Original Contribution
A FRET-based method to study protein thiol oxidation in histological preparations

https://doi.org/10.1016/j.freeradbiomed.2008.06.018Get rights and content

Abstract

Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique—which can be used in association with standard immunological staining procedures—to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool for providing further understanding of oxidative imbalance, a phenomenon common to many diseases.

Introduction

Thiols are the functional groups in the side chain of the amino acid, cysteine. Depending on the oxido-reductive (redox) status of the surrounding environment, thiols exist either in the reduced form of free thiols (-SH) or oxidized to disulfides (-S-S-). Disulfides are well known to play an important structural role, contributing to the maintenance of the proper folding in proteins. More recently it was found that the formation of disulfides in proteins also exerts critical regulatory functions: vital mechanisms such as protein import, regulation of signal transduction cascades, regulation of the activity of transcription factors, and proper function of the mitochondrial electron transport system rely on disulfide oxidation or reduction [1], [2], [3], [4]. Moreover, the formation of disulfides represents an early, reversible response to oxidative stress: it precedes higher forms of oxidation, e.g., carbonylation as well as the formation of sulfenic or sulfonic acids. Therefore, disulfide formation may play a protective role [5].

Increasing interest in the study of thiols has led to the development of a variety of technical approaches to detect thiols and disulfides. In particular, several spectrophotometry-based protocols were developed to measure thiol levels. More recently, proteomic methods to identify disulfides in proteins have been established [6], [7], [8]. However, means to image exact loci of thiol oxidation in cells and tissues are quite limited [9]. Imaging techniques would be particularly useful, since the redox environment differs between cell types and even subcellularly. In fact, the level of free thiols varies greatly in subcellular compartments: whereas the endoplasmic reticulum provides a very oxidizing milieu, the cytosol is highly reducing [10], [11]. Also, the intra- and extracellular environments are distinct, with more oxidizing conditions in the extracellular space. Finally, in heterogeneous organs, such as the brain, susceptibility to altered redox potential may differ between cell types because of their different metabolic properties.

Research into neurodegenerative disorders would benefit greatly from an imaging-based technology to evaluate thiol redox state in histological preparations. For example, although it is known that alterations in redox homeostasis contribute to the pathogenesis of disorders like Parkinson's disease, Alzheimer's disease and Huntington's disease, basic questions—such as whether the principal targets of oxidation are neurons or glial cells—remain unanswered [12], [13]. Current spectrophotometric and proteomic techniques rely on subcellular fractionation of total tissue homogenates. As such, they are not suitable for detecting differences in the redox state of single cell types in complex tissues.

To elucidate the roles of oxidative stress in neurodegenerative disorders, we developed a new protocol to study thiol oxidation in histological samples. This method can be conveniently combined with classical immunological methods. Therefore—used in association with cell-type specific markers—it provides information about the levels of oxidized thiols in specific cell types. Additionally, by measuring the fluorescence resonance energy transfer (FRET) between fluorescently labeled thiols and fluorescently labeled specific primary antibodies, the method can assess thiol redox state in a protein of interest.

Section snippets

Reagents

Reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified.

Rotenone treatment and animal care

All animal use followed University of Pittsburgh Institutional Animal Care and Use Committee approved protocols and was in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Lewis rats were treated with rotenone as previously described [14].

Primary antibody labeling

The process was carried out using the Alexa Fluor 488 antibody labeling kit (Invitrogen, Carlsbad, CA) according to

Results

Oxidized disulfides can be modified through a multistep process, involving the alkylation of free thiols, the reduction of disulfides, and the modification of the newly formed free thiols with fluorescent probes (Fig. 1). In this study, an important new negative control—in which the reducing step was omitted from the protocol—was included. Under these conditions, the signal detected by the instrument is due to background noise and can be used to set the zero level of the acquisition parameters.

Discussion

Here we described a new method to study disulfides in histological preparations. Our results show that the technique's sensitivity permits detection of variations in disulfides following treatment with oxidants. Additionally, used in combination with FRET analysis, the technique allows assessment of thiol oxidation in specific proteins of interest.

Although oxidative stress is an area of active investigation in many fields of biomedical research, no tools are available to image thiol oxidation

Acknowledgments

This work was supported by The Picower Foundation, the American Parkinson Disease Association Advanced Center for Parkinson Research at the University of Pittsburgh, and PHS Grants U54-ES012068 (J.T.G.) and K99-ES016352 (P.G.M.).

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