Dissertations / Theses: 'Serine hydrolases'

1

Fransson, Linda. "Enzyme substrate solvent interactions : a case study on serine hydrolases." Doctoral thesis, KTH, Biokemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4867.

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Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated. Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation. Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour. In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified.
QC 20100722

2

Elahi, AEM Rubayet. "Proteome-wide Functional Profiling of Serine Hydrolases in the Human Malaria Parasite." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/90181.

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The serine hydrolase (SH) enzyme superfamily is one of the largest and most diverse enzyme classes in eukaryotes and prokaryotes. The most virulent human malaria parasite Plasmodium falciparum has over 40 predicted serine hydrolases (SH). Prior investigation on a few of these have suggested their critical role in parasite biology. The majority of the SHs in P. falciparum have not been functionally characterized. Investigation of these uncharacterized SHs will provide new insights into essential features of parasite metabolism and possibly lead to new antimalarial targets. In this study, we have employed activity-based protein profiling (ABPP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to functionally characterize SHs. In our effort to profile plasmodial SHs using ABPP, we have identified a human erythrocyte SH, acylpeptide hydrolase (APEH) in the developing parasites. This finding is the first report of internalization of host hydrolytic enzyme by the parasite. Treatment of parasites with an APEH specific triazole urea inhibitor, AA74-1, caused growth inhibition in parasites with poor potency in the first replication cycle, however, the potency dramatically increased in the second cycle. We show that this unique growth inhibition profile is due to the inability of AA74-1 to inhibit parasite-internalized APEH in vivo. These findings suggest that internalization of active APEH by the parasite is essential for parasite survival. Lipases catalyze the hydrolysis of ester bonds of lipid species such as neutral lipids and phospholipids. Although roles of lipases in propagation, as well as virulence in various organisms, have been acknowledged, in P. falciparum lipases remain understudied. We combined LC-MS/MS with the SH-directed ABPP to identify lipases of SH superfamily in P. falciparum. We have identified 16 plasmodial SHs with putative lipase activity. Bioinformatics analysis of our identified lipases is consistent with our findings. We have screened a panel of various classes of SH inhibitors in a competitive ABPP. A plasmodial putative lipase was potently and specifically inhibited by human monoacylglycerol lipase inhibitor. This inhibition profile suggests it as a monoacylglycerol lipase which plays a role in releasing fatty acids from neutral lipid. This finding shows that how inhibitor screening can aid in building hypotheses on biological roles of an enzyme. Altogether, in this dissertation, we have presented a robust strategy of identifying and functionally characterizing SHs in P. falciparum, which opens the door to the discovery of new biological processes.
Doctor of Philosophy
Malaria contributed to nearly a half a million deaths in 2017. The vast majority of malaria-related deaths are due to the parasite Plasmodium falciparum. This parasite resides inside human red blood cells (erythrocytes) and grows rapidly during a 48 hour cycle. There are over 40 serine hydrolase (SH) superfamily proteins in the parasite. Biological functions of the majority of SHs in the parasite remains unknown. Study on these SHs will provide new insights into parasite biology, and possibly present new antimalarial drug targets. We used chemical biology techniques to identify and functionally characterize parasite SHs. In one study, we show the parasite intenalized a human erythrocyte SH, acylpeptide hydrolase (APEH). We used an APEH-specific inhibitor to investigate the biological significance of internalized APEH in parasite biology. Treatment of the parasite with the inhibitor resulted in parasite growth inhibition suggesting internalization of APEH is essential for parasite survival. Lipases are enzymes that aid in break down of lipids and have shown to be crucial for growth and pathogenicity in various organisms. Lipases and lipid catabolism remain understudied in the malaria parasite. We used mass spectrometry in our approach to identify 16 lipases in asexual parasites. We have also shown that screening with highly specific inhibitors can help in predicting biological function of a particular enzyme. In summary, in this body of work, we have presented an approach of studying SHs in the malaria parasite, which will provide new insights into parasite biology.

3

Galmés, Ordinas Miquel Àngel. "Molecular insights into the promiscuity of serine hydrolases. Towards a computationally guided protocol for the redesign of enzymes." Doctoral thesis, Universitat Jaume I, 2022. http://dx.doi.org/10.6035/14122.2022.725777.

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Two serine hydrolases, Candida antarctica Lipase B (CALB) and para-nitrobenzyl (Bs2) esterase from Bacillus subtilis, were used as a model to study enzyme promiscuity through QM/MM methods and experimental enzymes kinetics. Both, the catalytic and the substrate promiscuity were studied. The hydrolysis of amides and the epoxidation of alkenes catalyzed by CALB were explored. Moreover, a computational scheme for the redesign of the Bs2 was also proposed. The electrostatic environment around the active site was analyzed and a map of structural determinants in the vicinity of the active site pocket was done using 3D Convolutional Neural Networks. The proposed computationally guided protocol for the mutagenesis of enzymes based on the detailed analysis of the electrostatic environment of two structurally aligned trajectories using rotation quaternions was applied. A new mutant variant of the Bs2 was suggested as an improved catalytic variant by combining the best electrostatic features of both enzymes.
Programa de Doctorat en Química Teòrica i Modelització Computacional

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Kreuzer, Johannes Verfasser], Stephan A. [Akademischer Betreuer] Sieber, and Aymelt [Akademischer Betreuer] [Itzen. "The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids / Johannes Kreuzer. Gutachter: Aymelt Itzen ; Stephan A. Sieber. Betreuer: Stephan A. Sieber." München : Universitätsbibliothek der TU München, 2015. http://d-nb.info/1071651544/34.

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5

Yedji, Rodrigue. "Perturbateurs endocriniens de type phtalate et poisson zèbre Danio rerio : approche chémoprotéomique pour l'identification des cibles et recherche de signatures d'exposition." Electronic Thesis or Diss., Université de Lorraine, 2022. http://www.theses.fr/2022LORR0106.

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Les esters de phtalate constituent une famille de composés synthétiques très répandue du fait de leurs usages comme plastifiants. Ils entrent dans la composition de plusieurs produits plastiques tels que les emballages, les jouets, les produits cosmétiques, certains systèmes de toiture en plastique, ainsi que les matériaux de décoration de meubles. Les phtalates ne sont pas liés de manière covalente à la matrice des polymères et sont donc facilement rejetés dans l'environnement, entraînant par conséquent une exposition animale et humaine. En absence de produits de substitutions non-toxiques, les composés de type phtalate restent encore largement utilisés dans l'industrie en dépit de la classification de certains d'entre eux dans la catégorie des substances présumées toxiques par l'European Chemicals Agency (ECHA), en tant que perturbateurs endocriniens. De plus, ils sont cancérigènes et tératogènes. L'effet délétère des esters de phtalates sur les organismes est établi mais le caractère multiple des effets observés montre que les mécanismes d'action des phtalates ne sont que très partiellement élucidés. Nous avons utilisé deux approches de protéomique ciblée pour tenter d'éclairer nos connaissances sur les mécanismes d'actions des esters de phtalate. Pour cela, le dibutyl phtalate (DBP) a été utilisé comme phtalate modèle, et le poisson zèbre (D. rerio) comme organisme modèle. L'utilisation de la première approche de protéomique ciblée, le profilage protéique basé sur l'affinité (affinity-based protein profiling, AfBPP) a permis de montrer la perturbation fonctionnelle de protéines par le DBP avec des sondes photoactivables issues de la synthèse de types aryle azide. L'optimisation des conditions de fixation des sondes diazirine (Diazirine 2) devrait nous permettre de disposer d'une sonde pouvant être utilisée pour identifier les cibles protéiques du DBP dans le protéome du poisson zèbre. La deuxième approche, le profilage basé sur l'activité des enzymes (activity-based protein profiling, ABPP) a permis d'utiliser une sonde réactive spécifique des hydrolases à sérine (SHs) pour cartographier pour la première fois des SHs actives dans le protéome du poisson zèbre. L'identification des SHs dérégulées en présence de DBP chez les larves de poisson zèbre a également été rapportée dans cette étude. Nos résultats globaux indiquent que les approches de protéomiques ciblées telles que l'ABPP ou l'AfBPP peuvent être un atout pour comprendre les mécanismes d'action liés aux xénobiotiques en écotoxicologie
Phthalate esters are a family of synthetic compounds widely used as plasticisers. They are used in a number of plastic products such as packaging, toys, cosmetics, plastic roofing system and furniture decoration materials. Phthalates are not covalently bonded to the polymer matrix and are therefore easily released into the environment, resulting in animal and human exposure. In the absence of non-toxic substitutes, phthalate compounds are still widely used in industry, despite the classification of some of them by the European Chemicals Agency (ECHA) as suspected toxic substances and as endocrine disruptors. In addition, they are carcinogenic and teratogenic. The deleterious effect of phthalate esters on organisms is established, but the multiple nature of the effects observed shows that the mechanisms of action of phthalates are only partially elucidated. We used two targeted proteomics approaches to shed light on the mechanisms of action of phthalate esters. Dibutyl phthalate (DBP) was used as a model phthalate and zebrafish (D. rerio) as a model organism. Using the first targeted proteomics approach, affinity-based protein profiling (AfBPP), the functional disruption of proteins by DBP with photoaffinity probes from aryl azide synthesis was demonstrated. Optimisation of the binding conditions for diazirine probes (Diazirine 2) should provide us with a probe that can be used to identify DBP protein targets in the zebrafish proteome. The second approach, activity-based protein profiling (ABPP), used a reactive probe specific for serine hydrolases (SHs) to map active SHs in the zebrafish proteome for the first time. The identification of deregulated SHs in the presence of DBP in zebrafish larvae was also reported in this study. Overall, our results indicate that targeted proteomics approaches such as ABPP or AfBPP can be an asset for understanding xenobiotic-related mechanisms of action in ecotoxicology

6

Hamberg, Anders. "Serine Hydrolase Selectivity : Kinetics and applications in organic and analytical chemistry." Doctoral thesis, KTH, Biokemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-12831.

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The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved. In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase. In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches. In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance. In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.
QC20100629

7

Hamon, Nadège. "Synthese de nucleosides en serie carbocyclique à visée antivirale." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20087/document.

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Les analogues nucléosidiques constituent une famille importante d'agents thérapeutiques dans le traitement de maladies d'origine virale. Parmi ces composés, les nucléosides carbocycliques possèdent des propriétés biologiques intéressantes. Le premier chapitre de cette thèse est consacrée à la famille des neplanocines qui sont des carbonucleosides naturels. Nous avons détaillé l'interaction de ces composés avec leur principale cible, la S-adénosylhomocystéine hydrolase, ainsi que les différentes approches de synthèses de ces carbonucléosides et de leurs énantioméres avant de passer en revue leurs activités biologiques. Nous avons présenté dans le deuxième chapitre la première synthèse énantiosélective de la (éD)-néplanocine B. Le troisième chapitre est quant à lui axé sur la mise au point d'une synthèse de 3 '-halo-5'-norcarbonucléosides phosphonates ainsi qu’à l'évaluation de leurs activités antivirales
Nucleosides analogues constitute an important family of therapeutic agents in the treatment of viral diseases. Among these compounds, carbocyclic nucleosides have interesting biological properties. The first chapter of this thesis is dedicated to a family of natural carbonucleosides, the neplanocins. We have presented their mode of action against S-adenosylhomocysteine hydrolase, as well as various syntheses of natural neplanocins and their enantiomers before reviewing their biological activities. In the second chapter, we described the first enantioselective synthesis of (¨D)-neplanocine B. The third chapter is devoted to the development of the synthesis of 3 '-halo-5¡¯-norcarbonucleosides phosphonates as well as the evaluation of their antiviral activities

8

Ambrose, Timothy James William. "Serine hydrolase activity and roles for monoacylglycerol lipase in innate immunity and intestinal inflammation." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:f7a12796-ae8f-4121-ab1a-26778261ac78.

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Detection of evolutionarily conserved pathogen motifs by pattern recognition receptors (PRRs), particularly on dendritic cells (DCs), is crucial for adequate immune responses. Defects in DC function are known to be associated with inflammatory bowel disease (IBD). The endocannabinoid system (ECS) is the system through which exocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol signal. Regarding inflammation, cannabinoids generally exert anti-inflammatory effects, including on experimental colitis. However, most work has been performed in animal models and less is known about the function of this system in human immune cells, particularly DCs. Monoacylglycerol lipase (MGLL) is the key enzyme for hydrolysis of the endocannabinoid 2-arachidonoylglycerol, and a member of the serine hydrolase enzyme superfamily. This thesis defines the activity of serine hydrolase enzymes for the first time in human DCs upon stimulation by NOD2/TLR2 ligands using activity-based protein profiling (ABPP). MGLL is shown to be ubiquitously upregulated upon stimulation of DCs and in monocyte-derived macrophages. Through pharmacological inhibition studies, MGLL is demonstrated to regulate cellular and secreted lipids, not limited to endocannabinoids. However, overall DC function is independent of this enzyme suggesting that the effects of lipid modulation may be on bystander cells. Challenging the current literature, MGLL inhibition with a novel inhibitor worsens murine Citrobacter rodentium colitis. Finally, ABPP demonstrates a rich serine hydrolome in colonic tissue from human IBD with many enzymes previously undefined in this disease. Gene expression of ECS components suggests the enzymes ABHD12 and DAGLα/β may be potential markers of field change in IBD.

9

Ho, Cherry Pei-Yee. "Evaluation of a Serine Hydrolase Inhibitor JZL184 as an Immunomodulator against Avian Pathogenic Escherichia Coli O78 in Chickens." Thesis, Mississippi State University, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10788360.

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Studies with the serine hydrolase inhibitor JZL184 have suggested that enhanced 2-arachidonoylglycerol signaling could be strategized to stimulate innate immune cells to combat invading pathogens and improve host defense by prompting the systemic release of proinflammatory cytokines. Although the neurochemical effects of JZL184 were found to cultivate within 30 min in mice, its immune-regulating effects have been studied much later and its effects on chickens have not been clear. To explore the modulations in the chickens’ immune responses, we studied the effects of intraperitoneal injections of JZL184 in APEC O78-infected chickens on pathogenicity, histopathology, and IL-1β levels. The pathogenicity of the strain was assessed by isolating bacteria from livers, blood, air sacs, and hearts at 8, 28, and 56 h post-infection (p.i.). Air sacs, livers, and hearts were examined for histopathological changes at 8, 28, and 56 h p.i. Serum samples were collected at 8, 28, and 56 h p.i. and analyzed with a chicken IL-1β ELISA kit. Liver and spleen samples were homogenized for detection of serine hydrolases and carboxylesterases. Our work showed that 10 mg/kg and 40 mg/kg of JZL184 did not reduce the severity and progression of lesions produced in chickens challenged with 108 CFU of E. coli O78. The JZL184 treatments made colibacillosis lesions in the E. coli O78-challenged chickens worse and we did not find evidence of the injections increasing the serum cytokine levels of IL-1β at our sampling times.

10

Kornahrens, Anne. "Methodology development and synthesis of a biologically relevant natural product and targeted scaffold discovery for serine hydrolase inhibition." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:4cf5026f-e421-4b0b-9f62-707622f780b7.

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The phenomenal potential of synthetic chemistry to influence small molecule therapeutic development represents my overarching theme. The goal of designing or discovering a novel clinical candidate or biological target can be achieved through a variety of avenues available to synthetic organic chemists. The potential of natural product total synthesis and inhibitor development as impactful projects were pursued with the use and development of methodology. The natural product streptonigrin, a potent antitumor antibiotic, showed promise in clinical trials ultimately terminated in the 1970s. The underexplored mechanism of action prompted a reexamination of this potential therapeutic. The Donohoe group successfully designed a modular total synthesis accessing streptonigrin in 11 steps and 14% yield. The introduction of an asymmetric Suzuki-Miyaura reaction yielded a key intermediate in 42% ee and 65% yield, which represents the first example of purely synthetic access to enantioenriched late-stage streptonigrin analogues. The "hydrogen-borrowing" method allows the functionalization of ketones with methanol and transition metal catalysts, but examples lacked application to further α-functionalization. The successful development of a novel "interrupted- hydrogen-borrowing" method allowed the use of iridium and methanol to trap the reactive enone intermediates by preventing the subsequent "hydrogen returning". Thus, this enabled these reactive intermediates to be subjected to a 1,4-conjugate addition with a nucleophilic ketone enolate and subsequent cyclization and oxidation to form highly substituted pyridines from simple ketones. The exploration of serine hydrolases, an important mammalian enzyme class with a large portion still uncharacterized, requires inhibitors with new chemotypes that can covalently and irreversibly bind. The small library of urea- and carbamate- containing scaffolds with varied aromatic backbone substitutions was subjected to activity-based protein profiling to allow expedient examination of the whole serine hydrolase family. The predicted strong electronic effect of the p-substituent was confirmed, modulating the reactivity of the electrophilic urea or carbamate moiety. MudPIT analysis of a subset supported the demonstrated reactivity profile and identified the uncharacterized serine hydrolase PNPLA4 as a selective target of one inhibitor. This potent and selective inhibition was verified by in vitro and in cell assays and validates this chemotype as a family of serine hydrolase inhibitors.

11

Fransson, Linda. "Molecular modelling - understanding and prediction of enzyme selectivity." Licentiate thesis, KTH, School of Biotechnology (BIO), 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-10532.

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Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.

12

Veronique, Jacqueline. "Enzymes de la voie de l'acide shikimique chez les vegetaux : mecanismes mis en jeu par les hydrolases et synthese de substrats en serie quinique." Toulouse 3, 1988. http://www.theses.fr/1988TOU30100.

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13

Daâloul, Nabil. "Einsatz von Hydrolasen und Oxidoreduktasen zur Entfernung der vegetabilen Bestandteile aus der Wolle /." Aachen : Mainz, 2004. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=013110447&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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Benard, Stefan. "Chemisches Signal und biologische Antwort : Modulation der Generierung reaktiver Sauerstoffverbindungen aus neutrophilen Granulozyten /." Leipzig : AVA, Akademische Verlagsanstalt, 2000. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=009101788&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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15

Leppchen, Kathrin. "Anwendung von Saccharomyces cerevisiae in der Biotechnologie und Oberflächenchemie /." München : Verl. Dr. Hut, 2009. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=017317794&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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16

Hsieh, Yu, and 謝瑀. "Developing chemical probe for activity profile of serine hydrolases." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/28946423398059175235.

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碩士
國立陽明大學
生命科學暨基因體科學研究所
97
Serine hydrolases represent one of the largest and diverse families of enzymes comprising numerous proteases, lipases, esterases, and amidases. Serine hydrolases play important roles and also regulate numerous important functions in many organisms. Disorder of such serine hydrolases may cause different kinds of human diseases. To understand the functions of diverse serine hydrolases, it is necessary to develop a method to systematically detect or enrich serine hydrolases and thus the following studies of their biological significances. Fluorophosphonate/fluorophosphates (FP) is a well-known enzyme inhibitors of serine hydrolases. It has been demonstrated to covalently react with the catalytic serine residue of serine hydrolases and thus irreversibly inhibit the enzymatic activity. We collaborated with other laboratory to prepare an FP analogue with the function of biotin and polyethylene glycol (FP-peg-biotin) as a chemical probe to identify serine hydrolases. The following verification used SDS-PAGE, Western blot, and NeutrAvidin beads to separate, detect and enrich serine hydrolases. In summary, we anticipate this probe will help us to develop a high efficient and specific toll for studies of diverse serine hydrolases.

17

Huang, Yi-Long, and 黃義龍. "Qualitative analysis of fluorophosphonate-based probes targeting serine hydrolases." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/13313719297128260180.

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碩士
國立陽明大學
生物藥學研究所
98
Serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in human genome. However, up to 50% of these enzymes remain unannotated and their substrates are still unknown. In addition, activities of increasing serine hydrolases were shown associated with diseases like cancer neoplasia and invasiveness etc. Therefore, the systematic identification of serine hydrolases which are associated with diseases is of great importance. In this study, two novel hydrophilic fluorophosphonate (FP)-based chemical probes, including FP-edo-biotin and FP-edo-fluorescein, were synthesized and examined for their abilities to recognize as well as to pull down the serine hydrolases via covalent linkage. A recombinantly purified poly(3-hydroxybutyrate) depolymerase (PhaZ) was first used to determine the labeling kinetics and pull-down efficacy. Subsequent experiments using either site-directed mutagenesis of catalytic serine at 102 (S102A) or pre-heating treatment of PhaZ has shown an abolished labeling, indicating the activity-based labeling feature of these FP probes when targeting the active site serine. Mass spectrometric analysis of tryptic peptides from the FP-edo-biotin-labeled PhaZ has confirmed the existence of biotin-labeled peptide (99-106). In addition, I observed that in vitro β-elimination and Michael addition with 2-aminoethanethiol of probe-labeled PhaZ could detach the labeling probe and facilitate future identification of active site serine residues. Finally, the FP-edo-fluorescein has been applied to establish the serine hydrolase profiles of diverse mouse tissues and the results showed that such FP probe is capable to display cellular serine hydrolases efficiently. Further biotin pull-down and protein identification experiments are required in the future.

18

Harrison, Jenica Ledah. "The role of acanthamoeba culbertsoni serine proteases in abating microglial-like cell cytokines and chemokines /." 2009. http://hdl.handle.net/10156/2457.

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19

Hu, Rong-Chi, and 胡容綺. "Identification and Characterization of Serine Hydrolases in Senescence Using Chemical-based Proteomic Approach." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/77946475578088969137.

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碩士
國立臺灣大學
生物化學暨分子生物學研究所
104
Most of normal human somatic cells cannot divide indefinitely and eventually enter a state of irreversible proliferative arrest termed replicative senescence. Although previous researches have shown that telomere shortening is the major reason for inducing senescence in human somatic cells, factors that mediate and maintain senescence are largely unknown. Serine hydrolases is a large enzyme family which constitutes about 1% of the human proteome and many of them are involved in important physiological processes. In addition, many serine hydrolases have been shown to affect cellular senescence. Thus, the goal of this research is to identify and characterize novel serine hydrolases that participate in senescence. An activity-probe based chemical proteomic approach was applied to identify serine hydrolases with their activities been altered during senescence. Here the human lung fibroblast IMR-90 cell was used as a model in this research. Chemical probe LCL8027, which has an ethyl-benzylphosphonofluoridates reactive group, is used to label active serine hydrolases. Through comparative labeling of both young and old cells, several serine hydrolases were identified that showed alteration of labeling activities during senescence. Among them, both the activities of tripeptidyl peptidase 2 (TPPII) and protein phosphatase methylesterase 1 (PPME1) were found to be decreased during senescence. Further analyses showed that knocking down TPPII or PPME1 using small hairpin RNAs induced young IMR90 cells into senescence, suggesting a role of these two serine hydrolases in senescence. Together, this study successfully applied an activity probe-based proteomic analysis to identify novel serine hydrolases that might be involved in cellular senescence.

20

Carvalho, Luís Miguel Afonso Ramos de. "3-Oxo-β-Sultams and 4-Oxo-β-Lactams as chemical tools for activity-based protein profiling of serine hydrolases." Doctoral thesis, 2019. http://hdl.handle.net/10451/42844.

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Activity-based protein profiling (ABPP) is a technique that analyzes the dynamics in enzymatic activity in complex proteomes by using small molecular probes, deemed activity-based probes (ABPs), containing a reactive group to covalently bind enzyme catalytic residues, a tag for detection of labeled targets and a linker as a spacer and also specificity-enhancing. Different reactive groups have been developed to engage a wide range of enzymatic families but there is a constant need to create new chemical tools to expand the pool of engageable targets. In this work we evaluated two 4-membered ring chemotypes as new reactive groups for ABPP of serine hydrolases. The 3-Oxo-β-Sultam was revealed to be a highly reactive chemotype which labels a wide range of proteins with limited target occupancy. A crystallographic analysis of the reaction of 3-Oxo-β-Sultams with elastase enzymes revealed a previously unknown mechanism of inhibition of these enzymes by sulfonylation, suggesting 3-Oxo-β-Sultam compounds could be used to expand the pool of available sulfonylating tools in chemical biology. 4-Oxo-β-Lactams were shown to potently hit a selected group of serine hydrolase with high target occupancy, including human neutrophil elastase (HNE) and members of the ABHD and DPP families of enzymes. A competitive-ABPP approach revealed high potency of a library of 4-Oxo-β-Lactams to target these enzymes. 4-Oxo-β-Lactams were identified as a new chemotype for DPP8 and DPP9 inhibition. Crystallography experiments revealed a new binding mode of these enzymes with 4-Oxo-β-Lactams, highlighting that these compounds could be used to pursue selective DPP8 or DPP9 inhibitors, a highly pursued field in current medicinal chemistry.

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Guo, Jian-Liang, and 郭建良. "Conformational Analysis and QSAR for Serine Hydrolases Esterase of the Carbamyl C-N Bond of o,m,p-Di-N-Substituted Carbamyloxy Benzenes." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/68656801786077458974.

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Perez, Dianne Marie. "Structure-Function Studies of Serine Hydrolases: Synthesis of a Gene for α-Lytic Protease and the Purification and Characterization of a Mutant β-Lactamase." Thesis, 1988. https://thesis.library.caltech.edu/7523/5/Perez-dm-1988.pdf.

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The author has constructed a synthetic gene for α-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of α-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of α-lytic protease are preferred codons in E. coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the α-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The α-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating α-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β-lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_cat values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

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Franco, Flores Anna Kristyna. "The role of Alpha Beta Hydrolase 6 in the neuronal control of body weight, exercise and anxio-depressive behaviors." Thèse, 2018. http://hdl.handle.net/1866/22143.

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Wang, Yi. "Interaction of the third intracellular loop of the a2A-adrenergic [alpha-2A-adrenergic] receptor with the ubiquitin carboxyl-terminal hydrolase L1." 2006. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=016696167&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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Dissertations / Theses on the topic 'Serine hydrolases'

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