The use of fluorescent markers is a commonly used technique for detecting protein carbonylation. For this purpose organic chromophores such as DNPH have been standardly used. In this project we propose to further enhance the performance of such markers by exploiting cluster enhanced absorption and fluorescence. Such an enhancement effect is required in order to enable in vivo detection of the carbonylation. The advantage of using metal clusters is that they are biocompatible, soluble, robust in terms of optical properties and small enough to go through the cell membranes in contrast to quantum dots. Therefore, small clusters in combination with optical markers such as DNPH are potentially important for sensitive detection and quantification of carbonylation sites.

The aim of the above studies is to eventually allow in vivo monitoring and quantifying the carbonylation of proteins in cells as a measure for the progress of the aging process.

With the incurred intrinsic and environmental oxidative stress in the course of life, the steady-state between carbonylation as the non-reparable protein oxidation and the selective breakdown of carbonylated proteins by the dedicated proteases changes in favor of the accumulation of oxidized proteins (documented from humans to bacteria). Carbonylation of proteins in most cases leads to unfolding and a loss of protein function [1]. In normal conditions cell can deal with the load of moderately carbonylated proteins by targeting them to the proteasomal system for the breakdown. In instances of vehement oxidative stress, cell looses the capacity to efficiently degrade abundant amounts of carbonylated proteins [2]. Instead, the carbonylated polypeptides form high-molecular-weight aggregates that are resistant to degradation and usually interfere with the proteasome activity [3]. Accumulation of specific oxidized proteins is also associated with numerous age-related pathologies such as Parkinson’s disease, Alzheimer’s disease, atherosclerosis, cataract, etc. [2]. The exact cause-consequence relation between protein carbonylation and formation of oligomers or/and aggregates found in protein aggregation diseases has never been substantiated. It is thought that protein oxidative modifications are causative to such age-related pathologies and prevention or removal of altered proteins could delay their onset [4].

Thus far it is not possible to monitor protein carbonylation or carbonylation-induced protein damage on the cellular level. The only available method measures the amount of oxidized amino-acid residues in proteins (mainly lysine, arginine, proline and threonine) in the form of carbonyl (C=O) groups. In order to “detect” carbonyls in proteins, a dinitrophenol (2,4-dinitrophenyl-hydrazine, DNPH) is attached to the carbonyl groups and sequentially detected using spectrometry or anti-DNP antibody. This method requires preparation of cell free extracts and estimates the average carbonylation in samples normalized to 105 to 106 cells. The final detection is based on Western blot (that is hard to quantify) or ELISA methodology. However, while quantification of carbonylation with this method is possible, it is not possible to neither determine the distribution of damage among cells in a population nor detect protein damage on a single cell level. We expect that with the above described small clusters in combination with optical markers such as DNPH we will be able to measure the carbonylation on the level of single cells for the first time and detect the protein carbonylation as a marker of cell damage. The model protein we intend to investigate is alpha-synuclein, a protein associated with Parkinson’s disease when found in aggregated form [5]. Drs. Anita Krisko and Kristina Oresic at MedILS have found that mutations in alpha-synuclein that predispose to early onset of disease, render this protein highly susceptible to oxidative damage (carbonylation) that seems to precede aggregation (unpublished). We would like to use the combination of all the above described methods to detect carbonylated species of synuclein in vivo and finally elucidate molecular mechanisms that precede the initial events of protein aggregation in distinctive protein misfolding diseases (ie. molecular modifications of synuclein). In addition to the development of a novel method (detection and measurement of protein carbonylation in vivo), we will be elucidating some of the most puzzling events in the pathobiology of Parkinson’s disease such as: weather protein carbonylation of synuclein precedes or succeeds the process of protein aggregation and weather other synuclein modifications (such as ubiquitination, phosphorylation and/or acetylation) intensify protein carbonylation.

1. I. Dalle-Donne, D. Giustarini, R. Colombo, R. Rossi, A. Milzani, Trends in Molecular Medicine, 9, 169 (2003).
2. T. Nystrom, EMBO J, 24 (1311) (2005)
3. W. E. Balch, R. I. Morimoto, A. Dillin, J. W. Kelly, Science, 319 (5865): 916 (2008)
4. D. C. Rubinsztein, Nature, 443 (7113):780 (2006).
5. V. N. Uversky, J Neurochem, 103 (1):17 (2007)