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

Abstract

Cysteine residues in proteins have important biological roles. For example, disulfides 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 to provide further understanding of oxidative imbalance, a phenomenon common to many diseases. Keywords fluorescence microscopy; FRET; oxidative stress; cysteine; thiols; disulfides; Parkinson's disease Thiols are the functional groups in the side chain of the amino acid, cysteine. Depending upon 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 *Corresponding author: mastroberardinopg@upmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 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 t...

Oxidation and nitration in dopaminergic areas of the prefrontal cortex from patients with bipolar disorder and schizophrenia

“…The indirect method by Mastroberardino and colleagues, in which free thiols are blocked and disulfide linkages are reduced to be labelled with CPM, was used to increase sensitivity. 18 For our samples, the direct labelling method was sufficient to detect between-subject differences. In the methods described here, lower CPM labelling indicates greater oxidation of thiols.…”

“…7-Diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin (CPM; Invitrogen; D346) is a specific label for free thiols. 18 We completed CPM labelling using a previously published method by Mastroberardino and colleagues 18 with slight modifications. We used CPM to directly label free thiols without prior modification of the tissue to minimize alterations to the sections and introduction of modifications from the experimental procedure.…”

“…These sections were used as controls to account for nonspecific labelling of CPM. 18 For 3NT and TH double labelling, acetone-fixed sections were blocked with 10% goat serum in 0.3% phosphate-buffered saline Triton X-100 (PBS-T) for 30 minutes. Sections were then incubated with primary antibodies in PBS-T (48 h, 4°C): TH (Millipore; AB152, 1:250) and 3NT (Abcam; ab61392, 1:200).…”

“…Therefore, DAT/TH/3NT intensity = (average intensity of 5 fields from the section) -(average background intensity of 5 fields from the section). We determined CPM intensity using the sections treated with iodoacetamide and N-ethylmaleimide (negative control) to correct for nonspecific labelling, 18 which had significantly decreased CPM labelling, demonstrating its specificity for free thiols. Therefore, CPM intensity = (average intensity of 5 fields from the section) -(average intensity of 5 fields from negative control).…”

“…We performed apFRET with a CLSM (Olympus) using previously published techniques with small modifications. 18,25,26 We used a 60× water immersion objective. Image acquisition parameters were identical to those used for intensity analysis.…”