Oxidative cysteine catabolism in humans: characterization of the oxygenation and transamination reaction
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| Format: | Thesis Book |
| Language: | English |
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2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VI, 129 S. Ill., graph. Darst. |
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| adam_text | Titel: Oxidative cysteine catabolism in humans
Autor: Arjune, Sita
Jahr: 2014
Table of contents
List of figures............................................I
List of tables...............................................II
Abstract............................................V
Zusammenfassung...........................VI
1. Introduction..............................1
1.1 Importance of cysteine in biological processes........................................................1
1.2 Human cysteine catabolism.....................................................................................1
1.2.1 Oxidative cysteine catabolism..............................................................................3
1.2.2 Non-oxidative cysteine catabolism to hydrogen sulfide........................................4
1.2.3 Hydrogen sulfide and thiosulfate metabolism.......................................................6
1.3 Cysteine Dioxygenase.............................................................................................7
1.3.1 Structure and Mechanism of CDO.......................................................................8
1.3.2 Kinetic properties of CDO...................................................................................10
1.3.3 Regulation of CDO..............................................................................................11
1.3.4 Murine knockout of CDO.....................................................................................12
1.4 Aspartate Aminotransferase...................................................................................13
1.4.1 Localization and structure of AATs.....................................................................13
1.4.2 Function and substrate diversity of AATs............................................................14
1.5 Sulfite Oxidase.......................................................................................................15
1.6 Cysteine metabolism in health and disease............................................................17
1.6.1 Isolated Sulfite Oxidase and Molybdenum cofactor deficiency............................19
1.6.2 Biochemical characteristics of SOD and MoCD..................................................20
1.6.3 Treatment options for SOD and MoCD...............................................................22
1.6.4 Aims of work.......................................................................................................24
2. Results.........................................................................................................................25
Part I: Cysteine Dioxygenase............................................................................................25
2.1 Cloning, expression and purification of human CDO..............................................25
2.2 Kinetic characterization of human CDO..................................................................26
2.3 Impact of Cys-Tyr crosslink on CDO activity...........................................................28
2.3.1 Generation of CDO cross-linked variants............................................................28
2.3.2 Kinetic characterization of the CDO cross-linked variants...................................30
2.4 Mechanism of activity enhancement through crosslink formation...........................31
2.4.1 Iron chelation......................................................................................................32
2.4.2 Effect of iron on protein activity of wt and crosslink-free CDO.............................32
2.4.3 Determination of iron binding affinity using isothermal titration calorimetry.........33
2.5 Inhibition of CDO....................................................................................................34
2.5.1 Development of a well plate-based CDO assay..................................................34
2.5.2 Product-based inhibition of CDO.........................................................................36
2.5.3 Identification of CDO inhibitors............................................................................36
2.5.1 Characterization of the inhibition of CDO using HPLC........................................38
2.5.2 In silico modeling of CDO inhibitors....................................................................38
2.6 Role of CDO in the brain........................................................................................40
2.6.1 Expression pattern of CDO in mouse tissues......................................................40
2.6.2 CDO activity in different mouse tissues...............................................................42
2.7 Role of CSA in the brain.........................................................................................43
2.7.1 Toxicity of CSA in cortical mouse neurons..........................................................43
2.7.2 Investigation of CSA transport in cortical neurons...............................................45
2.7.3 Identification of a potential CSA transporter in cortical neurons..........................48
2.7.4 Influence of CSA and SSC on glutamate transport.............................................50
Part II: Conversion of CSA to sulfite..................................................................................51
2.8 Cloning, expression and purification of the two human AAT isoforms.....................51
2.8.1 Localization of human AAT isoforms...................................................................52
2.9 Expression of AAT isoforms in mouse tissues........................................................53
2.10 Kinetic characterization of human AAT isoforms....................................................53
2.10.1 Substrate specificity of AATs..............................................................................55
2.10.2 Kinetic characterization of cytosolic and mitochondrial AAT................................55
2.11 Characterization of the putative AAT reaction product ß-sulfinylpyruvate...............58
2.11.1 Development of a coupled enzyme assay of AAT and SO..................................58
2.11.2 Kinetics of sulfite formation from CSA.................................................................60
2.11.3 Stopped-flow experiment using coupled enzyme assay......................................61
2.12 Compartmentalization of sulfite formation between cytosol and mitochondria........62
2.12.1 Assay setup for sulfite identification through SSC formation...............................63
2.12.2 Characterization of the contribution of AAT isoforms to SSC formation...............64
Discussion..........................................................................................................................67
Cysteine Dioxygenase: biochemical and mechanistic aspects..........................................67
Role of CSA in the brain...................................................................................................72
Role of aspartate aminotransferase in sulfite synthesis.....................................................77
CDO inhibition: possible approach towards sulfite toxicity disorders.................................82
3. Materials and Methods................................................................................................85
3.1 Chemicals and Enzymes.......................................................................................85
3.2 Instruments............................................................................................................85
3.3 Strains and Vectors................................................................................................86
3.4 Molecular biological methods.................................................................................88
3.4.1 Cloning strategy..................................................................................................88
3.4.2 Generation of chemical competent cells.............................................................88
3.4.3 Heat shock transformation..................................................................................89
3.4.4 Plasmid amplification and purification.................................................................89
3.5 Biochemical methods.............................................................................................90
3.5.1 Protein Expression................................90
3.5.2 Protein purification........................................90
a. Immobilized metal ion affinity chromatography..........................................................90
b. Size exclusion chromatography.................................................................................90
c. Anion exchange chromatography..............................................................................91
3.5.3 Determination of protein concentration...............................................................91
3.5.4 In vitro generation of crosslinked ODO................................................................91
3.5.5 CDO activity assay.............................................................................................92
3.5.6 AAT activity assay..............................................................................................92
3.5.7 Quantification of amino acids through HPLC.......................................................92
3.5.8 Well plate-based CDO activity assay..................................................................93
3.5.9 Coupled-enzyme assay......................................................................................94
3.5.10 SDS-PAGE.........................................................................................................94
3.5.11 Immunoblotting...................................................................................................95
3.6 Biophysical methods..............................................................................................97
3.6.1 Isothermal titration calorimetry (ITC)...................................................................97
3.7 Stopped flow experiments......................................................................................9?
3.8 Cellular biological methods.....................................................................................98
3.8.1 Mammalian cell culture.......................................................................................98
3.8.2 Neuronal cell culture...........................................................................................98
3.8.3 Transfection of cortical neurons..........................................................................93
3.8.4 Immunohistochemistry........................................................................................99
3.8.5 Image analysis....................................................................................................99
3.8.6 MTT assay..........................................................................................................99
3.8.7 Amino acid transport assay...............................................................................100
4. Appendix....................................................................................................................101
4.1 Buffers and media................................................................................................101
4.2 Markers................................................................................................................102
4.3 Vectors.................................................................................................................102
4.4 Primers.................................................................................................................103
4.5 Sequences...........................................................................................................104
4.6 HPLC Calibration.................................................................................................106
4.7 Supplementary data of docking experiments........................................................107
Acknowledgement............................................................................................................108
References........................................................................................................................110
Erklärung..........................................................................................................................127
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| physical | VI, 129 S. Ill., graph. Darst. |
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| spelling | Arjune, Sita Verfasser (DE-588)1062974786 aut Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction vorgelegt von Sita Arjune 2014 VI, 129 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Köln, Univ., Diss., 2014 Zsfassung in dt. und engl. Sprache Katabolismus (DE-588)4234092-5 gnd rswk-swf Cystein (DE-588)4148687-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Cystein (DE-588)4148687-0 s Katabolismus (DE-588)4234092-5 s DE-188 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027923042&sequence=000004&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Arjune, Sita Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction Katabolismus (DE-588)4234092-5 gnd Cystein (DE-588)4148687-0 gnd |
| subject_GND | (DE-588)4234092-5 (DE-588)4148687-0 (DE-588)4113937-9 |
| title | Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction |
| title_auth | Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction |
| title_exact_search | Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction |
| title_full | Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction vorgelegt von Sita Arjune |
| title_fullStr | Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction vorgelegt von Sita Arjune |
| title_full_unstemmed | Oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction vorgelegt von Sita Arjune |
| title_short | Oxidative cysteine catabolism in humans |
| title_sort | oxidative cysteine catabolism in humans characterization of the oxygenation and transamination reaction |
| title_sub | characterization of the oxygenation and transamination reaction |
| topic | Katabolismus (DE-588)4234092-5 gnd Cystein (DE-588)4148687-0 gnd |
| topic_facet | Katabolismus Cystein Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027923042&sequence=000004&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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