Chemical Probe Design & Development for Assessing Protein Modification and Cross-Linking

Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the capacity of antioxidants in a cell. The CNS is particularly sensitive to oxidative stress and increased oxidative stress is implicated in the development and progression of neurodegenerative diseases. One of the results of increased oxidative stress is the peroxidation of polyunsaturated fatty acids (PUFAs). Lipid peroxides then undergo degradation and produce electrophilic lipid peroxidation products (LPx) which include 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE). These LPx contain two reactive functionalities that result in the formation of proteins adducts through different mechanisms. The aldehyde is capable of reacting with the terminal amino group of Lys residues to produce reversible Schiff base protein adducts. Additionally, the reaction of Cys, His, or Lys residues with the α,β-unsaturated aldehyde results in the formation of an irreversible Michael addition adduct. This bivalent reactivity has been shown to promote the formation of protein oligomers, implicated in neurodegenerative diseases, such as Aβ, tau, and α-synuclein. The methods commonly used to detect these protein adducts are chemical derivatization, immunoblotting, or LC-MS/MS analysis. These methods suffer from different limitations, therefore, we have developed a dual color near-IR imaging method that allows for the visualization and quantitation of the Schiff base adducts, Michael addition adducts, and protein crosslinking induced by these LPx. By adapting this method we were also able to enrich these modified proteins and use LC-MS/MS to identify HNE protein adducts in the lysates of neuronal SHSY-5Y cells. Several nucleophilic small molecules have been reported to protect tissues and cells from conditions of increased oxidative stress by behaving as chemical traps of LPx. It has also been reported that the trapping of LPx by these small molecules results in a decreased level of LPx protein adduct formation. We have shown that these trapping agents react with LPx in vitro and protect against the death of SHSY-5Y cells induced by the treatment of exogenous LPx. Our imaging method demonstrates that trapping agents containing a free thiol (glutathione, N-acetyl-cysteine) significantly decrease the formation of protein adducts. However, this method shows that the neuroprotective activity observed from certain aldehyde reactive scavengers (histidyl-hydrazide, hydralazine) is a caused by a prevention of protein cross-linking, not an overall decrease in protein adduct formation.