| dc.contributor.author | Mei, Yang | |
| dc.description.abstract | Autophagy, a conserved catabolic process required for cellular homeostasis in
eukaryotes, is regulated by many proteins. The central goal of my doctoral research is to
investigate conformational flexibility of autophagy proteins, with a special focus on BECN1, a
core component of the class III phosphatidylinositol-3 kinase autophagosome nucleation
complex that may serve as an autophagy interaction hub.
Our rigorous bioinformatics analysis predicts that 57% of 59 key human autophagy
proteins contain intrinsically disordered regions (IDRs), which lack stable secondary and tertiary
structure. The prevalence of IDRs suggests that IDRs play an important, yet hitherto
uninvestigated, role in autophagy. We confirm disorder of selected IDRs via biophysical
methods, and use additional bioinformatics tools to predict protein-protein interaction and
phosphorylation sites within IDRs, identifying potential biological functions.
We experimentally investigate four distinct BECN1 domains: (i) The IDR, which
includes a functional BCL2 homology 3 domain (BH3D) that binds BCL2 proteins, undergoing a
binding-associated disorder-to-helix transition and enabling BCL2s to inhibit autophagy. (ii) The
flexible helical domain (FHD) which has an unstructured N-terminal half and structured Cterminal
half forming a 2.5-turn helix in our 2.0 Å X-ray crystal structure. Our molecular
dynamics simulations and circular dichroism spectroscopy analyses indicate the FHD transiently
samples more helical conformations and likely undergoes a binding-associated disorder-to-helix
transition. We also show that the FHD bears conserved residues critical for AMBRA1 interaction
and for starvation-induced autophagy. (iii) A coiled-coil domain (CCD) which forms an antiparallel
homodimer in our 1.46 Å X-ray crystal structure. We have also built a atomistic model
of an optimally packed, parallel BECN1:ATG14 CCD heterodimer that agrees with our experimental SAXS data. Further, we show that BECN1:ATG14 heterodimer interface residues
identified from this model are important for heterodimer formation and starvation-induced
autophagy. (iv) A β-α repeated autophagy-specific domain which bears invariant residues that
we show are important for starvation-induced autophagy. Thus, we demonstrate that
conformational flexibility is a key BECN1 feature.
Lastly, we show that multi-domain BECN1 constructs have extended conformations with
no intra-domain interactions that impact structure of other domains, suggesting that BECN1
structure and conformational flexibility enable its function as an autophagy interaction hub. | en_US |
| dc.publisher | North Dakota State University | en_US |
| dc.rights | NDSU policy 190.6.2 | |
| dc.title | Structural Studies of BECN1, A Key Autophagy Protein, and Intrinsically Disordered Regions in Autophagy Proteins | en_US |
| dc.type | Dissertation | en_US |
| dc.type | Video | en_US |
| dc.date.accessioned | 2018-04-24T18:43:36Z | |
| dc.date.available | 2018-04-24T18:43:36Z | |
| dc.date.issued | 2016 | |
| dc.identifier.uri | https://hdl.handle.net/10365/28030 | |
| dc.title.alternative | Structure-based Functional Prediction of Intrinsically Disordered Region in Autophagy Proteins and Experimental Investigation of BECN1, an Interaction Hub in Autophagy | en_US |
| dc.identifier.orcid | 0000-0003-4357-9000 | |
| dc.rights.uri | https://www.ndsu.edu/fileadmin/policy/190.pdf | |
| ndsu.degree | Doctor of Philosophy (PhD) | en_US |
| ndsu.college | Graduate and Interdisciplinary Studies | en_US |
| ndsu.department | Cellular and Molecular Biology | en_US |
| ndsu.program | Cellular and Molecular Biology | en_US |
| ndsu.advisor | Sinha, Sangita | |
