Conformational Modulation of α-Helical N-Acetylated α-Synuclein

Author

Ricardo Fernandez, Virginia Commonwealth UniversityFollow

DOI

https://doi.org/10.25772/HX92-RC22

Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Heather R. Lucas, PhD.

Abstract

Human N-acetylated α-synuclein (^NAcαSyn) has long been accepted as an intrinsically disordered protein (IDP) predominantly found in terminal nerve regions in healthy subjects. ^NAcαSyn structural fluidity enables the formation of transient or dynamic assemblies. Some of these assemblies may serve functional, yet undefined purposes. ^NAcαSyn is highly susceptible to the surrounding environment, leading to misfolding under high cytosolic oxidative stress conditions, which in turn assemble in the form of toxic oligomers or amyloid fibrils. These ^NAcαSyn inclusions along with their accretion and migration within neurons, are pathological hallmarks of Parkinson’s disease (PD), dementia, and multiple system atrophy (MSA). Isolation protocols from human tissue samples have revealed the co-existence of a labile, yet physiologically relevant tetrameric conformation (^TαSyn) of 58 kDa. ^TαSyn is featured as α-helical and aggregation resistant, residing in dynamic equilibrium with its disordered monomeric counterpart. Isolation of ^TαSyn was initially challenged by its dynamic nature, hampering thorough and detailed studies of the influence of biometals on the complex, including its equilibrium with monomeric ^NAcαSyn. Our lab designed the first robust recombinant expression platform that enables purification of intact, human ^TαSyn from bacterial sources.Indeed, our purification platform using E. coli as a convenient expression host makes the elusive ^TαSyn conformer accessible for systematic biochemical studies needed to characterize its structure and biophysical features. Our studies corroborate previous reports that ^TαSyn is α-helical and retains its helical packing when challenged by environmental conditions that generally stimulate aggregation for the monomeric counterpart; this later feature is ^TαSyn notably appealing as a potential PD drug target. Rigorous characterization studies showed that ^TαSyn is concentration sensitive, allowing us to cater the purification platform to increase throughput. Advanced computational analyses suggest that ^TαSyn maintains resilience by packing its hydrophobic self-aggregating NACore region, stalling toxic oligomerization.The mechanism, however, for ^TαSyn formation and disassembly remains elusive.

Considering copper dyshomeostasis plays a fundamental role in a variety of neurodegenerative disorders, biometal binding may disclose an intimate relationship with ^TαSyn transient assembly. Our group has recently depicted (Cu^I/Cu^II) redox cycling to induce ^NAcαSyn tyrosyl radical coupling. The oxidative mechanism of ^NAcαSyn-Cu^I/O₂ to ^NAcαSyn-Cu^II-O₂^.- promotes radical centered tyrosyl residues and may involve a pathway that mimics copper oxidases. Herein, intermolecular and intramolecular dityrosine crosslinking occurs by radical coupling.Cu^I binding selectivity has been described at the thioethers of N-terminal methionine. Alternatively, Cu^II has been elucidated to possess two binding domains; the principal domain is found at the N-terminal region and the secondary domain at the C-terminal region. Studies have described the lower affinity towards the C-terminal region propels aggregation propensity. Based on the fundamental roles and a binding selectivity differences between Cu^I and Cu^II, we described the relationship of ^TαSyn in a Cu^I environment. Our studies describe that ^TαSyn is further stabilized upon Cu^I treatmentunder an anerobic environment. Furthermore, our findings gathered concrete evidence that ^TαSyn disassembles upon copper-bound redox cycling. Evidence suggests that stoichiometric (Cu^I/Cu^II) redox cycling mechanism causes folding rearrangement as a consequence of metal binding changes of +40 residues. Moreover, immunoassay and fluorescent studies suggest divergent folding/unfolding pathways that are guided by redox cycling, but not associated with PD related motifs. Our studies provide insight into metal mediated ^TαSyn structural modulation that is dictated by an environment under oxidative stress. Promiscuous metal coordination and subsequent structural modulation may enable a targetable approach to influence the monomer-tetramer dynamic equilibrium and asymmetry of early PD stages.

Employing photochemical crosslinking of unmodified proteins (PICUP) enables assessment of the folding dynamics between monomeric ^NAcαSyn and ^TαSyn in a metal-rich environment.PICUP is induced when aromatic sidechains are in proximity to each other and oriented for favorable ortho-ortho crosslink formation. ^NAcαSyn amino acid sequence hosts four aromatic, Y39, Y125, Y133 and Y136. These residues crosslink in a covalent ortho-ortho coupling between phenol groups leading to dityrosine formation. Detailed fluorescent characterization has facilitated in mapping the dynamic folding arrangements of ^NAcαSyn and provided a framework for future biophysical and chemical studies of ^TαSyn. Fluorescent studies unveiled that the main dityrosine contributors are Y39 and Y125, and Cu^I/Cu^II binding propels the formation of such. A detailed PICUP study of Cu^I and Cu^II, binding with ^TαSyn have been described. Studies unveiled ^TαSyn ability to isolate itself from the surrounding environment and susceptibility upon copper dyshomeostasis. Extensive characterization pathways of protein misfolding and aggregation are required to be studied for detailed biotherapeutic and biotechnology filing. To acquire a robust description of dynamic changes and dissociation, we stoichiometrically exposed ^TαSyn to PD relevant biometals, paving the way for future targets such as Fe^II/III and Mn^II.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-14-2021