Protein Oxidative Modifications in Neurodegenerative Diseases: From Advances in Detection and Modelling to Their Use as Disease Biomarkers

Abstract

Oxidation-reduction post-translational modifications (redox-PTMs) are chemical alterations to amino acids of proteins. Redox-PTMs participate in the regulation of protein conformation, localization and function, acting as signalling effectors that impact many essential biochemical processes in the cells. Crucially, the dysregulation of redox-PTMs of proteins has been implicated in the pathophysiology of numerous human diseases, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. This review aims to highlight the current gaps in knowledge in the field of redox-PTMs biology and to explore new methodological advances in proteomics and computational modelling that will pave the way for a better understanding of the role and therapeutic potential of redox-PTMs of proteins in neurodegenerative diseases. Here, we summarize the main types of redox-PTMs of proteins while providing examples of their occurrence in neurodegenerative diseases and an overview of the state-of-the-art methods used for their detection. We explore the potential of novel computational modelling approaches as essential tools to obtain insights into the precise role of redox-PTMs in regulating protein structure and function. We also discuss the complex crosstalk between various PTMs that occur in living cells. Finally, we argue that redox-PTMs of proteins could be used in the future as diagnosis and prognosis biomarkers for neurodegenerative diseases.

Keywords: Alzheimer’s disease; Parkinson’s disease; biomarkers; computational modelling; molecular dynamics; neurodegenerative diseases; redox post-translational modifications; redox proteomics.

Figure 1

Different types of redox-PTMs occurring in Cysteine residues of proteins (oxoforms), their interchangeability and reversibility. Cysteines (Cys) can undergo oxidation forming different oxidized species, which can be converted into other oxoforms. Generally, there are two primary levels of redox-PTMs on Cys residues of proteins based on cellular oxidative stress levels: reversible and irreversible Cys oxidations, as indicated by the coloured arrows. Reversible oxidation of Cys, such as S-nitrosylation (S-NO), S-sulfenylation (S-OH), S-glutathionylation (S-SG), and disulphide formation (S-S), occurs under low oxidative conditions and modulates protein function. At higher levels of oxidants (indicated in the orange box) or prolonged exposure to oxidative stress, the oxidative state of Cys becomes elevated and more difficult to reverse (S-sulfinylation, S-O₂H) or irreversible (S-sulfonylation (S-O₃H) or S-sulfinylation (S-O₂H) for all proteins except peroxiredoxins). These over-oxidized forms lead to loss of protein function and are commonly associated with cell death and disease pathophysiology. The blue box summarizes the chemical and enzymatical processes that can be used to experimentally reduce Cys. Adapted from [35,36,37,38]. TCEP: tris(2-carboxyethyl)phosphine; DTT: dithiothreitol; ROS: reactive oxygen species.

Figure 4

Crosstalk between redox-PTMs in the regulation of Parkin. The E3 ubiquitin ligase Parkin is post-translationally modified by multiple redox-PTMs that regulate its activity in a coordinated manner. Depicted are some examples of the complex interplay of redox-PTMs-driven regulation of Parkin activity. In physiological conditions, Parkin participates in the maintenance of the mitochondrial network homeostasis through the clearance of defective mitochondria by mitophagy. Mitophagy was shown to be promoted by increased S-sulfhydration of the deubiquitinase ubiquitin-specific peptidase 8 (USP8) [186], which in turn enhanced Parkin deubiquitination and recruitment to damaged mitochondria [186]. Additionally, direct hydrogen sulfide (H₂S)-induced Parkin S-sulfhydration leads to the promotion of its activity, increased mitophagy and consequent prevention of protein aggregation, enhancing neuroprotection. However, under increasing levels of reactive oxidative species and reactive nitrosative species (ROS/RNS), such as in NDDs, there is an increase in aberrant S-nitrosylation of Parkin and a concomitant decrease in Parkin’s S-sulfhydration that results in an overall decrease in Parkin enzymatic activity. This decrease in turn leads to impaired clearance of misfolded proteins and subsequent accumulation of toxic oligomeric species as well as impaired mitophagy, with consequent neurodegeneration [177]. Additionally, S-sulfinylation and S-sulfonylation of Parkin are associated with its aggregation [178]. Conversely, S-sulfinylation and S-sulfonylation of DJ-1, which lead to its inactivation [22,171,174], modulates Parkin S-nitrosylation, enhancing its activity and altering mitochondrial quality control with a predicted impact on PD pathogenesis [171,179]. Adapted from [47]. Figure created with BioRender.

Figure 2

Overview of a generic tag-based reductive switch-labelling method for monitoring reversible protein thiol oxidations. In a conventional method for studying cysteine oxidation, the first step (in blue) involves alkylation of the free thiols, which can be achieved using a conventional alkylating reagent (such as N-ethylmaleimide (NEM), iodoacetamide (IAM), iodoacetate (IAA), etc.) to block free thiols (corresponding to the most common method) or a modified version of the alkylating agents if there is interest in studying the reduced Cys. During Step 2 (in green), reversibly oxidized thiols are reduced with a reducing agent that can be unspecific (such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) for reducing all reversible Cys) or specific for a given modification (for example, arsenite for S-sulfenylation (S-OH), ascorbate for S-nitrosylation (S-NO), glutaredoxin for S-glutathionylation (S-SG)). The nascent thiols (reversible oxidized Cys) are then alkylated (Step 3 in purple) with an alkylating agent different from the one used in Step 1 or modified for use as probes or coupled with tags for purification/enrichment. The final step (Step 4 in grey) may involve separation (SDS-PAGE, HPLC) or enrichment (biotin affinity purification, antibody immunopurification) and identification of the alkylated Cys residues/alkylated proteins (typically by immunoblotting or MS analysis). Adapted from [35,36,37].

Figure 3

Essential steps in a molecular dynamics simulation workflow. Knowledge of an initial molecular structure is combined with established simulation parameters and—frequently—bespoke additional parameters to represent the PTM to produce a computational “system” that also represents the biologically relevant environment. From there, a variety of methods of molecular dynamics simulations may be employed—see text for details.