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Interactome of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells : INS-1 췌장베타세포 내 AMP-activated protein kinase (AMPK)-α1 및 -β1 단백질의 Interactome 분석
DC Field | Value | Language |
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dc.contributor.advisor | 김영수 | - |
dc.contributor.author | 문성윤 | - |
dc.date.accessioned | 2017-07-14T01:15:11Z | - |
dc.date.available | 2017-07-14T01:15:11Z | - |
dc.date.issued | 2015-02 | - |
dc.identifier.other | 000000024777 | - |
dc.identifier.uri | https://hdl.handle.net/10371/121769 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 협동과정 종양생물학전공, 2015. 2. 김영수. | - |
dc.description.abstract | Introduction: As a heterotrimeric enzyme, AMP-activated protein kinase (AMPK) is a major metabolic sensor in regulating cellular energy homeostasis, and is expressed on many tissues. In particular, AMPK contributes to insulin resistance associated with type 2 diabetes. Generally, cellular processes need tight control of protein kinases, which is influenced by through their formation of complex with other substrates. Despite their crucial function in regulation and pathogenesis, there are limited information on the interaction of protein kinases.
Methods: To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -β1 subunits. Prior to perform AP-MS, recombinant 6-myc tagged AMPKα1/β1 proteins were transiently transfected in INS-1. Immunoprecipitation was performed accompanied with two different beads: anti-myc antibody-conjugated agarose and magnetic beads. After in-gel trypsin digestion, peptides were analyzed in triplicate by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Through a large scale analysis using AP-MS approach, we represented that our approach identified 381 unique proteins in the AMPKα1/β1 interactomes: 325 interactors of AMPK-α1 and 243 for AMPK-β1. The result from the magnetic beads capturing shows a higher number of identified interacting proteins than that from the agarose beads capturing. Furthermore, we identified 196 novel interactors that have not been previously characterized in the existing protein-protein interaction databases. Notably, our bioinformatics analysis suggests that the novel interactors mediated functions that are related to the regulation of actin cytoskeleton. Specifically, several proteins were linked to pancreatic beta-cell functions, including beta-cell development, beta-cell differentiation, glucose-stimulated insulin secretion, and cell-cell communication. Conclusions: Our AMPK-specific interactome study suggests that our newly identified interactors could be valuable targets for the study of AMPK signaling pathways associated with pancreatic beta cell functions. * This work is published in Scientific Reports Journal. | - |
dc.description.tableofcontents | Abstract ………………………………………………………………………………………………… i
List of tables …………………………………………………………………………………………… iii List of figures ………………………………………………………………………………………… iv List of abbreviations and symbols …………………………………………………………………… vi Contents ……………………………………………………………………………………………… 1 I. Introduction ………………………………………………………………………………………… 3 II. Materials and methods …………………………………………………………………………… 5 2.1. Reagents and materials ………………………………………………………………………… 5 2.2. Cell culture ……………………………………………………………………………………… 5 2.3. Plasmid construction and transient transfection for AP-MS ……………………………………… 5 2.4. Preparation of cell lysates ……………………………………………………………………… 8 2.5. Pulldown assay using anti-myc coupled to agarose beads ……………………………………… 8 2.6. Pulldown assay using anti-myc coupled to magnetic beads …………………………………… 8 2.7. SDS-PAGE separation, in-gel digestion, and desalting ………………………………………… 9 2.8. Direct Immunoprecipitation (IP) ………………………………………………………………… 9 2.9. LC-MS/MS analysis …………………………………………………………………………… 10 2.10. Data processing ……………………………………………………………………………… 11 2.11. Bioinformatics analysis ……………………………………………………………………… 12 2.12. Immunoblot analysis and antibodies ………………………………………………………… 12 III. Results ( I ) ………………………………………………………………………………………… 13 3. Interactome analysis using AP-MS in mammalian system ……………………………………… 13 3.1. Overall scheme for profiling of AMPK-α1 and -β1 interactomes ……………………………… 13 3.2. Identification and Characterization of AMPK-α1- and –β1- interacting proteins ……………… 20 3.3. Functional classification of AMPK-specific interactors ……………………………………… 31 3.4. Interaction of proteins related to actin cytoskeletal organization with AMPK ………………… 35 IV. Results ( II ) ………………………………………………………………………………………… 37 4. Direct immunoprecipitation in INS-1 pancreatic beta-cell ……………………………………… 37 4.1. Overall scheme for profiling of AMPK-α1 and -β1 interactomes ……………………………… 37 4.2. Identification of novel AMPK-α1- and –β1- interacting proteins ……………………………… 42 4.3. Functional classification of AMPK-specific interactors ………………………………………… 49 4.4. Interaction of proteins related to actin cytoskeletal organization with AMPK ………………… 53 V. Discussion ………………………………………………………………………………………… 55 5.1. Comparison with other proteomics studies ……………………………………………………… 60 5.2. Relationship between AMPK and substrates in the AMPK -α1 and -β1 interactome ………… 62 5.3. Regulation of actin cytoskeletal organization …………………………………………………… 64 VI. Conclusion ………………………………………………………………………………………… 66 VII. References ………………………………………………………………………………………… 68 Abstract in Korean …………………………………………………………………………………… 75 | - |
dc.format | application/pdf | - |
dc.format.extent | 3081373 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | AMP-activated protein kinase (AMPK) | - |
dc.subject | affinity purification (AP)-mass spectrometry (MS) | - |
dc.subject | pancreatic beta-cell | - |
dc.subject | interactome | - |
dc.subject | proteomics | - |
dc.subject.ddc | 616 | - |
dc.title | Interactome of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells | - |
dc.title.alternative | INS-1 췌장베타세포 내 AMP-activated protein kinase (AMPK)-α1 및 -β1 단백질의 Interactome 분석 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Sungyoon Moon | - |
dc.description.degree | Doctor | - |
dc.citation.pages | vi, 76 | - |
dc.contributor.affiliation | 의과대학 협동과정 종양생물학전공 | - |
dc.date.awarded | 2015-02 | - |
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