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Functional characterization of BCL11B-a human transcription factor and UBR1-a human protein ubquitin ligase aided by proteomic analysis [Elektronische Ressource] / vorgelegt von Narasimha Kumar Karanam

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200 pages
Functional characterization of BCL11B-a human transcription factor and UBR1-a human protein ubquitin ligase aided by proteomic analysis I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Narasimha Kumar Karanam geboren am 16.07.1979 Nandyal, India Dekan: Prof. Dr. rer. nat. Klaus Fesser 1. Gutachter: Prof. Dr. rer. nat. Uwe Volker 2. Gutachter: Prof. Dr. rer. nat. Walter Halangk Tag der Promotion: 22nd November 2010 … … ..dedicated to my dear parents and wife Dissertation Content Content Content ............................................................................................................................................. 1 1. Abbreviations .......................... 5 2. Summary . 7 2.1. Characterization of the role of BCL11b in Human T cell lymphomas......................... 7 2.2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients‟ lymphoblasts cell lines .....................
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Functional characterization of BCL11B-a human
transcription factor and UBR1-a human protein ubquitin
ligase aided by proteomic analysis







I n a u g u r a l d i s s e r t a t i o n
zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald




vorgelegt von
Narasimha Kumar Karanam
geboren am 16.07.1979
Nandyal, India











































































Dekan: Prof. Dr. rer. nat. Klaus Fesser

1. Gutachter: Prof. Dr. rer. nat. Uwe Volker

2. Gutachter: Prof. Dr. rer. nat. Walter Halangk

Tag der Promotion: 22nd November 2010

























… … ..dedicated to my dear parents and wife

































Dissertation Content
Content
Content ............................................................................................................................................. 1
1. Abbreviations .......................... 5
2. Summary . 7
2.1. Characterization of the role of BCL11b in Human T cell lymphomas......................... 7
2.2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome
using UBR1 knockout mice and JBS patients‟ lymphoblasts cell lines .......................... 8
3. Introduction ........................................................................................................................... 11
3.1. Medical research and available technologies ............................... 11
3.2. Technical introduction: Proteomics .............................................................................. 11
3.2.1. Separation techniques: ........................... 13
3.2.2. Mass spectrometry: ................................................................................................ 16
3.2.3. Identification of proteins:....................... 18
3.3. BCL11b role in T lymphomas 19
3.4. Johanson Blizzard Syndrome ........................................................................................ 22
3.4.1. UBR1 protein .......................................... 24
3.4.2. N-end rule pathway of degradation ....................................................................... 25
4. Materials and Methods ......... 28
4.1. Materials ......................................................................................................................... 28
4.1.1. Chemicals................ 28
4.1.2. Enzymes and Substrates 30
4.1.3. Animals ................... 30
4.1.4. Antibodies ............................................................................................................... 31
4.1.5. Instruments.............. 31
4.1.6. Kits .......................... 32
4.1.7. Cell culture material ............................................................................................... 32
4.1.8. Softwares................................................. 33
4.2. Methods .......................... 34
4.2.1. Cell culture .............................................................................. 34
4.2.2. Cell stocks preparation ........................... 34
4.2.3. siRNA and cell transfections ................. 35
4.2.4. Determination of apoptosis: Annexin V binding assay ....... 35
4.2.5. Cytofluorometric analysis of mitochondrial transmembrane potential (Δψ ): .. 36 m
4.2.6. Cell lysate preparation ........................................................................................... 36
4.2.7. Bradford‟s method of protein estimation: ............................. 37
4.2.8. 1D SDS-PAGE: ...................................... 37
4.2.9. Fluorescent dye labelling for DIGE ...................................... 38
4.2.10. Rehydration ......................................... 39
4.2.11. IEF (Iso Eletric Focusing) .................................................. 39
4.2.12. 2D SDS-PAGE second dimension .... 40
4.2.13. DIGE scanning.................................................................... 40
4.2.14. Silver staining method of proteins visualization .............. 41
4.2.15. Image analysis .................................... 41
4.2.16. GeneSpring analysis ........................................................................................... 42
4.2.17. Assessment of protein phosphorylation by staining with Pro-Q® diamond
staining .................................................. 43
4.2.18. SYPRO® Ruby staining .................... 43
4.2.19. Protein identification .......................................................................................... 44
4.2.20. Western blotting ................................. 45
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4.2.21. DNA isolation ..................................................................................................... 48
4.2.22. Uracil N-Glycosylase (UNG) assay .. 48
4.2.23. Agarose gel electrophoresis ............... 49
4.2.24. Animal experiments ........................................................................................... 49
4.2.25. Induction of acute pancreatitis using Caerulein ............... 50
4.2.26. Measurement of enzyme levels in serum .......................... 50
4.2.27. Measurement of myeloperoxidase (MPO) activity in lung and pancreas
homogenates ......................................................................................................... 51
4.2.28. Detection of protease activity in pancreatic homogenates............................... 52
4.2.29. Histopathology ... 52
4.2.30. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling
(TUNEL) assay ..................................................................................................... 53
4.2.31. Acinar cell preparation and in-vivo elastase activity measurment .................. 54
4.2.32. Histological scoring............................ 55
4.2.33. Measurment of cytokines by CBA kit............................... 56
4.2.34. Simultaneous Calcium signal and protease activity measurements ................ 56
4.2.35. In vivo trypsin activity measurements using trypsin specific inhibitor S-124 56
4.2.36. In vivo trypsin activity measurements using RGS4 specific inhibitor CCG-
4986. ...................................................................................................................... 57
4.2.37. Data presentation and statistical analysis .......................... 58
5. Results ................................................................................................................................... 59
5.1. Proteome and functional analysis of BCL11b induced cell death in human T cells . 59
5.1.1. Induction of apoptosis by knockdown of BCL11B in Jurkat and huT78 cells but
not Jurkat BCLxL .... 59
5.1.2. Analysis of the effects of the reduction of Bcl11b levels on the protein pattern of
Jurkat and huT78 cells - BCL11B knockdown predominantly triggers increases
of protein levels ...................................................................................................... 61
5.1.3. Identification of Bcl11b regulated proteins by mass spectrometry ..................... 65
5.1.4. Global protein degradation upon BCL11B knockdown ....... 71
5.1.5. Pro Q® diamond staining/ Phosphoproteome analysis ....... 73
5.1.6. Influence of reduction of BCL11B levels on protein phosphorylation in Jurkat
cells ......................................................................................................................... 74
5.1.7. Influence of reduction of BCL11B levels on protein phosphorylation in HuT
cells 76
5.1.8. Analysis of the effect of reductions of BCL11B levels in Jurkat BCLXL cells . 77
5.1.9. Analysis of differentially expressed proteins in Jurkat BCL11B overexpressing
cells ......................................................................................................................... 82
5.1.10. ERM-proteins (ezrin, radixin, moesin) are overexpressed and
hyperphosphorylated upon BCL11B knockdown ............... 87
5.1.11. BCL11B knockdown reduces the mitochondrial membrane potential ............ 89
5.1.12. BCL11b knock down results in up regulation of dUTPase enzyme and uracil
misincrporation in DNA....................................................................................... 90
5.1.13. Validation of other target proteins by Western blot analysis .......................... 92
5.1.14. Validation of regulated proteins in BCL11B overexpression cells ................. 93
5.2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome
using UBR1 knockout mice and JBS patients‟ lymphoblasts cell lines. ....................... 94
5.2.1. Development of a protein based assay to detect subclinical Johanson-Blizzard-
Symdrome ............................................................................................................... 94
5.2.1.1. Proteome analysis of JBS patients lymphoblasts ............. 94
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5.2.1.2. Analysis of differentially expressed proteins by 2D-DIGE in JBS patients
lymphoblast ........................................................................................................... 95
5.2.1.3. Principle component analysis (PCA) and cluster analysis ............................... 96
5.2.1.4. Quantification of UBR1 by western blot ........................................................ 101
5.2.1.5. Hierarchical clustering of JBS patients and controls ..... 102
5.2.2. Characterization of mechanisms of pancreatic insufficiency in a murine model...
……………………………………………………………………………………….106
5.2.2.1. Comparative analysis of protein patterns of the pancreas of wild type and
UBR1 KO mice without or with pancreatitis.................................................... 106
5.2.2.2. Time-resolved analysis of the effects of caerulein induced pancreatitis ...... 124
5.2.2.3. Measurement of protease activity .................................................................... 125
5.2.2.4. Cytokines measurement ................... 128
5.2.2.5. Measurement of intracellular elastase activity and necrosis rate in isolated
acini…… ............................................................................................................. 129
5.2.2.6. Histology/ HE staining ..................... 131
5.2.2.7. TUNEL assay.... 133
5.2.2.8. Identification and targeting of probable UBR1 substrates responsible for
UBR1 knockout phenotype ................................................................................ 135
5.2.2.9. Impaired trypsin degradation and hereby prolonged activation of protease
activity does not influence the phenotype ......................... 135
5.2.2.10. Identification of RGS4 as a direct substrate of UBR1 ............................... 137
5.2.2.11. Influence of loss of UBR1 on calcium signalling ....................................... 138
5.2.2.12. Inhibition of RGS4 rescues the pancreatic phenotype of UBR1 knockout mice
…………………………………………………………………………...140
6. Discussion ........................................................................................................................... 143
6.1. Characterization of the role of BCL11b in Human T cell lymphomas..................... 143
6.2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome
(JBS) using UBR1 knockout mice and JBS patients‟ lymphoblasts cell lines.................... 148
6.2.1. JBS-desease mechanisms and development of a diagnostic proteomic signature
……………………………………………………………………………… 148
6.2.2. Characterization of mechanisms of pancreatic insufficiency in a murine model
………………………………………………………………………………...151
7. References ........................................................................................................................... 161
EIDESSTAATLICHE ERKLÄRUNG ...................... 187
Curriculum Vitae ......................... 189
Scientific Contributions .............................................................................................................. 190
Acknowledgements ..................... 193

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