Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - VISQC (Visualizing cell maintenance: Chemical tools to investigate the microenvironments of misfolded proteins)

The capacity of the cell to maintain protein quality and function diminishes upon aging and disease. Genetic mutations may result in protein malfunction or structural defects due to an alternation of an essential amino acid. The cell has evolved many mechanisms to handle misfolded proteins; some proteins are refolded by molecular chaperones while others are targeted for degradation by the ubiquitin proteasome system and yet others are sequestered in deposits at a specific cellular location. How cells make decisions on a specific protein destination is only hardly understood and progress in this field is slow due to the lack of useful chemical and biological tools.
In this project we are addressing this question by using a methodology that allows visualization of the cellular response towards an unfolded protein of which the physical properties are well known. Up to this point, we have developed a novel chemo-enzymatic labeling strategy to identify (unknown) protein-protein interactions that is based on a spatially-restricted arylamine N-acetyltransferase (NAT). The NAT enzyme activates chemically synthesized hydroxamic acid probes to nitrenium ions, which react fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. We have shown that spatial localization of the NAT enzyme to specific cellular compartments allows the labeling of proteins only in close proximity of the expressed NAT enzyme. Purification of the labelled proteins is done by the use of bioorthogonal chemistry with the reactive groups present on the synthesized hydroxamic acid probes. This enzymatic labeling strategy is generic and can be implemented to address many biological questions concerning subcellular molecular dynamics.
We are currently preparing stable cell lines that express the NAT enzyme that is fused to a variety of mutants of alphaB-crystallin conferring different stabilities and aggregating behavior. Insight in the maintenance of these alphaB-crystallin aggregates by the QC system as identified by mass spectrometry will provide valuable information on the cellular dynamics and recognition of misfolded proteins. We are additionally exploring the QC response in a variety of cellular compartments such as the cytosol, the nucleus and the mitochondria with the alphaB-crystallin constructs. Understanding these processes will help in the development of novel diagnostics and therapeutics for diseases that are related to misfolded proteins or to a defect in the cellular protein quality control machinery.

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Life Sciences
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