vitro the existence of the NO-ADMA-DDAH pathway was established in the 3T3-L1 cell line before investigating the effect of chronic ADMA exposure of their biology. ADMA was found to cause adipocyte hypertrophy through mTOR signalling in an NO independent manner. The effect of elevated ADMA on adipocytes in vivo was investigated through an adipocyte specific DDAH1 knockout. These mice developed visceral obesity with adipocyte hypertrophy with an increase in mTOR signalling. On a high fat diet there is a large increase in intracellular adipocyte ADMA in both knockout and control mice. Adipose vasculature is an important regulator of adipose expansion; to investigate the role of ADMA in this process an endothelial specific DDAH1 knockout mouse was developed. Angiogenesis in these mice is reduced restricting adipose growth in obesity. This thesis establishes that the NO-ADMA-DDAH1 pathway to be important in adipose physiology. Elevated ADMA in adipocytes and endothelial cells is detrimental to their function and increases various factors associated with the metabolic syndrome.

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Bernges, Isabel [Verfasser], and Elke [Akademischer Betreuer] Oetjen. "Die Metabolisierung der Dimethylarginine durch die Enzyme AGXT2 und DDAH1 und deren Anwendung für den Prozess der Drug Discovery / Isabel Bernges. Betreuer: Elke Oetjen." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2014. http://d-nb.info/1064076769/34.

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3

Tran, Cam Thanh Lucy. "Molecular analysis of human DDAH genes." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408021.

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Tommasi, Sara. "Design and synthesis of human dimethylarginine dimethylaminohydrolase (DDAH) inhibitors and development of a novel DDAH activity assay." Thesis, University of Aberdeen, 2015. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=227616.

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Nitric oxide (NO) is a key physiological messenger, but an excessive production of this molecule can be detrimental, leading to the onset or worsening of many pathological conditions. Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme in the NO pathway, involved in the metabolism of asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), which are both endogenous inhibitors of NO synthesis. Two isoforms of DDAH have been identified in humans, namely DDAH-1 and DDAH-2. DDAH inhibition represents a promising strategy in the treatment of NO overproduction under pathological conditions without affecting the homeostatic role of this messenger. In this work I described the design and synthesis of 12 novel potential DDAH inhibitors together with the development of a new UPLC-MS based assay to measure the activity of HEK293T cell lysates overexpressing recombinant human DDAH-1 in metabolizing ADMA into dimethylamine and L-citrulline. The same assay was used to assess the potential of the novel compounds, as well as of the well-known DDAH inhibitor L-257, to inhibit DDAH-1 catalyzed L-citrulline formation from ADMA. Three of the novel molecules (compounds 10a, 14a and 14b) showed very interesting inhibitory activity: in particular, the methylacylsulfonamide analogue of L-257 (10a) resulted in 13-fold higher inhibition potency than L-257 itself (98% of inhibition at 1mM, IC50 = 3±3 μM and Ki = 1±0 μM). This molecule was chosen for molecular dynamics simulations to study the putative mechanism for 10a inhibition of DDAH-1 activity. Furthermore, DDAH-1 and DDAH-2 were engineered introducing a FLAG-tag at the C-terminal of the proteins to allow their purification from the lysate components by immunoprecipitation. Although the purification protocol requires some further improvement, the fusion proteins did not show to be functionally affected by the modification.

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5

Kelly, P. D. "Elucidation of the biochemical and physiological role of DDAH2." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1317766/.

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The asymmetric methylarginines monomethyl-L-arginine (L-NMMA) and asymmetric dimethylarginine (ADMA) are endogenously occurring inhibitors of the nitric oxide synthase (NOS) enzymes. Elevated plasma ADMA has been identified in a range of human cardiovascular disorders, some of which are associated with impaired NO signaling. The dimethylarginine dimethlyaminohydrolase (DDAH) enzymes are responsible for the degradation of asymmetric methylarginines in vivo. The physiological link between DDAH, ADMA, and NOS was demonstrated by a mouse genetic knockout of DDAH1. Ddah1+/- mice had elevated ADMA concentrations and impaired NOmediated vasoreactivity which resulted in increased systemic vascular resistance and increased blood pressure. In 1999, a second isoform of DDAH was identified, DDAH2. This thesis reports the biochemical characterization of a novel mouse genetic knockout of DDAH2. Although ADMA is considered the most prominent methylarginine species in vivo, this study finds significant concentrations of L-NMMA are also present. Previous work has hypothesized a possible immune function for DDAH2, independent of DDAH1. Mouse peritoneal macrophages were found to contain DDAH2 and exhibit DDAH enzymatic activity, but no DDAH1 was detected. NO generation by cytokine treated peritoneal macrophages from ddah2-/- mice was significantly less then from ddah2+/+ macrophages. Conversely, pharmacological inhibition of DDAH in macrophages did not result in indirect NOS inhibition; this may be due to isoform specificity of the previously developed DDAH inhibitors. To determine the functional significance of the decreased NO generation capacity of ddah2-/- macrophages, their in vivo and in vitro antibacterial properties were investigated. Ddah2 genotype did not have any effect on the ability of peritoneal macrophages to kill S. aureus in vitro. But, in a mouse model of polymicrobial sepsis, ddah2-/- mice showed a significant increase in mortality. Follow-up characterization of this model showed a decrease in bacterial clearance by ddah2-/- mice along with a significant increase in asymmetric methylarginine concentration and changes in the concentration of several cytokines.

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Pullamsetti, Soni. "Role of Dimethylarginine Dimethylaminohydrolases (DDAH) in pulmonary arterial hypertension." Giessen VVB Laufersweiler, 2006. http://geb.uni-giessen.de/geb/volltexte/2006/2892/index.html.

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7

Tomlinson, James. "The role of DDAH and ADMA in kidney disease." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/24541.

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Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis and elevated plasma levels associate with poor cardiovascular and renal outcomes. The dimethylarginine dimethylaminohydrolase enzymes (DDAHs; 1 and 2) metabolise ADMA. A DDAH1 gene variant associates with higher kidney tissue mRNA expression, lower plasma ADMA but counter-intuitively, a steeper rate of eGFR decline. This indicates that renal DDAH1 activity may be deleterious and circulating ADMA does not necessarily reflect the NO-ADMA balance (or severity of disease) within kidney tissue. This study tests the hypothesis that reduced renal DDAH1 activity protects against the progression of kidney function decline, independent of circulating ADMA. Renal DDAH1 expression predominates within the proximal tubule. A novel proximal tubule-specific DDAH1 knock-out (PTD1KO) mouse was developed, which demonstrated tubule-specific dysregulation of ADMA and NO that was not evident systemically. Phenotyping studies in PTD1KO mice did not identify consistent alterations of urinary biochemistry at baseline or after salt loading, however, proteomic analysis revealed significant alterations of urinary peptides at baseline; including down-regulation of uromodulin and collagen. At 12 weeks following folate renal injury, the PTD1KO mouse exhibited less kidney function decline, collagen deposition and pro-fibrotic gene expression (Col12alpha, TGFbeta and ET-1) than controls. Furthermore, ADMA and DDAH1 inhibition reduced tubular sodium and fluid reabsorption in rat microperfusion studies, although studies in PTD1KO mice failed to reproduce this effect. Finally, in vitro studies using a PT cell line and primary PT culture indicated an inhibitory effect of ADMA upon PT cell proliferation. Consistent with recent human genetic studies, these data provide experimental evidence indicating a reduction of renal tubule DDAH1 activity can protect against progressive kidney fibrosis and function decline, independent of plasma ADMA. This work provides novel insights into the role of the NO-ADMA-DDAH axis within the kidney, particularly the tubule.

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Pullamsetti, Soni [Verfasser]. "Role of dimethylarginine dimethylaminohydrolases (DDAH) in pulmonary arterial hypertension / vorgelegt von Soni Pullamsetti." Giessen : VVB Laufersweiler, 2006. http://d-nb.info/98866240X/34.

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9

Hicks, Diana. "English language teaching teacher's guides : a critical discourse analysis of three texts." Thesis, University of Bristol, 2000. http://hdl.handle.net/1983/a13246cc-dda1-4a94-b061-7c3a415ee82e.

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10

Khanom, N. "Evaluation of novel arginine based inhibitors of DDAH and investigations into radical hydroacylation of vinyl sulfonates." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/192842/.

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The thesis is in two main sections. In the first section, studies on methylarginine processing enzymes are presented. Dimethyalrginine dimethylaminohydrolase (DDAH) is a class of enzymes involved in the metabolism of methylarginines ADMA and L-NMMA, which indirectly regulate physiological nitric oxide levels. It is desirable to inhibit excess NO in pathological situations, and the arginine mimetic L-257 is a DDAH inhibitor which reduces levels of NO. Synthesis of ester analogues of L-257 proved to be troublesome with a low yielding key guanidine forming reaction. However, amide analogues were readily synthesised, and testing for DDAH inhibition showed the dimethylamide analogue possessed similar activity to L-257. Further design and synthesis of a 7-membered cyclic analogue, based on the crystal structure of huDDAH1 with L-257, provided a novel analogue with no significant inhibition for rat kidney DDAH. Purified and isolated huDDAH2 protein showed activity after incubation with substrate L-NMMA. In the second part studies on aldehyde auto-oxidation are presented. Aldehydes autoxidise to their acids, via an acyl radical, which can undergo addition reactions with electron-deficient acceptors in a radical hydroacylation reaction. An α- iodo and α-chloro hexanal failed to autoxidise, however 7-hydroxycitronellal readily autoxidised and added to pentafluorophenyl(PFP)-vinyl sulfonate. Further studies on hydroacylation of butanal with PFP-vinyl sulfonate led to functionalised β-ketosulfonates which undergo elimination to form an enone and can then undergo further conjugate addition in situ by nucleophiles. Conjugate addition was carried out using carbon, nitrogen, oxygen and phosphorus nucleophiles, providing a method of obtaining products which are challenging to make via hydroacylation of electron-rich alkenes. Decarbonylation of pivaldehyde to the t-butyl radical, via auto-oxidation, was optimised and the alkyl radical captured by a number of electron-deficient acceptors, providing a complementary method to current methods of t-butyl addition using metal reagents.

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11

Sonnenberg, Derek M. "Design and Synthesis of Novel Chloroacetimidine Inactivators as Potential in vivo Activity Probes of DDAH-1 for Regulation of NOS." Thesis, Southern Illinois University at Edwardsville, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10247909.

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Nitric Oxide, NO, is an important neurotransmitter and signaling molecule in almost every system of the human body. Its regulation is equally important as a dysfunction in regulation leads to pathophysiologic conditions and disease states. There are many ways that NO is regulated, but one of the more common methods is to regulate the enzyme that creates it, Nitric Oxide Synthases or NOS. Again, many ways to regulate this enzyme exist, but this work focuses on inhibition by an endogenous molecule produced via the normal process of protein degradation, Asymmetric Dimethyl Arginine or ADMA. Another enzyme in the body is responsible for metabolism of ADMA called Dimethylarginine Dimethylaminohydrolase or DDAH. By regulating the activity of DDAH, we can control concentrations of ADMA and therefore the inhibition of NOS, which in turn regulates the production of NO. It is the purpose of this project to create a probe that can be used in vivo for the DDAH enzyme family using organic synthesis to produce a product that, when effectively bound to the enzyme target, can also undergo a click chemistry reaction to tailor the utility of the probe itself. The Dixon Lab has designed such a molecule and the synthesis is what follows.

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12

Ratz, Patrick. "Synthesis of 4-[(2-chloroethanimidamido) methyl --N- (prop-2-yn-1-yl) benzamide, a possible in vivo activity probe of DDAH-like enzymes." Thesis, Southern Illinois University at Edwardsville, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=1602062.

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Nitric Oxide is an important molecule in human cells. It is responsible for multiple functions in various systems including the cardiovascular, immune, nervous, digestive, and reproductive systems. Nitric oxide is synthesized by the enzyme nitric oxide synthase, and its activity is controlled by the levels of asymmetric dimethyl arginine (ADMA) which in turn are controlled by Dimethylaminohydrolase (DDAH). DDAH is an important enzyme for study due to its ability to indirectly control nitric oxide synthase. 2-chloroacetimidine is an inhibitor of DDAH. Synthesizing molecules that contain a 2-chloroacetimidine moiety and a retrievable chemical tail could prove to be instrumental in further studying DDAH and other enzymes with similar reactivity in their active sites.

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13

Gaber, Mohammed Bakr Adel [Verfasser]. "Hypoxia-dependent mechanisms in the pulmonary circulation : Role of dimethylarginine dimethylaminohydrolase 1 (DDAH-1) in acute, sustained and chronic hypoxia / Adel Gaber Mohammed Bakr." Gießen : Universitätsbibliothek, 2011. http://d-nb.info/1063109906/34.

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14

Carmann, Christina [Verfasser], Thomas [Gutachter] Lücke, and Stephan [Gutachter] Volker. "Das DDAH/ADMA/NO-System und die nitrit-abhängige renale Carbonanhydrase-Aktivität bei Kindern und Jugendlichen mit Diabetes mellitus Typ 1 / Christina Carmann ; Gutachter: Thomas Lücke, Stephan Volker ; Medizinische Fakultät." Bochum : Ruhr-Universität Bochum, 2018. http://d-nb.info/1154307794/34.

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15

Burstein, Gayle Diane. "An investigation of the irreversible inhibition of human N[superscript ω], N[superscript ω]- dimethylarginine dimethylaminohydrolase (DDAH1)." Thesis, 2014. http://hdl.handle.net/2152/31281.

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Nitric oxide synthases (NOS) are responsible for the production of nitric oxide (NO), an essential cell-signaling molecule, in mammals. There are three isoforms of NOS with widely different tissue distribution. The overproduction of NO is marked in many human disease states and cancers, however due to the similarities of the enzyme isoforms, targeting NOS for inhibition has proven challenging. Endogenously, the methylated arginines, N[superscript ω]-monomethyl-L-arginine (NMMA) and asymmetric N[superscript ω], N[superscript ω]-dimethyl-L-arginine (ADMA), inhibit NOS. N[superscript ω], N[superscript ω]-Dimethylarginine dimethylaminohydrolase (DDAH1) metabolizes these methylated arginines and thus relieves NOS inhibition. The role of DDAH1 in the regulation of diseases such as cancer and septic shock is still being elucidated. It is thought that targeting DDAH1 for inhibition rather than NOS may circumvent many of the current problems with the treatment of NO overproduction such as isoform selectivity. My PhD studies focus on the synthesis of a series of irreversible inhibitors of DDAH1, an extensive study of their in vitro mode of inhibition, a comparison of analytical fitting methods, and the viability and efficacy of the inactivators in a human cell line. I also studied a potential endogenous inactivator of DDAH1, nitroxyl (HNO), a one-electron reduction product of NO.
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16

Chen, Shu-Fan, and 陳書樊. "The biomarker for the early stage of diabetic nephropathy and microRNA miR-30c affects kidney fibrosis through regulating expression of DDAH1 and IRS1 proteins." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/z6mx6c.

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碩士
國立交通大學
生物科技學系
105
Diabetes mellitus (DM) is a chronic disease that can result in many complications in people. One majority of them is kidney disease, which may finally lead to the end-stage renal disease (ESRD). However, the precise mechanism is still unclear. Hence, the better understanding of the disease may provide us novel therapeutic targets. MicroRNA is a short non-coding RNA that plays an important role in cell growth, proliferation, differentiation and immune response. Furthermore, it also has the potential to regulate several physiological and pathological processes. We found that many specific miRNA can regulate the expression of TGF-β in the kidney. Recent studies have highlighted the importance of miRNA in the regulation of glycaemia and the reduction of hyperlipidemia in DN patients. It can also modulate the abnormal extracellular matrix thickening, and prevent the chronic kidney disease from ESRD. First, according to the values of ACR, we separated the participants into four groups, H, DN1, DN2, and DN3. We detected the serum levels of β-trophin and urine levels of NO and found that β-trophin and NO were higher in DN3 and DN4 groups than in Healthy group. In order to explore the regulation of specific genes in diabetic nephropathy patients, we used micrroarray analysis to find the miRNA, which can regulate the β-trophin and NO production. In recent study, mir-30c has been found that can downregulate the lipid synthesis and the secretion of lipoprotein. It is also associated with the NO production, inflammatory response, and apoptosis. Most importantly, we found that miR-30c strongly upregulated in the development of DN. To further investigate the role of mir-30c in renal dysfunction, we overexpressed miR-30c in the podocyte cell line (E11). Upregulation of miR-30c can inhibit the DDAH1 and IRS1 expression in transcriptional levels. Our results suggest that miR-30c may play a critical role in the DN development and blockade of miR-30c may be a potential therapeutic option in DN.

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Yang, Yin, and 楊茵. "Characterization of DDA1, a p53-regulated gene." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/06664281071119188252.

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碩士
國立陽明大學
生物化學研究所
87
Wild type p53 expressed from a temperature-sensitive (tsp53) construct induces both G1 cell cycle arrest and apoptosis in the p53-negative IW32 mouse erythroleukemia cell line. Using PCR-based differential display analysis, we previously identified a new p53-inducible gene, DDA1, whose mRNA was upregulated in tsp53-transfected IW32 cells following induction of wild type p53 expression by temperature shift to 32°C. The DDA1 mRNA induction was detectable within 1 hour after temperature downshift, and rapid degradation was observed when the temperature was shifted back to 37°C, suggesting that the expression of DDA1 is dependent on the continuous presence of p53. The DDA1 mRNA was also induced in DNA damaging reagent-treated NIH3T3 cells. Previous studies from this lab have shown that mouse DDA1 cDNA predicted to encode a protein of 498 amino acid residues containing 12 transmembrane domains. The loop region between 6th and 7th transmembrane domains was used as immunogen to produce rabbit polyclonal antibodies, and antibodies that can recognize E. coli expressed mDDA1 protein was obtained. Immunofluorescence analysis indicated that DDA1 protein is located in the cytoplasm. Overexpression of mDDA1 cDNA in H1299 cells inhibited cell growth，as shown by the colony formation assay. The cDNA of human DDA1 that is 73 % identical to mouse DDA1 was acquired by library screening and database searching. The two amino acid sequences share 90 % identity.

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CHIAHUIWU and 吳佳惠. "Characterization of a p53-regulated gene DDA1." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/64211439442454738164.

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碩士
國立陽明大學
生物化學研究所
88
Abstract p53 tumor suppressor is a transcription factor that causes cell growth arrest and induces apoptosis. Identification of the p53 downstream target genes is therefore important to unravel the mechanisms underlying p53 actions. We have previously identified and cloned a p53-regulated gene, DDA1, by the RNA differential display of an IW32 erythroleukemia stable clone (1-5) that contains a temperature-sensitive p53 mutant gene, tsp53val135. Sequence comparison revealed that mDDA1 shares 73% and 90% identity in its nucleotide and protein sequences, respectively, to the newly identified human thiamine transporter gene (hTHTR-1). To further investigate the subcellular localization and function of DDA1, we established DDA1 expressing clones under the control of the tetracycline inducible promoter. After 24 hours treatment of 2 mg /ml Doxycycline, clone 118 transfectant cells could be induced to express DDA1 mRNA and protein. Immunofluorescence analysis indicated that DDA1 was present on the plasma membrane. Growth of DDA1 stable transfectant was partly inhibited in the presence of Doxycycline. Ability of the clone 118 cells to uptake thiamine increased 2-fold in the presence of Doxycycline. These data demonstrated that mDDA1 possesses thiamine transporter activity. hTHTR-1 mRNA was induced by DNA damage in a p53 dependent manner. Induction was detected in 293 cells expressing endogenous p53, but not in 293T cells whose p53 was inactivated. Together these results indicate that the high affinity thiamine transpoter is a p53 regulated gene. 中文摘要 ..................................................... 1 英文摘要 ..................................................... 2 緒論 ..................................................... 3 實驗材料 ..................................................... 16 實驗方法 ..................................................... 18 實驗結果 ..................................................... 30 實驗討論 ..................................................... 36 參考文獻 ..................................................... 40 附圖 ..................................................... 47

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19

Lüneburg, Nicole [Verfasser]. "Molekularbiologische Untersuchung der DDAH-Defizienz / vorgelegt von Nicole Lüneburg." 2008. http://d-nb.info/990534472/34.

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Stone, Everett Monroe. "The catalytic mechanism of dimethylarginine dimethylaminohydrolase (DDAH) from pseudomonas aeruginosa." Thesis, 2006. http://hdl.handle.net/2152/2934.

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Chobanyan, Kristine Tsikas Dimitrios. "Entwicklung, Validierung und Anwendung von GC/MS-Methoden für die Bestimmung der DDAH-Aktivität in vitro und in vivo : Evaluierung der Bedeutung von S-Nitrosothiolen als potentielle Inhibitoren der DDAH-Aktivität /." 2007. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=017048731&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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22

Tan-Andresen, Jing [Verfasser]. "Entwicklung und Validierung eines Dimethylarginin-Dimethylaminohydrolase (DDAH)-Aktivitätsassays in Gewebehomogenat / vorgelegt von Jing Tan-Andresen." 2008. http://d-nb.info/991805550/34.

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Chang, Shih-Chieh, and 張仕杰. "Elucidating the effects of adenovirus-mediated DDAH on vascular endothelial and smooth muscle cells function." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/34758008288652431087.

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碩士
中國醫藥大學
醫學研究所
93
Restenosis is a major clinical problem that occurs in 30% to 50% of patients who undergo a Percutaneous Transluminal Coronary Angioplasty (PTCA) procedure and limits its long-term success. Accumulating evidence has indicated that Nitric Oxide (NO) is a major regulator of cardiovascular physiology and can reduce vascular and cardiac contractility. It is found that an endogenous inhibitors, asymmetric dimethylarginine (ADMA), may regulate NOS activity. A vascular endogenous enzyme, dimethylarginine dimethylaminohydrolase (DDAH), has been found to be the major mediator to metabolize ADMA in vivo. In order to test if the ADMA-DDAH pathway plays a role in restenosis, we used adenovirus as a vector to overexpress DDAH gene in cell and animal models. In the present study, we aim to elucidate the effect of adenovirus-mediated DDAH overexpression in endothelial, smooth muscle cells, and rat vessel to determine its potential therapeutic indication on balloom injury-mediated neointima formation. The translational regulation of DDAH on VCAM-1 was also thoroughly investigated in this thesis.

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Chobanyan, Kristine [Verfasser]. "Entwicklung, Validierung und Anwendung von GC-MS-Methoden für die Bestimmung der DDAH-Aktivität in vitro und in vivo : Evaluierung der Bedeutung von S-Nitrosothiolen als potentielle Inhibitoren der DDAH-Aktivität / vorgelegt von Kristine Chobanyan." 2008. http://d-nb.info/990164217/34.

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Wang, Yun 1981. "Controlling nitric oxide (NO) overproduction : N[omega], N[omega]-dimethylarginine dimethylaminohydrolase (DDAH) as a novel drug target." 2010. http://hdl.handle.net/2152/14048.

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Nitric oxide (NO) overproduction is correlated with numerous human diseases, such as arthritis, asthma, diabetes, inflammation and septic shock. The enzyme activities of both NO synthase (NOS) and dimethylarginine dimethylaminohydrolase-1 (DDAH-1) promote NO production. DDAH-1 mainly colocalizes in the same tissues as the neuronal isoform of NOS and catabolizes the endogenously-produced competitive inhibitors of NOS, N[omega]-monomethyl-L-arginine (NMMA) and asymmetric N[omega], N[omega]-dimethyl-L-arginine (ADMA). Inhibition of DDAH-1 leads to elevated concentrations of NMMA and ADMA, which subsequently inhibit NOS. To better understand DDAH-1, I first characterized the catalytic mechanism of human DDAH-1, where Cys274, His173, Asp79 and Asp127 form a catalytic center. Particularly, Cys274 is an active site nucleophile and His173 plays a dual role in acid/base catalysis. I also studied an unusual mechanism for covalent inhibition of DDAH-1 by S-nitroso-L-homocysteine (HcyNO), where an N-thiosulfoximide adduct is formed at Cys274. Using a combination of site directed mutagenesis and mass spectrometry, we found that many residues that participate in catalysis also participate in HcyNO mediated inactivation. Following these studies, I then screened a small set of known NOS inhibitors as potential inhibitors of DDAH-1. The most potent of these, an alkylamidine, was selected as a scaffold for homologation. Stepwise lengthening of the alkyl substituent changes an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-L-ornithine for neuronal NOS (1.7, 3, 20, >1,900 [mu]M, respectively) and DDAH-1 (990, 52, 7.5, 110 [mu]M, respectively). X-ray crystal structures suggest that this selectivity is likely due to active site size differences. To rank the inhibitors' in vivo potency, we constructed a click-chemistry based activity probe to detect inhibition of DDAH-1 in live mammalian cell culture. In vivo IC50 values for representative alkylamidine based inhibitors were measured in living HEK293T cells. Future application of this probe will address the regulation of DDAH-1 activity in pathophysiological states. In summary, this work identifies a versatile scaffold for developing DDAH targeted inhibitors to control NO overproduction and provides useful biochemical tools to better understand the etiology of endothelial dysfunction.
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Lluis, Matthew Wayne. "Crystal structures of dimethylarginine dimethylaminohydrolase-1 (DDAH-1) from Homo sapiens bound to the inhibitors N⁵-(1-iminopentyl)-L-ornithine and ebselen and functional studies of the translin●trax complex from Mus musculus." Thesis, 2009. http://hdl.handle.net/2152/ETD-UT-2009-12-423.

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Nitric oxide (NO) is reactive, radical gas that is involved in a myriad of cellular signaling pathways including the regulation of blood flow and immunodefense. NO is produced from the oxidation of L-arginine to L-citrulline by nitric oxide synthase (NOS). The activity of NOS and by default, the production of NO, is regulated by the arginine derivatives N[omega],N[omega]-dimethyl-L-arginine (ADMA) and N[omega]-monomethyl-L-arginine (NMMA) which arise from the proteolytic degradation of post translationally methylated proteins. The cellular concentrations of ADMA and NMMA are regulated by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catabolizes these compounds to L-citrulline and dimethylamine or methyl amine, respectively. Because over and under production of NO has been implicated in several pathophysiological states, compounds that control NO production by inhibiting NOS or DDAH may prove useful as treatments. In this study, the crystal structures of human DDAH-1 with the inhibitors N⁵-(1-iminopentyl)-L-ornithine (L-IPnO) and ebselen were solved to 2.9 and 2.0 Å resolution, respectively. L-IPnO was observed to inhibit DDAH-1 in essentially the same manner as another amidino-containing inhibitor: docking to the enzyme via hydrogen bond and ion pair interactions and forming a covalent adduct with the active site cysteine. Ebselen was also observed to covalently attach to the active site cysteine, however, the docking mechanism was absent of hydrogen bond and ion pair interactions. The work presented here contributes to the design of compounds that may effectively regulate the production of NO for therapeutic purposes. Translin is a highly conserved mammalian RNA and DNA binding protein known to be involved in DNA recombination and repair, RNA trafficking in neurons, and post-transcriptional regulation of gene expression in male germ cells. Although crystal structures of the mouse and human orthologs of translin have been solved, they do not provide details on the structure-function relationship of the protein. Studies have identified a partner protein for translin, translin associated factor x (trax), which is believed to have a crucial role in assisting translin with its cellular functions. It is believed that trax regulates translin’s affinity for certain RNA and DNA sequences. In this work the binding affinities of translin and the translin●trax complex were investigated. It was observed that translin preferentially binds to G-rich RNA sequences, most likely recognizing a secondary structure intrinsic to these sequences, whereas translin●trax preferentially binds G-rich DNA sequences. The results from these experiments provide insight into the cellular functions of translin and trax and their respective roles in mRNA trafficking.
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