Biochemical and functional characterization of giant ankyrin-G

Abstract

Ankyrins link diverse ion channels and cell adhesion molecules to the spectrin-based cytoskeleton which stabilizes membranes and regulates physiological processes in various excitable and mechano-resistant tissues and cells. They are regarded as key scaffold proteins at these domains. Three members can be found in vertebrate ankyrin family: ankyrin-R/B/G (AnkR/B/G), encoded by ANK1/2/3, respectively. Ankyrins are subjected to extensive alternative splicing events in a spatiotemporal manner, thus further increases the functional diversity. 480 kDa giant ankyrin-G (AnkG) is a neuronal-specific isoform, containing a 2600 amino acids insertion encoded by a single exon 37. It is specifically enriched at the Axon Initial Segment (AIS) and acts as a master organizer to coordinate high density of ion channels and cell adhesion molecules for AIS assembly and maintenance. It has also been linked to a variety of psychiatric disorders including bipolar disorder, schizophrenia and intellectual disability. However, how do the 480 kDa AnkG, especially its large insertion, functions at AIS remains to be elucidated. In my dissertation presented here, the large insertion of the 480 kDa AnkG was systematically studied using a combination of biochemical, structural and biophysical methods. Firstly, I characterized the potent interaction (K_d = 2 nM) between a 26 amino acids peptide derived from 480 kDa AnkG giant insertion and GABARAP, which belongs to Atg8 family and plays indispensable roles in autophagy. The crystal structure of AnkG peptide in complex with GABARAP revealed an additional C-terminal α-helix contributing to this strong interaction. This binding mode can be applied to other Atg8 family members and to a peptide identified in the giant insertion region of 440 kDa AnkB. Overexpression of these ankyrin-derived peptides can occlude autophagy in living cells and animals spatiotemporally. This study not only generated an effective and genetically encodable autophagy inhibitor but also shed lights on how ankyrins might regulate autophagy in nervous system. Secondly, AIS region that segregates the somatodendritic region and the axonal region might play an important role in neuronal polarity maintenance through acting as a diffusion barrier. 480 kDa AnkG is considered as the master organizer at AIS and the top candidate for fulfilling the diffusion barrier function. Here I purified the AnkG giant insertion and tested its effect on protein diffusion in vitro through single molecule tracking and FRAP. The intrinsically disordered AnkG insertion can indeed restrict protein diffusion in solution and on supported lipid bilayers. Further study revealed that this diffusion barrier is predominantly based on intermolecular collisions, and biochemical interactions may contribute additional specificities. In silico simulation mimicking protein diffusion in AnkG coated tube-like AIS structure supports this observation and demonstrates that a hollow AIS, without AnkG filling up the inner of the tube, can still be an effective diffusion barrier. Collectively, AnkG-based diffusion barrier was reconstituted in vitro which shed lights on how AnkG might serve as a diffusion barrier at AIS in living neurons for polarity maintenance.

Date of Award	2020
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology