Hyperphosphorylation of Intrinsically Disordered Tau Protein Induces an Amyloidogenic Shift in Its Conformational Ensemble

Agnesa Shala,Alexandr Bezginov,Adnan Sljoka,Gerald Audette,Derek J Wilson,Shaolong Zhu

doi:10.32920/14668836

Abstract

Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.

Highlights

Full length tau protein and it’s splice variant isoforms [1] were originally identified in their normal biological role as promoters of cytoskeletal stability through specific interactions in microtubules [2]
Time-Resolved ElectroSpray Ionization (TRESI)-Hydrogen/Deuterium Exchange (HDX) was implemented on a custom microfluidic chip that supports a ‘bottom-up’ workflow (Fig. 1A) [26]
In order to examine the low protection factors associated with residual structure in intrinsically disordered protein (IDP), the chip was operated at flow-rates resulting in HDX labeling times from 42 ms to 8000 ms, and an average residence time in the digestion microreactor of 6.6 s

Summary

Introduction

Full length tau protein (htau40) and it’s splice variant isoforms [1] were originally identified in their normal biological role as promoters of cytoskeletal stability through specific interactions in microtubules [2]. Great interest in tau was briefly ignited when it was found to be one of the two main amyloidogenic species in Alzheimer’s [3], the other being the Amyloid-β (Aβ) peptide. The mechanisms driving tau pathology are poorly understood, with often conflicting evidence for splice variant ratios [1,6], total concentration [7], non-physiological interactions with vesicles [8,9], misprocessing [10] and erroneous post-translational modification [11]. The role of mis-phosphorylation, hyperphosphorylation by GSK-3β has emerged as a significant occurrence in tau pathology [12–14]. Without an understanding of the structural implications of hyperphosphorylation, it is unclear if it is a causative agent [11,12,15], a cellular response to amyloid formation [14], or even a protective mechanism against pre-fibrillar aggregate toxicity [16]

Methods

Results

Conclusion