Full metadata record
| DC Field | Value | Language |
|---|---|---|
| dc.contributor | Department of Applied Biology and Chemical Technology | en_US |
| dc.contributor.advisor | Zhao, Yanxiang (ABCT) | - |
| dc.creator | Qiu, Xianxiu | - |
| dc.identifier.uri | https://theses.lib.polyu.edu.hk/handle/200/10398 | - |
| dc.language | English | en_US |
| dc.publisher | Hong Kong Polytechnic University | - |
| dc.rights | All rights reserved | en_US |
| dc.title | Biochemical and structural studies of beclin2 in autophagy regulation and GPCR signaling | en_US |
| dcterms.abstract | Autophagy is an evolutionarily conserved cellular catabolic process that plays a critical role in maintaining cellular homeostasis through lysosomal degradation of aged or dysfunctional cytoplasmic materials. A recent study reveals that Beclin2, a novel homolog of Beclin1 in mammals, plays a dual role in autophagy modulation and lysosomal sorting of GPCR. Similar to Beclin1, Beclin2 acts as an essential autophagy regulator by interacting with a variety of autophagy-related proteins such as Atg14L and UVRAG. Additionally, via its interaction with GASP1, Beclin2 modulates the lysosomal degradation of a subset of GPCRs in an ubiquitination-independent pathway. However, the molecular mechanism underlying this dual action of Beclin2 in autophagy modulation and GPCR degradation remains unclear. In this study, recombinant human Beclin2 was cloned from the human cDNA library, and expressed in Esherichia coli. We conducted biochemical characterization to confirm that the coiled coil domain of Beclin2 is a metastable homodimer and functionally inactive. In a similar manner to Beclin1, Beclin2 homodimer can bind to autophagy enhancers like Atg14L or UVRAG via their respective coiled coil domains and convert to the more stable and functionally active Beclin2-Atg14L or Beclin2UVRAG heterodimer. We determined the structure of Beclin2 coiled coil domain at 2.2 Å resolution by x-ray crystallography. The structure reveals an anti-parallel coiled coil homodimer with multiple "perfect" hydrophobic pairings at the dimer interface to stabilize the dimeric structure. However, there are also several "imperfect" pairings at the dimer interface involving polar or charged residues. Similar to Beclin1, these "imperfect" pairings render the Beclin2 coiled coil homodimer metastable. | en_US |
| dcterms.abstract | Comparison between Beclin1 and Beclin2 coiled coil regions for Atg14L/UVRAG bindings reveals that Atg14L is the preferred binding partner for Beclin2 while UVRAG is preferred by Beclin1. The structure of Beclin2-Atg14L complex was solved at 2.5 Å resolution via x-ray crystallography, which reveals a parallel coiled coil heterodimer. This heterodimer retains the canonical hydrophobic pairings and gains additional stabilizing interactions at the interface, thus render it notably stable compared with Beclin2 homodimer. In addition, structure-based mutations were generated to further delineate molecular determinants involved in Beclin2-Atg14L complex formation. Furthermore, we also evaluate the potency of Beclin2 in modulating the endosomal trafficking of D2 dopamine receptor (D2R). Recombinant human Beclin2 N-terminus and GASP1 C-terminus were purified individually, followed by interaction investigation by a variety of assays. Our current in vitro data did not observe direct association between Beclin2 and GASP1, however, we indeed find that Beclin2 variants, and Beclin2-targeting peptides, exert a functional role in mediating D2R degradation. Further work is required to investigate its underlying molecular machinery. In summary, by focusing on biochemical and structural studies of Beclin2 coiled coil domain and Beclin2-Atg14L assembly, these findings presented in this thesis provide valuable clues to elucidate the molecular mechanism underlying the role of Beclin2 in autophagy regulation. In addition, the biochemical studies on Beclin2GASP1 interactions also provide insights to understand the dual action of Beclin2 in autophagy and GPCR trafficking. | en_US |
| dcterms.extent | xiii, 180 pages : color illustrations | en_US |
| dcterms.isPartOf | PolyU Electronic Theses | en_US |
| dcterms.issued | 2017 | en_US |
| dcterms.educationalLevel | Ph.D. | en_US |
| dcterms.educationalLevel | All Doctorate | en_US |
| dcterms.LCSH | Hong Kong Polytechnic University -- Dissertations | en_US |
| dcterms.LCSH | Autophagic vacuoles | en_US |
| dcterms.LCSH | Cellular signal transduction | en_US |
| dcterms.accessRights | open access | en_US |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| 991022378659803411.pdf | For All Users | 4.93 MB | Adobe PDF | View/Open |
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