A FRET-based method to study protein thiol oxidation in histological preparations.
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.
Mastroberardino
Pier G
PG
Pittsburgh Institute for Neurodegenerative Diseases and Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15260, USA. mastroberardinopg@upmc.edu
Orr
Adam L
AL
Hu
Xiaoping
X
Na
Hye Mee
HM
Greenamyre
J Timothy
JT
D000818:Animals; D015153:Blotting, Western; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D011506:Proteins; D013438:Sulfhydryl Compounds