Understanding signal transduction systems in prokaryotes and eukaryotes Open Access

Jia, Xin (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/9s161641g?locale=en
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Abstract

The ability of cells to adapt to dynamic and diverse environment by altering their cellular behavior is crucial for adaptation and survival. However, the biochemical mechanisms and signaling transductions pathways involved in adaptation and survival are still poorly understood. In this dissertation, various analytical and biochemical techniques were employed to understand novel signal transduction pathways in both prokaryotic and eukaryotic systems.

A novel putative stressosome complex from the Gram-negative marine bacterium Vibrio brasiliensis that contained a heme-bound sensor protein and had the ability to sense environmental O2 was described. The novel stressosome altered its signaling pathway in response to various environmental O2 levels. The described complex presents an opportunity to interrogate the effects of ligand-dependent stressosome signaling both in vitro and in vivo, as well as provides further insights into its potential role in regulating virulence gene expressions in limited O2 condition in the Vibrio genus.

Secondly, I reported our recent progress toward development of an efficient protocol to identify enzyme members of the 3',5'-cCMP signaling pathways. Following previously published protocols, we partially purified a putative soluble cytidylate cyclase that synthesizes 3',5'-cCMP from CTP in rat livers. Additionally, we also isolated a potential 3',5'-cCMP-binding protein, THO complex subunit 5, using 3',5'-cCMP affinity chromatography. The THO complex subunit 5 is involved in cellular proliferation by controlling transcription and mRNA export, suggesting 3',5'-cCMP may be involved in regulating transcription through THO subunit 5.

Lastly, we developed a sensitive and versatile method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cells. This protocol allows for comparison of multiple cNMPs in the same systems and was used to examine the relationship between tissues levels of cNMPs in various systems. Utilizing this analytical method, I reported the first identification and quantification of 2',3'-cIMP in mammals. The developed analytical method offers a tool for quantification of cNMPs levels in cells and tissues of varying disease states, which will provide insight into the roles of cNMPs in vivo.

Table of Contents

Chapter 1: General Introduction…………………………………………………………. 1
1.1 General introduction…………………………………………………………. 2
1.2 Prokaryotic Signaling Transduction…………………………………………. 3
1.2.1 Introduction…………………………………………………………….. 3
1.2.2 Heme-Based Sensor Proteins in Bacteria……………………………… 4
1.2.2.1 Introduction………………………………………………………. 4
1.2.2.2 Heme-Based Globin-Coupled Sensors (GCSs)………………….. 6
1.2.3 Prokaryotic Kinase Pathway…………………………………………… 9
1.3 Mammalian Second Messenger Signal-Transduction Pathways…………… 12
1.3.1 Introduction…………………………………………………………… 12
1.3.2 The Second Messenger Signaling Pathway…………………………... 14
1.3.3 Adenylate Cyclase……………………………………………………..16
1.3.4 Discovery of Guanosine 3',5'-Cyclic Monophosphate (3',5'-cGMP) and
Guanylate Cyclase……………………………………………………………………… 19
1.3.5 3',5'-Cyclic Nucleotide Phosphodiesterase (PDE)…………………… 21
1.3.6 3',5'-cAMP-Dependent Protein Kinase (PKA)………………………. 22
1.3.7 Cytidine 3',5'-Cyclic Monophosphate (3',5'-cCMP)………………… 23
1.3.8 Early Characterization of Cytidylate Cyclase………………………… 24
1.3.9 Discovery of 3',5'-cCMP Phosphodiesterase (PDE) and Potential 3',5'-
cCMP-Dependent Protein Kinase………………………………………………………. 25
1.4 Eukaryotic 2',3'-cAMP-Adenosine Pathway………………………………. 26
1.4.1 Introduction…………………………………………………………… 26

1.4.2 2',3'-cAMP-Adenosine Pathway……………………………………... 27
1.5 Aim and Scope of the Dissertation…………………………………………. 30
1.6 References…………………………………………………………………... 32
Chapter 2: A Novel O2-Sensing Stressosome from a Gram-Negative Bacterium……… 48
2.1 Introduction…………………………………………………………………. 49
2.2 Results and Discussion……………………………………………………... 54
2.2.1 Expression and Purification of the Stressosome Components………... 54
2.2.2 Kinase Activity of RsbT in Controlling Stressosome-Regulated
Pathways………………………………………………………………………………... 58
2.2.3 Sensing in Fully Reconstituted Stressosome Complexes…………….. 60
2.2.4 Stressosome Complex Structural Analysis…………………………… 68
2.2.5 Conclusion……………………………………………………….…… 69
2.3 Experimental…………………………………………………………………70
2.3.1 Cloning and Site-directed Mutagenesis………………………………. 70
2.3.2 Protein Expression and Purification…………………………………... 72
2.3.3 Serine kinase autothiophosphorylation and thiophosphorylation assay
using ATPγS.…………………………………………………………………………… 72
2.3.4 Western blot analysis……………………………………………….… 74
2.3.5 O2 dissociation rate…………………………………………………… 74
2.3.6 Stressosome complex structural analysis……………………………... 74
2.3.7 RsbR autooxidation experiment………………………………………. 75
2.4 References…………………………………………………………………... 75

Chapter 3: Study of 3',5'-cCMP as a Putative Mammalian Second Messenger- Progress
Toward Identification of 3',5'-cCMP-related Enzymes in Mammalian Tissues……….. 79
3.1 Introduction…………………………………………………………………. 80
3.2 Results and Discussion……………………………………………...……… 83
3.2.1 Approach I: Identification of cCMP-Related Enzymes through Protein
Purification……………………………………………………………………………… 83
3.2.1.1 Partial Purification and Properties of Cytidylate Cyclase Activity
……………………………………………………………………………………………83
3.2.1.2 Partial Purification and Properties of cCMP-Specific
Phosphodiesterase (PDE) Activity……………………………………………………… 91
3.2.2 Approach II: 3',5'-cCMP Matrices for the Affinity Purification of
cCMP-binding Protein………………………………………………………………….. 95
2.3 Conclusion………………………………………...……………………… 104
3.4 Materials and Methods……………………………………..……………… 106
3.4.1 Materials…………………………………………………………..… 106
3.4.2 Partial purification of cytidylate cyclase…………...………………... 107
3.4.2.1 Soluble cytidylate cyclase preparation………………………… 107
3.4.2.1 Membrane-bound cytidylate cyclase preparation……………... 108
3.4.3 Partial purification of cCMP-specific PDE………………………….. 108
3.4.4 Cytidylate cyclase activity assay……………………………………. 109
3.4.5 cCMP PDE activity assay…………………………………………… 110
3.4.6 HPLC optimized conditions…………………………………………. 110
3.4.7 HRMS optimized conditions………………………………………… 111

3.4.8 Size exclusion chromatography optimized conditions……………… 111
3.4.9 Synthesis of intermediate 2 in Scheme 3.1………………………….. 111
3.4.10 cCMP-phosphate probe synthesis………………………………….. 112
3.4.11 Synthesis of N, N'-dicyclohexyl-4-morpholinecarboxamidinium cCMP
salt 3 in Scheme 3.2…………………………………………………………………… 112
3.4.12 Synthesis of imidazole-activated cCMP 4 in Scheme 3.2…………. 113
3.4.13 Synthesis of 2'-OH-cCMP probe…………………………………... 113
3.4.14 Determination of coupling efficiency of cNMPs with DADPA resin
using spectrophotometric analysis……………………………………..……………… 114
3.4.15 Preparation of enzyme fraction for cCMP affinity chromatography. 115
3.4.16 cCMP matrices for the affinity purification of cCMP-binding protein in
rat brains……………………………………………………………………………….. 115
3.4.17 Preparation of in-gel tryptic digest samples………………………... 116
3.4.18 MALDI-MS optimized conditions……………………………….… 116
3.4.19 Statistical analysis………………………………………………..… 116
3.5 References…………………………………………………………………. 117
Chapter 4: Study of 3',5'-cCMP as a Putative Mammalian Second Messenger- A Facile
and Sensitive Mass Spectrometry-Based Method for Quantification of Cyclic Nucleotide
Monophosphates in Mammalian Organs……………………………………………… 121
4.1 Introduction……………………………………………………………….. 122
4.2 Results and Discussion………………………………….………………… 126
4.2.1 Optimization of LC-MS/MS Analytical Method……………………. 126
4.2.2 LC-MS/MS Method Calibration and Limits of Detection…………... 131

4.2.3 Optimization of Extraction Method………………………….……… 131
4.2.4 Method Validation and Reproducibility Tests………………………. 138
4.2.5 Applications……………………………………………………….… 141
4.2.6 Versatility of the Method……………………………………………. 152
4.2.7 Discussion…………………………………………………………… 153
4.2.8 Conclusions…………………………..……………………………… 155
4.3 Experimental………………………….…………………………………… 155
4.3.1 Materials…………………………..………………………………… 155
4.3.2 Calibration Curves…………………………………………………... 157
4.3.3 NIH-3T3 Cell Growth……………………………………………..… 157
4.3.4 cNMP Extraction from Rat Organs…………………………..……… 157
4.3.5 cNMP Extraction from NIH-3T3 Cell Line………………….……… 158
4.3.6 LC-MS/MS Sample Preparation………………….…………………. 159
4.3.7 LC-MS/MS Optimized Conditions………………….………………. 159
4.3.8 Statistical Analysis………………….……………………………….. 160
4.3.9 Method Validation Test………………….………………………….. 161
4.3.10 Method Reproducibility Test………………….…………………….161
4.3.11 Synthesis of 2',3'-cIMP………………….…………………………. 161
4.5 References………………….………………………….…………………... 162
Chapter 5: Conclusions and Perspectives………………….………………………….. 172
5.1 Summary………………….………………………….……………………. 173
5.2 A starting point for studying a novel stressosome-regulated pathway in Gramnegative
bacterium Vibrio brasiliensis………………….…………………………….. 173

5.3 Continuing to purify and investigate the functions of 3',5'-cCMP-related
enzymes in mammals………………….………………………….…………………… 174
5.4 Development of a useful tool for identification and quantification of various
typical and atypical cNMPs………………….………………………….…………….. 175
5.5 References………………….………………………….…………………... 176

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