Format

Send to

Choose Destination
Free Radic Biol Med. 2016 Jan;90:59-74. doi: 10.1016/j.freeradbiomed.2015.11.010. Epub 2015 Nov 11.

Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analog of 2-chlorohexadecanal.

Author information

1
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria.
2
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria.
3
Christian Doppler Laboratory for Flow Chemistry, Institute of Chemistry, University of Graz, Austria.
4
Institute of Chemistry, University of Graz, Austria.
5
BioTechMed Graz, Austria; Institute of Molecular Biosciences, NAWI-Graz, University of Graz, Austria; OMICS-Center Graz, BioTechMed Graz, Austria.
6
Institute of Forensic Medicine, Medical University of Graz, Austria.
7
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria.
8
BioTechMed Graz, Austria; Institute of Molecular Biosciences, NAWI-Graz, University of Graz, Austria.
9
Weill Medical College of Cornell University, New York, NY 10065, USA.
10
Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes MK7 6BJ, UK.
11
Institut Cochin, Inserm, U1016, CNRS UMR 8104, Paris Descartes University, Sorbonne Paris Cité, Paris, France.
12
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria. Electronic address: wolfgang.sattler@medunigraz.at.

Abstract

Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analog is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.

KEYWORDS:

Blood–brain barrier dysfunction; Click chemistry; Hypochlorite; Myeloperoxidase

[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center