Poldip2: An investigation of structure and signaling pathways to elucidate the function of a novel cell cycle regulator. Open Access

Brown, David Ingram (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/gm80hv697?locale=en
Published

Abstract

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication and ROS production via Nox4. My goal is to clarify Poldip2 function by analyzing protein structure and Poldip2 regulated signaling pathways.

An in silico analysis of Poldip2 structure uncovered a highly conserved region on the surface of the protein that resembles a binding pocket. This region is solvent accessible, opening the possibility of Poldip2 binding an ion, small molecule or having enzymatic activity.

A gene microarray analysis comparing wild type and Poldip2 knockout samples uncovered key pathways such as circadian rhythms, cell cycle, mitochondrial function and metabolism that are affected by Poldip2 depletion. A further analysis of Poldip2 function in the cell cycle was performed.

Data presented in this study support a novel role for Poldip2 in the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2-/- embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2-/- mouse embryonic fibroblasts exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to senescence, as measured by p16 and p19 expression. There was also no change in apoptosis by Annexin V staining. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2-/- cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Cdk1 and CyclinA2 are downregulated in Poldip2-/- cells, and these changes are reversed by transfection with SV40 large T-antigen (SV40 LTA), suggesting that Poldip2 may target the E2F pathway. In contrast, p21 expression is unaffected by SV40 LTA transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative diseases such as cancer and atherosclerosis.

Table of Contents

Acknowledgements
Table of Contents
Index of Figures
List of Symbols and Abbreviations
Chapter 1: Introduction...1 Poldip2 Overview...2 Poldip2 background 2
Poldip2 influence on Nox protein function...7 Poldip2 and Proliferation...8 Proliferation and mitosis...8
Cyclins and Cdks...11
Cell Cycle inhibitors...12 Senescence...13 Replicative senescence/telomere...14
Redox/stress-induced senescence...16
Oncogene-induced senescence (OIS)/Tumor suppressor loss senescence...17
Immortality: MEFs...17
Biomarkers of Senescence...18
Senescence and the cell cycle...19 Proliferative disease...19
Pharmacological inhibitors of the cell cycle...21
Dissertation Aims...23
Chapter 2: Poldip2 Structure...24 Introduction...25

Methods...27

Sequence alignment...27
In silico protein folding...29
Site directed mutagenesis...29

Experimental Results...29

Poldip2 Domain Structure...29
In silico Poldip2 modeling: YccV domain...30
In silico Poldip2 modeling: ApaG domain...33
In silico Poldip2 modeling: Full length Poldip2...36
Sequence and structural comparison between bacterial and human ApaG...38
Investigating Poldip2 function and binding to p22phox...38

Conclusions...41


Chapter 3: Poldip2 gene array and pathway analysis...46

Introduction...47
Methods...47

Preparation of cDNA from mouse aortas...47
Preparation of cDNA from mouse embryonic fibroblasts...48
Gene array...48
Statistics: Significance Analysis of Microarrays...48

Results...48
Array Results...48

Circadian clock genes...49
Cell cycle genes...53
Mitochondrial function genes...55
Metabolism...56

Conclusions...56


Chapter 4: Poldip2 in the cell cycle...60
Introduction...61
Methods...62

Animals...62
Preparation of mouse embryonic fibroblasts (MEFs)...63
Cell culture...63
Growth curve/Doubling curve...63
Cell cycle analysis...63
Apoptosis...64
MEF immortalization...64
Western blot...64
RNA extraction/qRT-PCR...65
Statistics...65

Results...66

Poldip2 knockout results in reduced fetal weight and perinatal lethality...66
Poldip2 knockdown causes reduced cell growth and doubling time in MEFs...66
Poldip2 knockdown does not alter expression of senescence markers or increase apoptosis...69
Poldip2 knockout arrests growth in G1 and G2/M...71
p53 phosphorylation and downstream targets are altered in Poldip2-/- MEFs...73
E2F target genes exhibit reduced expression in Poldip2-/- cells...73
SV40 immortalization restores Poldip2 growth to wild type levels...76

Discussion...79


Chapter 5: Conclusion...84
Overview...85

Summary and Significance...86
What can we learn from the structure of Poldip2?...87
What do Poldip2 knockout gene arrays uncover about Poldip2 signaling?...91
How does Poldip2 regulate the cell cycle?...93

Concluding statement...96


Appendix A: Nox proteins in signal transduction...98
NADPH oxidases: a brief history...99

NADPH oxidase activation...101
Physiological targets of Nox-derived ROS...101
Compartmentalization...101
Physiological roles...103
Nox proteins in disease...104

Nox-derived ROS...104

Superoxide...104
Hydrogen peroxide...105

Nox proteins in signal transduction...106
Nox1...106

Tissue distribution and physiological function...106
Mechanisms of activation...108
Subcellular localization...108
Signal transduction...109

Nox2...113

Tissue distribution and physiological function...113
Mechanisms of activation...113
Subcellular localization...114
Signal transduction...114

Nox3...116

Tissue distribution and physiological function...116
Mechanisms of activation...117
Subcellular localization...118
Signal transduction...118

Nox4...118

Tissue distribution and physiological function...118
Mechanisms of activation...119
Subcellular localization...120
Signal transduction...120

Cell and tissue specificity of Nox proteins...123

References...125

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