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Title: Biochemical and structural studies of p62/SQSTM1 and the beclin1-UVRAG interaction in autophagy and endosomal trafficking
Authors: Wu, Shuai
Degree: Ph.D.
Issue Date: 2016
Abstract: Autophagy is an evolutionarily conserved cellular process that traffics cytosolic contents to lysosomes for degradation and recycling. This process plays a critical role in maintaining cellular homeostasis and its malfunction has been implicated in the pathogenesis of multiple human diseases. There is intense interest to understand the molecular mechanism of autophagy execution and regulation with the long-term goal to target this process for novel disease-modifying therapies. My thesis reports the biochemical and structural studies of two important components of the autophagy machinery, i.e. the autophagy receptor protein p62/SQSTM1 and the Beclin1-UVRAG interaction within the VPS34 lipid kinase complex. The first part of my thesis focuses on p62/SQSTM1, a scaffolding protein that serves as a receptor in the selective autophagy process by binding to ubiquitinated substrates. This activity relies on its C-terminal ubiquitin-associated (UBA) domain, a small motif that binds to ubiquitin with moderate affinity. Cell biology study from our collaborator's lab reveals that ULK1, a serine/threonine kinase critical for autophagy induction, phosphorylates the UBA domain at S409. Such phosphorylation enhances p62's association with ubiquitinated substrates to promote their clearance through autophagy. My thesis has summarized our biochemical and structural studies to delineate the molecular details of how S409 phosphorylation leads to enhanced UBA- ubiquitin (Ub) binding. Our data reveal that the phosphorylation mimicking mutation S409E destabilizes the UBA dimer interface and promotes its transition to the monomeric form that suitable for Ub binding. Furthermore we have identified K422 as another critical residue for UBA dimer interface and our preliminary data shows that post-translational modification at this site may lead to enhanced Ub binding in similar manner to S409 phosphorylation. Thus modulating the UBA dimer stability may be a strategy to regulate p62-dependent selective autophagy.
The second part of my thesis reports our work on the Beclin1-UVRAG interaction. These two proteins are tightly associated to form the regulatory arm of the mammalian Vps34 Complex II. They serve to stabilize the otherwise structurally dynamic Vps34-Vps15 catalytic arm and up-regulate its lipid kinase activity to promote autophagosome biogenesis. Our structural study of the Beclin1-UVRAG complex reveals a tightly packed coiled coil interface that is strengthened by complementary features on Beclin1 and UVRAG respectively. The notable high affinity of the Beclin1-UVRAG interaction is further confirmed by in vitro and in vivo studies. The functional significance of this strong association in regulating autophagy and endosomal trafficking has been investigated by cell-based autophagy assays and EGFR degradation experiment. Our results show that the affinity of the Beclin1-UVRAG interaction is particularly important for the endosomal trafficking process. Furthermore, using the Beclin1-UVRAG structure as guidance we designed a series of stapled peptides with the aim to promote the Beclin1-UVRAG interaction and to enhance autophagy and endosomal trafficking. Our data show that these rationally designed stapled peptides can bind to Beclin1 in vitro, enhance autophagic flux and accelerate EGFR degradation in cell-based assays. These findings highlight the exciting possibility that modulating the Beclin1-UVRAG interaction can potentially serve as a novel approach in quest for autophagy-targeting therapies.
Subjects: Hong Kong Polytechnic University -- Dissertations
Autophagic vacuoles
Pages: xvi, 167 pages : color illustrations
Appears in Collections:Thesis

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