Next Generation Proteomics in the Nervous System 公开

Mertz, Joseph (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/44558d79n?locale=zh
Published

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 List of Figures and Tables Figure 2.1. Sequential elution strategy of Mib1 affinity purification. .................... 28 Figure 2.2. Grouping proteins by their sequential elution profiles. ...................... 33 Figure 2.3. Mib1 interaction partners participate in several important signaling pathways.............................. 35 Figure 2.4. Interconnectivity in Mib1 Ubiquitin Proteasome System interactome.......................................... 36 Figure 2.5. Mib1 colocalizes with Usp9x (FAM) and 3 members of the catenin family.................................... 38 Figure 2.6. Mib1 colocalizes with and downregulates CDKL5. ........................... 41 Figure 2.7. Mib1 inhibits dendritic spine outgrowth and limits pro-outgrowth effects of CDKL5 in neuronal culture..................... 43 Figure 3.1. Cloning of the BirA sequence into the HA-Mib1 construct results in HA tagged BirA-Mib1...................... 54 Figure 3.2. BirA-C985S ligase dead negative control produced similar levels of positive signal to active form............. 55 Figure 3.3. BULLS gene constructs included a full length test protein and two truncations used for negative controls...... 56 Figure 3.4. Western blot examination of truncation mutants shows decreased but still substantial positive signal....................... 57 Figure 3.5. Modified of the AP sequence decreases signal in both test and negative control samples................................... 58 Figure 3.6. Examination of multiple incubation periods shows increases in signal from both test and negative control............. 60 Figure 3.7. Proposed models of technical pitfalls. ................................................... 63 Figure 4.1. DEEP analysis utilizes multiple comparisons of APP IP-MS proteomics from human Alzheimer's disease tissue. ................................... 75 Figure 4.2. DEEP Co-Immunoprecipitation an LC-MS/MS Analysis ................... 78 Figure 4.3. Histogram Analysis and Gaussian Fitting of Data from Ctl1 and Ab1 Comparisons........................................ 79 Figure 4.4. Histogram Analysis and Gaussian Fitting of Data from Differential Enrichment Comparisons for both Ab1 and Ab2............. 81 Table 4.1. Summary of Key DEEP Analysis Values ........................................... 83 Figure 4.5. Multiple DEEP analysis .................................................................... 85 Table 4.2. Highlights of Pathways and Contained Proteins from Tiers 1 and 2. . 87 Figure 5.1. Spliceosome subunits are enriched in Alzheimer's disease. ................. 95 Figure 5.2. U1-70K is a subunit of the U1 snRNP complex and contains several defined domains...................................... 97 Figure 5.3. The N40K transgenic mouse exhibits decreased full length U1-70K as well as deficits in spatial learning and working memory............ 99 Figure 5.4. N40K mouse brain lysate samples used in this study exhibit even levels of transgene and decreased U1-70K levels........................ 104 Figure 5.5. LC-MS/MS analysis detected 9 peptides from U1-70K and all originated from the N40K region. .................................................... 105 Figure 5.6. Overview of the comparisons in this study demonstrate most proteins cluster around zero change in abundance............................ 107 Figure 5.7. Proteins specific to U1, and no other spliceosome subunits exhibit roughly 2 fold increases in Tg over WT...................................... 109 Figure 5.8. 492 proteins were significantly altered in our core age-specific analyses and many others altered in overall and age-related comparisons..111 Figure 5.9. There is significant, but incomplete overlap of proteins altered in both hippocampus and cortex at 12 mos........................... 112 Figure 5.10. Several biological functions are enriched in comparisons from multiple time points and similar functions exhibit age-related changes. .... 114

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