Structural and biochemical studies of two key components within the autophagy molecular machinery : the Beclin1-VPS34 complex and p62

Abstract

Autophagy is an evolutionarily conserved cellular pathway that clears long-lived or dysfunctional cytosolic components in response to metabolic stresses and other related signals. The key role of autophagy in maintaining cellular homeostasis leads to its implication in a variety of human disorders. Full understanding of the autophagy process can offer new opportunities for therapeutic intervention of autophagy-related diseases. Our studies mainly focus on the structural and biochemical mechanisms of two essential components within the complex autophagy molecular machinery, i.e. the p62/SQSTM1 adaptor protein and the Beclin1-Vps34 complex. P62 is a multidomain protein which has been proposed as the adaptor between ubiquitinated proteins and autophagosomes. The issue we seek to address about p62 is to understand the structural mechanism of how the phosphorylation of serine 409 of p62 leads to enhanced association with ubiquitinated protein and its effective clearance by autophagy. The Beclin1-Vps34 complex is the core lipid kinase machinery responsible for the nucleation and maturation of autophagosome. Our work on the Beclin1-Vps34 complex covers two aspects. The first aspect is to understand how the interaction between Beclin1 and UVRAG, an important autophagy modulator, affects Vps34-mediated processes including autophagy and endosomal trafficking. The second aspect is to identify the key structural domains of Beclin1 and Vps34 that are critical for their association to form the Beclin1-Vps34 complex. For the p62 project we characterized the interaction between p62 UBA domain and mono ubiquitin. Our data reveals that the p62 serine 409 phosphorylation mimic mutant S409E shows about 10-fold stronger binding affinity to ubiquitin than that of wild type. Furthermore, the S409E mutation leads to lower thermal stability, presumably by destabilizing the dimeric form as seen in the wild-type structure. NMR data of the S409E mutant suggests that the S409E mutation leads to conformational changes in p62 UBA domain. Such changes may promote the binding of p62 to poly ubiquitin as in the physiological conditions. For our study on the Beclin1- UVRAG interaction, our crystal structure of the Beclin1-UVRAG complex reveals a parallel coiled coil with a "perfect" interface consisting of five "leucine-zipper" pairs to render the Beclin1-UVRAG interaction highly stable. Our structure-based functional studies show that this interaction is important for endocytic trafficking like the EGFR degradation but less so in starvation-induced autophagy. For the Beclin1-Vps34 project we conducted preliminary screening to map the key domains within Beclin1 and Vps34 that are critical for their interaction. Our experiments have uncovered a fragment in the C2 domain of Vps34 that is indispensable for the Beclin1-Vps34 complex association and full activation of the complex. E.coli expression system based solubility screening thorough the C2 domain of Vps34 has revealed a soluble but unfolded fragment containing the key Beclin1 interaction region. For future studies, more proper approaches will be applied to identify the structural and functional features of the Beclin1-Vps34 interaction.