Next Generation Proteomics in the Nervous System Open Access
Mertz, Joseph (2016)
Abstract
Classical approaches to protein signaling pathways in the nervous system disregard the vast complexity of biological systems in general, and the brain especially. Large gaps remain in our understanding of the physiological and pathogenic roles of critical proteins such as the E3 ubiquitin ligase Mind bomb 1 (Mib1), the Alzheimer's disease (AD) hallmark Amyloid progenitor protein (APP), and the spliceosome subunit, U1-70K. To examine the proteins and signaling networks that interact with Mib1, APP, and U1-70K, and how they might relate to disease, we developed two novel interactomics methodologies exploring the affinity-stratified brain interactome of Mib1 and a highly controlled and affinity-stratified in vivo AD interactome of APP. Further, we thoroughly characterized the hippocampal proteomic perturbations at 3, 6, and 12 months in a newly developed transgenic mouse expressing N40K, a truncated form of U1-70K found in abundance in AD. Our findings greatly expanded the known interactomes of Mib1 and APP, established a role for Mib1 in dendritic spine development, and characterized its antagonistic interaction with the potent neurodevelopment regulator, CDKL5. We found numerous overlaps between N40K proteome perturbations and those seen in neurodegeneration (both in animal models and human), including strong decreases in the synaptic vesicle protein Synaptophysin. These methods represent improvements to the system-wide study of protein-protein interactions and the results greatly increase our understanding of the roles of these molecules, in addition to providing numerous avenues for future investigation.
Table of Contents
Acknowledgments. vi
Table of Contents. vii
List of Figures and Tables. ix
CHAPTER ONE. 1
Introduction. 1
Quantitative Proteomics. 3
Interactomics. 5
Ubiquitin Proteasome System, and the E3 Ligase Mind bomb 1. 7
Alzheimer's Disease and the Roles of APP. 10
This dissertation. 12
CHAPTER TWO. 14
Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals A Novel Role In Dendritic Spine Outgrowth. 14
MATERIALS AND METHODS. 18
Plasmids and Antibodies. 18
Affinity Purification and Sequential Elution. 18
TMT Labeling of Digested Peptides. 19
Long Gradient LC-MS/MS Analysis of TMT Labeled Peptides. 20
Protein Quantification by TMT Labeled Peptides. 21
Interaction Network Analysis. 22
Protein Preparation and Western Blot Analysis. 22
In vitro Ubiquitination Assay. 23
Primary Hippocampal Neuron Culture and Transfection. 23
Immunocytochemistry. 24
Dendritic Spine Morphological Analysis of Primary Hippocampal Neurons. 25
RESULTS. 25
Mib1 Affinity Purification from Rat Brain and Sequential Elution. 25
Sequential Elution Profiling of Mib1 Affinity-Purified Proteins by Isobaric Labeling. 29
Mib1 Interacts with Usp9x and Catenin Family Members. 36
Mib1 Ubiquitinates CDKL5 and Alters Its Localization, Abundance, and Functional Effects on Neuron Morphogenesis. 39
DISCUSSION. 44
CHAPTER THREE. 49
Biotin and Ubiquitin Labeling of Ligase Substrates (BULLS) Proteomics Analysis of Mib1 Substrates. 49
MATERIALS AND METHODS. 51
Plasmids and Antibodies. 51
Cell Culture & Transfection. 52
Protein Preparation & Western Blot. 53
RESULTS. 53
Biotinylation and Ubiquitination by BirA-Mib1, BirA-C985S, and Free BirA. 53
Biotinylation and Ubiquitination by Mib1 Truncation Mutants. 55
Modification of the BirA Acceptor Peptide Sequence. 57
DISCUSSION. 60
CHAPTER FOUR. 65
Differential Enrichment and Elution Proteomics (DEEP) Analysis of the APP Interactome. 65
MATERIALS AND METHODS. 70
AD Brain Tissue Lysate Preparation. 70
Co-Immunoprecipitation (Co-IP). 70
10-plex TMT-based Quantitative LC-MS/MS Analysis. 71
Protein Quantification by TMT Labeled Peptides. 73
Interaction Network Analysis. 74
RESULTS. 74
LC-MS/MS Analysis of APP Co-Immunoprecipitation. 74
Enrichment. 78
Differential Enrichment. 79
Differential Elution. 80
Multiple Antibody DEEP Analysis. 83
Pathway and Network Analysis. 86
DISCUSSION. 87
CHAPTER FIVE. 93
Perturbations in the N40K Transgenic Mouse Proteome. 93
MATERIALS AND METHODS. 100
Mouse Brain Tissue Lysate Preparation and Western Blot. 100
10-plex TMT-based quantitative LC-MS/MS analysis. 100
Protein Quantification by TMT Labeled Peptides. 102
Interaction Network Analysis. 103
RESULTS. 103
Western Blot and LC-MS/MS Analysis of the N40K Brain Proteome. 103
Quantitative Analysis of the N40K Proteome. 105
Examination of Spliceosome Proteins and Others Altered in Multiple Comparisons. 108
Cytoskeleton, Mitochondria, Ciliogenesis, and Other Pathways Important to Neuronal Function Exhibit Perturbations in N40K Mice. 112
DISCUSSION. 114
CHAPTER SIX. 119
General Discussion. 119
Interactomics. 120
Mounting Problems with Antibody-Based Assays. 122
General Conclusions and Looking Forward. 123
References . 126
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